Citation
Identification of Neurosecretory Molecules in Aplysia Californica and Related Molluscs

Material Information

Title:
Identification of Neurosecretory Molecules in Aplysia Californica and Related Molluscs Genomic Approaches
Creator:
Sloan, Jinnie
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (229 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Medicine
Committee Chair:
Moroz, Leonid L.
Committee Members:
Anderson, Peter A.
Denslow, Nancy D.
Graduation Date:
8/8/2009

Subjects

Subjects / Keywords:
Complementary DNA ( jstor )
Exons ( jstor )
Gene banks ( jstor )
Generally accepted auditing standards ( jstor )
Genomics ( jstor )
Modeling ( jstor )
Molecules ( jstor )
Neurons ( jstor )
Neuropeptides ( jstor )
Signals ( jstor )
Medicine -- Dissertations, Academic -- UF
aplysia, bioinformatic, genomic, in, invertebrate, learning, memory, mollusc, neuron, neuropeptide
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, M.S.

Notes

Abstract:
ABSTRACT OF THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IDENTIFICATION OF NEUROSECRETORY MOLECULES IN APLYSIA CALIFORNICA AND RELATED MOLLUSCS: GENOMIC APPROACHES By Jinnie Amber Sloan August 2009 Chair: Leonid L. Moroz Major: Medical Sciences Several major questions related to the molecular underpinnings of neuronal identity and function such as, What makes a neuron a neuron? What is the genomic basis of unique neuronal phenotypes? How different is the transcriptional profile of one neuron from another? Can molecular techniques be used to determine neuronal identity? remain at the center of research in neuroscience. As a first step to answering these questions, and providing a list of molecular markers to identify specific neurons types, we have attempted to identify and quantify nearly all transcripts likely to be uniquely expressed in different neuronal classes (such as neuron-specific secretory products) and play a crucial role in neuron identity and function. This includes transcripts that encode neuropeptides, prohormones and related secretory signal peptides. Molluscs have served as powerful model organisms for cellular and system neuroscience for more than 40 years (McPhie and Miller, M., 2006; Kandel, 1970). Their nervous systems consist of simplified networks of large identified neurons, allowing unprecedented opportunities to study the principles of organization of neural circuits as well as learning and memory mechanisms. As our major experimental models, we chose Aplysia californica and related species, sea slugs belonging to the class of Gastropod Mollusca and species belonging to 14 cephalopod molluscs. Our long-term goal is to identify neurosecretory molecules of peptide nature using a combination of comparative and genomic approaches. We have chosen neurosecretory molecules as putative molecular markers because they are highly abundant in neurons and are responsible for cell signaling and modulation. Originally, the major limitation in using Aplysia californica, as well as other molluscs, has been the lack of genomic information available for these model species. To overcome this limitation, we have aimed to bridge the gap between genomic and non-genomic models. The Moroz lab has sequenced > 980,000 ESTs/cDNAs from eight key mollusan species (Gastropods: Aplysia californica, Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea stagnalis, and Cephalopods: Octopus vulgaris, Nautilus pompilius). These sequences were assembled and cross-annotated using the extensive transcriptome and genomic information from Aplysia californica. My thesis deals with comparative analysis and identification of both evolutionary conserved and novel transcripts that encode neuropeptides, prohormones and other predicted secretory products. As a part of the project, we have also employed computational approaches to predict novel signal molecules based on shared predicted protein motifs that are conserved across all secreted signaling proteins. Through this work, I identified a selected list of candidate transcripts predicted to encode secretory molecules across selected molluscs and identified putative neuropeptides present in individual neuronal classes including motor neurons, sensory neurons, and interneurons. This work also led to the identification of a set of neuropeptides that is co-expressed in sensory and motor neurons. The developed molecular resources and the ability to map gene expression has allowed me to provide a detailed study of the genomics of identified cells and provide a critical bridge between genes, circuits and behavior in the broad evolutionary context. Overall these 15 identified neurosecretory products may be the first step to creating a comprehensive list of gene products expressed in individual neurons that can be used for molecular identification of neuron types. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2009.
Local:
Adviser: Moroz, Leonid L.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31
Statement of Responsibility:
by Jinnie Sloan.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Sloan, Jinnie. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2010
Classification:
LD1780 2009 ( lcc )

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peptides: identification, cloning processing, distribution, and action. J Neurosci. 19(21), 9618-
34.

Myomodulin-like Neuropeptides Precursor3*
Gene Bank Accession Number: EU934739
Reference: n/a
CDS:
AGT CA GGGAAT CGTTT GCA T GGCGA GA CGCGGACTATA GAA GGA GAT GGCAAT CT GT GT TT CACAT CA
ACACAAACACA GGA GGAAAAC CATTAAAAGATCAAACAT GGC GCAA GCATAAAATGA GAT GT CGCA G
TACCCTATCACACATAATTGTATAGAATAGGCTTTTTGAAAAGATAAATATACCCAAACATGGCGAAG
CT GGA CACAA GA CGT GGCA GAAAAACAACA GCA GA GAATT GTA CAAAAAAGTCCTAT CAT CCACTT T
GTGA CGTCATGGGGAGTGT GTGAGACGT GAGA GATCCGCCT CCTGTAACTCGTGTTA GGTTTCAGTCA
T GCGA CAAT GGCGA CGGT CACGTT TT GCTT GA GGCT GAAA GCTT CCTA GGAACA CCCGGGAT TCGTT C
CCT GTTT CCTT GA TCTCT CTACA CTAAACCACATTA GA CCT GT CA GTATT CT GTTAACATATT GGAA GCT
TACCATTCCT TTATTT CTTT CTA GAAAAATGAA GTATT GGT GT CAAT GCGATTTTAAAAT GT CCACTAA
CTTTTAA CCCTAACCCT CAT GGTAA GA GGATATAATAATTCTAT CGTT CAAAGATAACCA CTT CCT CCA
T GTTTT TTTAA GGGGGAT GT CATATTATAT GAT GT GT GT CACTT T GAAAACT GTTTAAACT CCCGAA GG
TATTACA GATAAATTT GACTT TT GATT GAA GAT GT T GT TATTTTTT CTTT CA GA GCA GTTT GTATTTT CG
CATTT CGGCA GA GGTAA CCAT GAA CAT GA CCAATCT CCA CTT GCATT GTA CAA GTACGACTAAGAAA G
AAAAATGCT CTTTATA CGCCTATT GCA CCGTACA CGGCT CCTA CGGTATACGCT GAAT GT GGCAAAAT
GGT TTTT GGCT CCAT CATAATACAAAAAGTTAACGTCCT GA GTCTTT CTT CAATCCCCCT CTTT GGGT TA
GTT GGC TA CATTAA GCT GA CA GA CTTT CATAA GCAAAGC CAC T GGAAAAAAATAAACAAACAAATCC
ACCCCCCAACCCCCCCCCCCCCCAACAAAACAACAAGAACAATTTCA GTTAGGACTTGAAA GAACAA
AAGGGTAATAGTGAAACATTGTTTCGAGGATTCAGGGAGTAATTCGATTTTATCGGCTTTGGTAACA G
TAGCGT CGT GA GC T CTT CTT GGT TTTA CAAAAAAAAAAAAAAAAAAAAAAAAAGA GGACA GGGCT CG
GGAT CT GGGGCA CGGA GA GT GA GA GAAACA CAAT GT GCT TT GGGGAAAAAAGCACGTAA GT CCCACT
CGTCCCCAGACACGAAGGAGCAAACAGACATCTGGATGGGCGTGGCCACGATCTGTGGTGACATCAC
GGCTGGCGGCAATTAAAACTTT GGA GACATTGACTGGACGCAA GCAAA GCTGGAA CCAAATTATGTC
AACTTTT CAAAGGAA CT GA CGAA GGT GT CCAT GTT CTAT CGGAT CACGTTT GA CTACA CGT CT GCTT CC
TCTA CT CT GCCT T CACGTTTTATTTAAT CGT CTAAGGAACGCGGGGGAA GGCAAA GT CGATT T CAAAA
ACGTAACTCAAGCTTGTTAACACCTGACCCCT CTTCGTCCCCTCT CCCTGATATAATTTAGTAGCCTAG
GACA GACAAAGTCAACACACATATCT CAGTGGCA TTT GGAATTAAAGGGTTTTCCGAGGAGTGTCTTT
ATCT CA GAT CTTAA GGA CAAAC GA CA GGAAAT GT GAAACA CAAAAGGGAA GCAACA CACGT GCGAAA
GGAAAAGATAAATAT GTGAATTGAAAAATACACTGGCGAGGAGCAAAGTTCAATAATGCTCTGAAAA
AGAAACGA CA GCCGGGGGCT GAT CTT GTTAAAAGA CAAACAAACCCGAAA CACAA GAA GA GA GGA G
AGGAAAAGA GCT GA CTAAATGA CTAAAACCAAATTAAAACAAAGGT GA GAA GA GGA GAA GATTT GA
CGATT T CGTTAAATA GACCAATACATTTT TCATTT TT GAAACA CATT GTA CGTTTTAT CA GCGT GA CCT
AGCA GCT GA CCCCA CT CCCTTTA CTAATCGTTTT CT T GTT GAACT TT GGCA GTT TTATTT CCGCT GA GA C
ACAATAATGTTTGC GTTATCCTCCCAAAGACGGCATACATTTTCGTGCA GCAATAATATTCAAGGTTCT
TTAAAAGCACAAATGAAAGAGGATATTGAGGC GTACATTT GTCTTGAAATATAAAGT CGAATTTTAGA
ATGA GAAAAAGA GCGA GA GA GA GA GA GACGA GAA GA GAAAAAGCGTACAA GCGGTAAATTTTATT G
ATTTTACAAT GTAATAATGCT TTT CC CCA GACT CAA GAAAAAACAAACAGA CAAATAACAACAAACGA
ATGCTTTGACAACTAAAATAACATTTCCTGAAACTCAACAACAAAGCTAAAT GCACCCCACACCCCCA
AACCCCCCTTCCTTTGCAATCACAAAAAGAAAGTGA CACAAAACAAAGCAAAAAGCACACGC GACAA
GACA GCATTAGGAGAAAACGAGCGAAA GCATTACCA GGGAAATAATTTTGTTTCT GT TTCATCACA GA
TTAACTATTTGTAACACGAGGGAAAATTTGAAAAGATTCCTTCCCTCACCTCACCCGTCCCCTTCCCGG
AGCT CT CCATTT CGT CTT CATAACAT GATA GCAT GA TA GTTAATATTATTTTTT CCCT CCTTTTTT GT CCC
CACCA CACA CCTTAT GAA CATA GGTACCATTTATAACA GGA GAATCTTT GAT CT CCT CT TA GACT GCAG
CAGGAAAGGCAAATCGA GAAAAAAAAATCACTTT CT CCGTTT CCGTTTTTATATTTTTATACATGTACA
T GTAATTCTTTTT GTTT CAATA GCATTT CAACCCT CCT CTTT CTTAAATAAAGCACGAATAATTTT GAAA
ATAACATAATAGCGCA T GTTTCTTATTCGCGGGCCTTTAGTAAGTACCGTAATAGAATATAACAGCAG
CGTGTATTCTTGT GAATGGT CTTT GAA CCTTTATGTTTACT CAGGGA GTGTCACT CTTTCT TTACACCTT
CGTATT CTACAAGA CTGAAACAAACGGGATTGTTTAAAAATGCTTTATCGGGCTCTCATTATGAAACG
TCAGTTCAGGATGTTCGATCTTGAGACACCAAGTCTCTACACTCCTCTTTGCACCCTCCCCCTGCCCCC























U \ L7 Frequency
-, ,-I i'- l .. |'.|l l~' .d SN Frequency
SO MCC Frequency








Neurosecretory Protein 1 20 22 23 24 25 26 27 28 29 3031




Figure 4-2. Digital expression of neurosecretory products predicted by traditional cloning.









In addition to providing the ability to identify and isolate individual neurons, the giant

polyploidy neurons ofAplysia contain vast amounts of genetic material with a chromosome copy

number up to 100,000n (Gillette, 1991) and RNA content estimated at up to 0.2 pg in some

cases. This makes Aplysia an attractive model for gene expression profiling as large amounts of

RNA can be made readily available for sequencing.

Limitations of Sequencing Technology

Over the last several decades, our understanding and application of high- and low-

throughput methods to study gene expression has continued to grow. Each method has different

sensitivities as well as advantages and disadvantages due to basic technological constraints and

the absolute number of each mRNA molecule to be measured. One major obstacle to using

high-throughput sequencing to measure gene expression is the amount of data generated by this

method. For research scientists, the complexity of the transcriptome becomes overwhelming, if

not impossible, to understand without the use of bioinformatics approaches.

As the sequencing technology continues to grow, and EST coverage begins to reach

genomic scales, many researchers are faced with a daunting task of sifting through generated

data to find transcripts with function relevant to their research aims. To address this problem,

our lab has devised a method for quantifying transcript expression and abundance, in a user

friendly output, to create a digital expression profile (DEP) for each transcript. We have applied

this method to individual neurons in the memory-forming network ofAplysia to determine what

transcripts are relevant for the function and maintenance of specific cell types, including motor

neurons (MN) L7 and R2, sensory neurons (SN) and MCC interneurons.

Here, we applied the method to a selected list of transcripts previously predicted by our lab

to encode secreted signaling molecules to quickly screen hundreds of transcripts from individual









Cross-Species Analysis

Using the analysis of predicted Lottia SSMs, a cross-species transcriptome analysis of

Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea

stagnalis, Octopus vulgaris, and Nautilus pompilius revealed homologous predicted SSMs in all

related molluscs (Table 2-2). For each species, the percentage of all Lottia secretary proteins for

which homologs exist varies across species (Figure 2-5), suggesting different evolutionary rates

between species. Species with more homologous proteins may be closer common ancestors to

Lottia than those with few homologs. O ne precaution to such conclusions however, is that the

number of transcripts in the EST collection for each species varies (i.e. 273,922 inLymnaea vs.

10,209 in Melibe) and fewer homologs may be present simply as a function of low coverage.

My analysis also shows that classically and non-classically secreted proteins evolve at different

rates across species (Figure 2-5). There is a consistently higher percentage of non-classically

secreted proteins than classically secreted proteins found in each species, suggesting non-

classically secreted proteins are more highly conserved.

Genomic Organization of Selected Neurosecretory Products

Our preliminary comparison of predicted neurosecretory products across species revealed

they are likely subject to great evolutionary divergence. To determine if variability in

evolutionary divergence may be due to differences in the genomic organization, the intron/exon

boundaries of selected neurosecretory products were analyzed using the recently released Aplysia

genome, available on trace archives ofNCBI. Most vertebrate genes are composed of seven to

eight exons, however the number ofintron/exon boundaries found in the selected neuropeptides

fromAplysia is relatively small, an average of four exons (Table 2-3). While there is some

variation in the number of exons between selected neuropeptides (most have 3, 4 or 5 exons),









downloaded and added to the above results in FASTA file format. All redundant sequences were

removed and the master list was aligned with cDNA ESTs fromAplysia as described below.

To ensure that all results represent SSMs, all Aplysia homologs were screened using

SMART (Schultz et al., 1998), and any sequences with a transmembrane domains or enzyme

protein family domains were removed.

Genomic approach h

To facilitate prediction of the Lottia secretome, 23,851 full length protein sequences were

downloaded from the Joint Genome Institute (JGI) website (www.jgi.doe.gov, May 2009).

These sequences represent the best filtered protein-coding gene models as determined by JGI's

genomic assembly. The latest release ofNCBI's non-redundant protein database, NR, was

downloaded to provide annotation for any predicted secretary products. NR contains protein

sequence entries from the following databases: GenPept, Swissprot, PIR, PDF, PDB, and NCBI

RefSeq.

Classical Secretory Prediction*

The original data set of 23,851 full length protein sequences from the Lottia genome was

analyzed with TargetP 1.1. Batches of 1000 sequences were fed to TargetP via a custom

wrapper script (Citarella, M.R. unpublished), written in Perl using the 'short' output option for

TargetP. Sequences were considered to be targeted to the secretary pathway if they had a SP

secretaryy pathway) score > .80. All other sequences were discarded from the prediction. The

selected sequences were then analyzed with SignalP 3.0 for the presence of a signal sequence

using both Neural Network and Hidden Mark Model methods and the 'short' and 'no-graphics'

output options. Proteins were determined to have a signal sequence if SignalP returned a "Y" for

four out of the five scores for the Neural Network method a nd the Hidd en Markov Mod el

method predicted that it contained a signal peptide. Transmembrane domains were then











Feeding circuit activating peptide precursor
Gene Bank Accession Number: 22947345
Intron/Exon Boundaries:
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Placement in
Number Sequence Length Sequence
1 GACCAGACAG CCTCAACTTC CCACTGCAAG 87 GTAAGAAAAC -18-69
2 TCTCGTTCAG ACACCGGAAC AAATGTAAGG 2162 GTCAGTTACA 70-2231




Intron 1


Exon 1 2




FMRFamide neuropeptide precursor
Gene Bank Accession Number: 84551
Intron/Exon Boundaries: Coding Region: 149-1942 nt
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Placement in
Number Sequence Length Sequence
1 tcaaatct ag AGTTCTTCAA CCGACTGATC 137 gt aagttgct -20-117
2 caatctgc ag GCGCCCGTGA CTGTGCGAGA 140 gt gagtaccc 118-257
3 ggtatctg ag ATTTGGCCGG AATACAACCA 1695 gt gatatttg 258-1952




Intron


Exon 2 3




L11 neuropeptide precursor
Gene Bank Accession Number: 155779
Intron/Exon Boundaries :
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Placement in
Number Sequence Length Sequence
1 ggaccccc ag GTCATCATGC CTGCACGTGA 117 gt tgtcgttg -6-110
2 cccacagg ag GTTTGITTTCG CCTACAACAG 141 gt gggctata 111-251
3 ttttccacag AGTTTTCTTA ACGAAACTAT 87 gt caatagac 385471


Intron

UI


No oovesae In genom


Exon 1










GGTA CA GGGAA GA GGGCGGT CT CCA CT GTCA CCGA GAA GGAAATCCCA CT CT GCT GGCA GAT CGCGG
ACAAAGAAATCAT CGATAT CAT GCTT TTAATACA GCGAA CCGCAAA GAAACTCTCTT CCT GCCT GAAT
ACCTGCCCAGAACTGTGACGACATGTGTGAAAGCATTGGCTCAGCTGCGGCAGAGTTGATTTGATGAT
GCTGATCGCTTGGTCTTTATACGCCTGAGAGCTCTACACTTTCGCCAAAGAACGCCAGAC

Hypothetical protein 2 secretaryy, pheromone like peptide similar to seductin)
Gene Bank Accession Number: AAN83922
Reference:
Cummins, S. F., Nichols, A. E. et al. (2004). Characterization ofAplysia enticin and temptin, two
novel water-borne protein pheromones that act in concert with attraction to stimulate mate
attraction. J BiolChem. 279(24), 25614-22.

Insulin-like 2+
Gene Bank Accession Number: 19850964
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGGAACCACA GT CTACCCTCGATTTAT GT GT GACCTT GA CT CTT CT T GT TT CGA CATT CGGA GTAAT G
ACA GCTCAGGGA CAACTCGTGTGTGA CAGAGA CTCAAGGCCACACCCAAGAGGTATCTGCGGCTCTCG
TCT GA CTCGGGCCCA CAACAACCT GT GCT TCCT GTT GA GT CAAACCTA CCCCGA GTA CTT CCCGGGCAA
GA GAT CGGCCT CACCA GA GCTACA GGC GT CT CAAAACAAACGT GTT CCCAA CCAAA GCCT CATT GA CG
GAT CTA CA GCAT CCT CT GT CT GT GCT GCCGACCTT GACCTAT CTA GGGACGA CT GGGCT CGCT CGCCT T
CAA GAGACCTTTGGC GGGCCCAGCCCT GCCAGGA CCCTGAGGGTAGA GTAAGA GTCCGCGGCGACCC
AGA GGCCA GT GCCAA GCT CAATCGGT TCA CGGCCAT GAT GCTA GA CT CAAT GGCA GGGA GAA GGCT G
AAGA GA GGA GGAACT CA GTCCAA CAT GGT CT GCGACT GCT GCTA CCATAT GT GCT CA C CTA GA CAACT
CGCCA CTTA CT GTTA G
Protein:
MEPQSTLDLCVTLTLLVSTF GVMTAQGQLVCDRDSRPHPRGICGSRLTRAHNNLCFLLSQT YPEYFPGKR
SASPELQASQNKRVPNQSLIDGSTASSVCAADLDLSRDDWARSPSRDLWRAQPCQDPEGRVR VRGDPEA S
AKLNRF TAMMLDSMA GRRLKRGGT QSNM VCDC C YHM CSPRQLAT YC

Insulin-like 7+
Gene Bank Accession Number: 94434859
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGACAGGAAACCTCGACAGTGGACTGGCATTCACACTACTGCTGGTGACCTCGACGATGACCTTCAC
ACATGGAGAGACT CGATATTGCCGA CCTGGATTTTCCAGGCCTCATCCCAGAGGCTTCTGCGGCTCAG
CTCTAGCACGTCTGCA CGCCAACTTCTGCCTGCTACTCCGCTGGGCCTACCCA GAACACTTTCCCATGG
GAAAACGATCTGCTGACCGGGCAGCCCACCATCCGCCCA CCACTCCGCCCACCATCCACGGC
ATCCCA CTGTCCGCTCT GGCCGAT GTGTCA CTTACTGACCACGAGCA GCTCAGAACGATGCTATACCCC
GACGCCTCAGGTGAGCTCAAGAAACCATTGCTGAGTGAGCATGAAATTAATGAGGTCTGGATCCAAG
ACAACGCACCATCTTCTTCCCCTGCAAATCCCTTCGTTCGACGCTTGCGCAAAAGAGCTGCGCGGGGA
AAACGTAGTCTTGTTTGTGACTGTTGCTACAGTGCGTGCGATGAGCGGAAACTGGCAGTGTATTGA
Protein:
MT GNLDSGLAFTLLL VTSTMTFTHGETRYCRPGF SRPHPRGF CGSALARLHANF CLLLRWA YPEHFPM GK
RSADRAAHHPPTTT SSPTIHGIPL SALAD VSLTDHEQLRTMLYPDAS GELKKPLL SEHEINEVWI QDNAP
SSSPANPF VRRLRKRAARGKRSLVCDCCYSACDERKLA VY








































Figure 2-1. Summary of model organism and number ofESTs/cDNAs collected. More than
980,000 ESTs/cDNAs were collected from the CNS of five key model species (Pleurobranchaea
californica, Clione limacina, Tritonia diomedea, Melibe leonina, andLymnaea stagnalis). Here,
the number of ESTs for each species is shown under their name.









D digital E xp session P rofling ..................................................................... ..................69

APPENDIX

A MODERN VIEW OF ANIMAL PHYLOGENY ....... ......................................... 220

B M O LL U SC A N C L A SSE S ......................................................................... ....................222

L IS T O F R E F E R E N C E S ............................................... ............................... .........................223

B IO G R A P H IC A L SK E TC H ............................................................................. ....................229












































7

















0.0005


005

0 I C II







I I11"11 1 |

LLI















elk 6S1 ev2O \9iU 0- d
OMCC Frequency 0o y~'O St
Predicted Neurosecretory Product \G




Figure 4-4. Digital expression of neurosecretory products predicted by genomic approach.









Isolation of Fulicin in the CNS of Aplysia

454 sequencing revealed higher expression of transcripts aligning to the coding regions of

the MIP gene than real time PCR (RT-PCR) experiments (Table 3-1). We found that MIP

showed strong sequence similarity to another gene, Fulicin, in its 5' region (Figure 3-4) as

supported by previous work in Mytilus edulis showing MIP and F ulicin have a structurally

similar C-terminal region (Hirata et al., 1988; Kim et al., 1991).

MIP and Fulicin share 5' UTR and coding region

We postulate that the shared 5' untranslated region may serve as a molecular mechanism

underlying the observed co-localization of these two neuropeptides. To understand the

functional role of this shared region, we extended the sequence of the coding region into the 5'

UTR of each of these genes to check if the shared sequence continued upstream of the protein

start site.

Using 5' RACE, we extended the non-coding region of both Fulicin and MIP to reveal a

480 nt region, called TxFrag 2, that includes the 5' UTR and the 21 amino acid signal peptide of

MIP and Fulicin (Figure 3-1A, blue and Figure 3-5).

TxFrag2: Structure and Function

Alignment of TxFrag2 to genomic data available onNCBI reveals that the 480 nt region is

transcribed from four defined fragments of DNA separated by three intergenic splice sites,

characteristic oftransgenic noncoding RNA. The fourth defined fragment contains the signal

sequence shared by both MIP and Fulicin. At the end of the signal sequence there is a splice site

in both MIP and Fulicin, the start of Exon 2 marks the beginning of the unique coding region of

both genes.

Previous work mapping short noncoding RNAs (sRNAs) indicates sRNAs cluster at the 5'

and 3' of genes. Similar to these results, Transfrag2 is located at the 5' of MIP and Fulicin. Like










Gene Bank Accession Number: n/a
Reference: n/a
Full Length Clone:
CDS:
GAACTTTTGCACT CCC GCGGAAACT GGAT CGAGGCAGAAATA GAAGCAGA GAGT CACCCAAAACCAT
TCA CACAT GAGCGAAAACTTCCTACCAAACAAAACTATTACGT CATA GCAACT CGGCT GC GGGTATAT
ATAAGCGCGGA CGGAATCA CGGT CAAACATA GGGC GCTACAGAATTGATTAAAGA CGGACTTAA GCT
TTGACTA GGTTGTAAGTGCTTGCCT CCCTCCTTGCATT CTCAA CAAAAACAACTACAATAACAACGCAA
ATTCT GCTTTA GA CT GAAAAAAAAGTATTCA CA GTATA GGA GA CCCTCCGTT CCGGC GAT CAACAA GA
AAACGAACTT CTGAAAACGAACAAGGAAAGGC TCA C GTGTTCAGAC T GAGGCTACGAAACAGAAACA
ACATATATACA GA GACGGA GA CT GTTA GATT GAA CCA GT GAATT GAAA GA CCA GGAA GAA GAATATT
GAGGCCCTTGCAACGAACGACTTTAGAGCGAAAGAAAAGTTTCTCTGAACTGCAAATTGTAACTTTCG
TCCCTCCTGCCAAAAACACTCCTGCACAGTCATGGAACCCCAACTTCTCAGCGTCGTCATTCTCGTTCT
CTGCGCATGTGCCGCGTCTGCCCTTCCGACTAACAGCAGACGAGAGGACCTGAGTCATCTGGTGACTC
T GCT GGGGAAACT GAA GAACATT GA GCGA GCA CGCCA GAATA GCGA GGGGT CA GCA CA GGCGCAT GC
ACAAGAGGCATGGGAAGGTCCCA GAGTGTCCAGCTTCCCA GAAACT GAGGTCA CAGCTGA CGTCATA
CCA GA CGAA GT GT CA GCGGCCAA GGGGTAT CT CGGA CA GA GGCCGGCGA CT GGC GT GGTAAACAA GC
GACA GGGGGCGTGGTCTTA CGATTACGGTCTGGGC GGTGGCA GGTTCGGGAAGAGAAGCTACGGGGA
TTACGGTATCGGAGGTGGACGGTTCGGCCGTGACGTGGACCATGTAGATCTGTCGGACGCCACCGAGA
ATGA GAT TACGT CAT GAA GGGC GA GCA GGGT CCGA GT GGGT CAAGTA GAA GT GA CGGC GTAA GAA GA
AATATCTCCATCTTTTTGTGACATCACAAGACA CATTTGCTGA GGAATGATGCTTGAA GAGGTCGCTTC
TACTTGCAAT CGGGT CGTGAATCGTTGAGAACGAT GAGATAGGATTAATTAAACAGAAATGAAACCCT
TAAAAAAAG
Protein:
MEPQLLSVVILVLCA CAASALPTNSRREDLSHLVTLLGKLKNIERARQNSEGSA QAHAQEAWEGPRVSSFP
ETEVTAD VIPDEVSAAKGYL GQRPAT GVVNKRQGAWS YD YGL GGGRF GKRS YGD YGIGGGRF GRD VDH
VDLSDATENEITS

Leptin precursor 1
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
ctgaattttggtcttcctaataaaacatctggtaggaccaatacttcagaaaggagcagaatgtcccccaaatgtgcatttctaaacaggaacactttttt

Leucokinins precursor
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
GGCTGGGACTCTACCACAA GCGTAGGA CAGAA GGGATTTTTTCCATACGTGATCTA GCTTCCCAGTTTT
AGAATCATTTCCATTGAGGAACGAATCATTTATCTTGTGGAACCAGGTCCGATTATATTATGTCAGCAG
AAAATTCAAATTCATACGAAAACCT CAAA GAATTT GT GT CATTTT GAA CCA GAACGGA CT GA CAACTT
GAAATTCTGAATATTTTAAATTATTAGTTACGGCAAAAATAAATTATAGCGCAAAGAAGAAATACGGA
TTTT GCAT GT CA GGA CTTT TAAACCCATAACA GAT GGT TAT GCAAA GGAATTTTATTT CAATTT GA GCA
CCGCATGTGAGGAAGTTT CAAAACCTGT CAACTCAACATTT CGAAAAAAACAAATGACTTAAAAATCT
TAGTACTGCGTGAAAGGAGAAAGTGAAATCATGGAGAAACCATTTTCCATATTGGGTGCTTATAAAGT
GTTTT CGA GCA GAAACA GGCCCA CTAA GGA TA GTT GTTT CCGTTTT CCAAACT CAAAT GT GT T GCGGAT
AGCTA CT GT CTTT CT GA TTT GT CTACT GGGT TCA CAA GACTAT GCA GTT TTAT GCGAA GTT CA GGA CT C
GGCAGTCCGTGA TTTGACTACATCT GTACACACTTTTACCAAAAGATCCAGTGGGCCTTTAGACTCCTT
AGAAATATTGCACTTCGGGAACGACGGGAAGTTT GGAGAATTTTCTGTGAAAAGGTCGGATAACGTTG
ACCACGAAAACGAAATTGTTGTTGTGAATACAGAAGTGCCAGAGTTTTCGGAAGGTGTTGTTTCTAAA
AGCGA T GT TCTA GAAAAACTT GCA GA CGA CTTT CTT GAA GGGGA GGGGACAAATATTATTCCT GAAAA









LIST OF ABBREVIATIONS

BLAST Basic Local Alignment Search Tool

CNS Central Nervous System

ER Endoplasmic reticulum

ESTs Expressed Sequence Tags

DEP Digital Expression Profile

IN Interneuron

MN Motor Neuron

NCBI National Center for Biotechnology Information

ncRNA Noncoding RNA

NR protein database for Blast searches, compiled by NCBI

PCR Polymerase Chain Reaction

RACE Rapid Amplification ofcDNA Ends

SN Sensory Neuron

sRNA Short Noncoding RNA

SSMs Secreted Signaling Molecules

UTR Untranslated region



Symbols

* Cloned by Jinnie Sloan

+ Cloned by the Moroz lab











Phase II


TargetP

23,851 3,640

SignalP

I 1,853

THMHH
1,034


Predion Results Phase III Honllog Resilts
Blast against ESTs
--0 181-

24

-- -- 75

+- 50

----- 38

S--- 296

p 94
.)I. 128


Final


167 101
Predicted
Expressed in 2 Secreted
or more species Signaling
Molecules


Figure 2-4. Workflow for Computational Prediction of SSMs. The filtered gene models for
Lottia gigantea were downloaded from JGI. After filtering the original 23,851 predicted proteins
through four protein prediction software programs, 859 proteins were predicted to be classically
secreted signal molecules. That is, contained signal sequences, no transmembrane domains, and
were targeted for secretion outside the cell. Using BLAST alignments, 400 c lassically-secreted
homologs were identified inAplysia ETSs.










GCTAAATTTT GTAATTCT GAATT GATTT GA GGGTATTTTTT CGAA CATAATATCAATAT GACCA CTT GG
ACT CATATAAAAAATTCGTT CT CGATA CGCCAT GA CAAAAAGCT GTAAAT GAACTAAAT GCA GA GTA G
AAAAGAGAAGAGCTAAATTGCGAT GCATGTTTCAAGTCGGAGATAAAACAAATCGAGTGCTGGTCAA
CA GAT CT GCCA GC CGTTAT CTTAA CGGT GC TACTA GGC GTTTA CT GGCAACAAT GA GCGT GA TA GT GTT
TATTGTCAGTAGTAGCGTGACGTGAGATGGGGGAGCCGATCAATGTGTTGCTTTATTAATAAGGAAGA
GAAAAAAAAGAAGGGGGT GGGT CT GCGT GGGAA GGAAATTT GT GGGA GCA GGCA TTTTT GTTTT CGC
GCGAACCCATT GGCCA GTGTATAAATCAT CGTCTCATT TAAGACAGAAGTGTCTAGAAGCAAGACCGC
ATT GGT CA GAT CTT CGA GGGT GTAATTA CTACT GGCTT CCT CT CTTTT CT T GTAAAAT GCGT CAAATTA
GAAA GCCT CT GAATAAATTCCTT T CGT CCGGT TT GT GATT GCCGT TATTT GCCATTATTACCCGGGGCTT
GCCGAGAAGCACGGGAGTTCGACAACGATAAAACCATCGGGCGAGCTGTTGGAGAGAGGGAAACGA
ACCGCT CGGGGAA CGGT CGCA GAA CT GT GCGT GA GAA GAAAAAGACGA GCT CGT GGT CA CTTT GT GG
TCATCTTCATTGTCTGCTGACCCCCA CGGAGATTT CATTCGACTCCTCAGACTCATAATAACATCTCCT
GT GTCT GA CAAACA GGTTAAAGGAT CA GT GAA GCTT GAA CGA GT GT TTT CTTA CCATT TCGT CT CTA GT
GA CCAA GA GCT CGTAACTT GGCT CAAAGA GTA GACCGGT CTT CT CAATATATAGTATTTAA GT GA CTA
CA GTA CAAATTTCA CTTTAT CTA GA CA GCCTTT CCCCCA GA GA CCACT GT GA TAAAACTATTAGGT CAA
TTTACTT CA GAT CAT CT CATT GA CGTTA GT CT GGCATT GAAATACT CGCT GT GCT GGAT CTATT GCTCGT
GT GGTT GCATTTT GGT GCTA GTATTACT CT GCGT CTACGCCA CT GAAACAACA GCAT GA CTT GCGCCA G
TTATTTGA GGACTATCCCCGCT CT CGT CGTTGCAGT CCTATGTCTGGGAAGTGCGCTGAGCTCACCGTA
CAGAGACGAGGACCTGAGACCATTAATTGCGAGCTCGGCCCTTGACTCGGACACAGGTGTCTTCCTGG
ACATGCGCACACGGCGTGGGAGTGGCCAACAGCTGTATTACTCACCGCATTCTTTCACCAGCAAGAGG
ATGAATCAGATATCGTGCAGCTGCTGTT CCAATTCCTT CAATAGCAACT GTT GCTTT CAGTGCATGCA G
AGGCTT GGAAA GA GAA GCGGGGCAA GAT CA GA CAACAAAAATGA GGCT CCT GAA CT CCT GA GGAACG
GACCA GAGCTATATTATAACGAAGGCGGCCGCA GCAGTGA CCATGA CCTCCTT CCAATGCCCCTT CAA
CCCGA GGTCAGCGATTGGC GAGCCAAAGGCA GAGCTTGCT CGTGCTGTCTTTATCAGATGCACCAAGA
GCCGTT CGCT CA GT CTT GGCCTT GCT GTA GATT GT GCAA GA GAT CCAAGCGAT CT GTT GGA GCCGT GCC
CGCACCAGCTGAGACCAGCAAGAAAGAAACGTGAAGAAAGTCTTGTAGTTGTAGCATTTCAGACTGA
TTATAATGA CTAAATACAACACGA GTAACCT CAAT CA GCGCT GATT GGAATAATCA GAATACGAA GA C
ATGTT CT GTAT GCCT CCT CCATCATATTAT GCT CGAAAATTCATCTATATTTTA GGT GGAAAGTA GAAT
T GAAAGGACA GTTTA

FIRFamide related neuropeptides precursor*
Gene Bank Accession Number: n/a
Reference: n/a
Partial Clone:
GA GTAATCTCCCATTTT TCTTACT GT GT GT GT GGTA GAT GA GGT CT GTCT CTATCTT CAAAAAACGGTA
GAT CATT GT CGT GGGGGACGACTTTAAACAAATAGT CAT GACGA GT CTT TCTCA GA GA GAT GGT TTTA
ACA GATT CACGA GAAA GT CT GA GCCA GT GGGA CA GCGA GA GCGA GCGA GA GA GCGA GA GA GA CA GA
GAGA GAGA GAGAGA GAGAGAGAGAT GGGAGA GAGA GAGAGA GAGA GATGGGA GAGA GAGAGA GAG
AGA GAGAGA GAGAGAGAGAGAGACA GAGA GAGAGA AGACA GAGAGAGAGAA GA CA GA GAGA
GA GACA GA GA GA GA GA GA GA GA GA GA GA GAAA GA CA GA CA GACA GA CA GTTTA GTT TTA GTAA GGT
CTTAAGGTGTTCACAGTTCTGTTTAAGGTCATATGAGCACCAAGGGGTGGATGTCAACAGAGAAGT C C
AAACGGGGAAT CA GGC TTACT CT GCT CA CCT CT CTA GC CTTAACA GCATAAA GT CT GATT T GA CTAT CA
AAGCAC C C C GGAAATAGAAATTCAC CAGAGTAACAAACATACAAAACAAGAAACGGAAAAACGACG
ATAGTAAGGA TGAGGAAAAGAAAAGCA GCTGCAGTT CCCCCTATGTTGTTCCTGCACA GTATGAGAAT
GGCATT GA TAAATCA GCCCAT CTATAATTCTACT CATT CTA CTTT GCATAA GAA CAT GTTAATACCCCC
CCCCCCCCCCCCCCCC TT GGTAATGGCCTAAACACAACTT CGGTCAGCAGCACTGCCCTGGTGGGATG
GTCCTCT GTGCCACACCGGAACAAACA CA GTTTTGTGGATATTGAAAAGGAAACAAGCAGTTGCAAAC
GA GA GCA GGT CGT GTT CTACGAACT GTAAA GCAAA GATT GTT CT GAT GAACCAACGT GT GGA CCAT CA
TCAT CTT CAT CATAATTTATAGTAATAT GTATTATTATT GTTATTATTTATTACTATTATTATTATTATTATTAT
TATTATTATTATTATTATTATTATTATTATTATTATTACT GTT GTT GTT GTT GTT GCTT TT GTT GT TATAAT
AATAATACTAATTATTATTAGTATCCTTTGTATTAATATCATCATTATCAAGACAATCTCAGTATCAAA
ACAATTAATGAT GATAA CTACT GTAA GT C GTAACGTTATATTTATTACT T GTT TT CA GAAAGT GA CGC G
TCAGA CAAGA GAGACTACCTCCGCTTTGGCCGCAGCACTGACGGCAGT TTCCTGCGCTTTGGCCGAGA
CGCTCAACAGAACGGTGACAGCACCAGCGATGACGTCAATGACGACCTAAAACGGGACCCCATTATG
CGGTTTGGAAGAGGGGATGGTTTCCTCCGATTCGGCAAAGGGGATCCGTTTATCCGGTTCGGGAAAAG










GKSGMDNAADGEGALSSDKRASNFVRIGKALSSPGAPAQPSSVGS S SSMSSLSSPSSSSSSHSDSSSSEESLG
DGVLFDDDKPISVASRAHAFVRIGKIPSSAFVRIGKNSDVMVEPALGRRAGYRRGSRGGQSSFVRIGK

Neuropeptide CP2 precursor
Gene Bank Accession Number: 18844703
Reference:
Vilim, F. S., Alexeeva, V. et al. (2001). Cloning, expression and processing ofthe CP2
neuropeptide precursor ofAplysia. Peptides 22(12), 2027-38.

Neuropeptide Y mRNA, complete cds
Gene Bank Accession Number: 155793
Reference:
Rajpara, S. M., Garcia, P. D. et al. (1992). Identification and molecular cloning of a neuropeptide
Y homolog that produces prolonged inhibition in Aplysia neurons. Neuron 9(3), 505-13.

Neurotoxic peptide caeron-like precursor
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
CGCA GTCGGTACTTTTT TTTTTT GAA GAGTTTGCAGATATGACA CTTTATTTCCGCGCTCAGTTCATCAT
CTCTCGCGACGGACTCATCTAATTCCGCCATTGGTACTGTCGCTTGACGGACTCATTGAGCAGATTCTA
GCGTCATGAAGAATGTCGCGTGACTGACTCGTCTAGTAAATGATGTGAGGGTTAATGTCGAAACGTTG
CTCACATGCGCTCTTGA CGCGA CAGGT GCCCTTGAAAATAGGGA GGTTGAA GTCTTGTCTTTTACTGA C
TGCCA GGTT GAA GTAA CA GCATT GGT CTTT T CCA CA GT CGCT GTT GCTA GTA CA GGTCGT GGC GGCCTT
GCGTTTGACGA CCACCGGTCTCTGACAGGTGCCCATTTGACTGAAAGGCGTGTCCGAATCCTTCACAA
GCT GGCA CTT GA GA CCTTT TT GGCA GTT GCA GGAACT GAA CAA GTT GCA GGA CT CT CCCT CA GCGCT G
AGT TT CT GGCAAAATCCGGGGATACCGT CCA GGTT GA GCGCT CT TCT GCT GGGC GT GA CGCGCAT GCA
GCACT CACCCTTCCCGCACTGGTCTT CACGCACACAGTTGTCACCCTGGGCCGCAAA GGCCAGA CCGA
GGCCCAGAGCCAAGACA GCGATA GT

Neurotoxin-like-1+
Gene Bank Accession Number: 71148939
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGATGAAGGTTCTCCTGTGTGTGTCTGCTCTCGTCCTCGTCAGCGTGAACGCAAAGGTGGGTGA CGA
GTGTT GGAACGAAAGCA CGTGCGACCCTGGAGAATGTTGTTTGAAGAA CGAGGGCTCTATGATCGTCA
GCAA GAA GA GAT CT GA CTTT GGA CCCATCT T GGGA GCAA CGAAA GGGA CGT GCCAAAACTATCGGTT
GGAA GGCGA CAACTGCCA GAGA GAAGA CATTTGGGA CGAAGTAACAATGT GTCCCTGTGA GACCGGC
TTGAGGTGTGTCTCGA GGGAA GAGCCGAGATT CGTT CCAGGATT CTTTTCAAAGA GAGTTATTATAGC
GCCTGCCAAACCAGGATACATTTGGCGGGAAAGGTGTGAGAGAATCGAGAGAAACCAGGATTTTTTC
GTCAGCTAA
Protein:
MMKVLLCVSALVLVSVNAKVGDECWNEST CDPGECCLKNEGSMIVSKKRSDF GPILGATKGT CQNYRLE
GDNCQREDIWDEVTMCPCETGLRCVSREEPRFVPGFFSKRVIIAPAKPGYIWRERCERIERNQDFFVS

Neurotoxin-like 2+
Gene Bank Accession Number: 71148941










TCAAATTCT CAAATTA GGCCCT GA GCA GATA GAACTT GT GATT CT GGA CAATAATCGT CCA CTTATTTT
CATAAGCTTGGGCTTTCCCCCTCGTTT

R3-14 peptide 2
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
ATTAGGAAGGGGGGCCGATCGCAGCGGCCATTTACCCCACTATAGGGGACCAAAAGGGGGGTCCCCG
CCCCCCCCCCTA GGTT GGA CGGATAT GTTTAACTT T GAATT GAATTT CTT CGGGGCGGA GGCGGT CGGG
GTTTAAAT GCAA GAAACCCATAA GGCTTT CCA GGTTATT CCCCCT TCGGTTTT GGGATTT CCCT CCCCC
CTGAGGATTATAATTTGGCAGCCCCAGTGAACTTAACTGGTCGGC GGGGGCT TAAAACATCCGCAATT
AAAATGAATGGAAGGGCCCTAAAATTGCCCTCACCTAGTTTAAACAACCGTATGCCCTGC T CACCGTC
GAA CGCT CCGTATACCGGA CTT TA GCAT GGA GATTAT CA GGGGA GAAA GGGGT GGT GTTTT GGGGTTT
TCCTCTCTACAGCTTTTTTGACGCACCGAGCCCCCTCA CCGATGGGGGGGCTCCAAGTTAGTTTGGCCG
TTTTCCTCCGCCGCCAACCTTTGGATTCATTCCCGCTTGGGTCCTCTCGGTTCAACCAGAACCAAAGAC
TGGCTTTTTTTGCTGTTTCCCGAAGGGA GGTTGTGA GTTTGTTTCTTTTTTTTGCGGGAAATCCTGCCCC
TTTCAT CCCCTTT CGGGGGCTT GGT GGTA CAAAACCT CCGCAT CCCA CCCCTAAATTAATT GTTTT TA GT
AGTTTTATAGGGGTATACAATTCATTTAATTTTTCTGGGGACGTTTTTTTTCCCTTTGACAGTTTTGGCA
GAT TTTAAAAACAAACGGGAA GCA GAA GAA CCCT CCGCCTT CAT GA CCCCCCTTT CCA GA CAAATTGG
GCAAAAGCACCTTTGGGGTGCCGTCAACCCCGACCGTAAACCCGGCCCTGGGT GGCCGT CACGGCC
GCTTCCTCATTTGTAACCGAAACCGATACGGGGGGGGGCATTTTTCCGGGGC

Schitos omin-like
Gene Bank Accession Number: 56200042
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGAT GAGACAACTGTTCGTCCT GGTCATCGTGGCCGGAGCCGTGCTCGCCGACAACTACTGGTGCCC
CAA GGCCGGC GAGGCTTTCGAGTGCTTCGAGTCGTCCCCCACAGAAAGGTT TT GCCTGACCAACGGA C
GCGA GACCGCTGTCATCTGCTCCAAGTGCA GGAA GAAGTTCGA CTTCTGCCGTAACGACTT CGTCAAA
TCCAAGAGAGCCCACACTGACTGTGGAGCCGGTTGGGAAAGCACTCCCTGCACCCACGACAACTCACA
CGTGCCCGCTGTCTTCCCCGGGAAACTTTAA
Protein:
MMRQLFVLVIVA GAVLADNYWCPKAGEAFECFESSPTERFCLTNGRETA VICSKCRKKFDFCRNDFVKSK
RAHTDCGAGWESTPCTHDNSHVPA VFPGKL

Second (short fragments obtained from MS data)*
Gene Bank Accession Number: n/a
Reference: n/a
Partial Clone:
NTGGGATTCGCA GTCGGTACT TTTTTTT TTTTTAGCCTCTT CTTTTTA GCCATTCCTAAAGA GTGAGAGT
TTTTTTATAATTTAATTTTTTAT GAACTT CAAAAGGT GGAA GGA CAA GATT GAAT GATTTA GT GCA GAG
GACA CT CAAAAAAAAAAAAAAATTCATCTCTGCTAATGAGCGATTTCTGCGAGACTAAAATTTGTTTT
ATT GT GGGGT T GATTT GTA GCT CTA CGTT CT GGGA TT GTACGCCT CAAGA GTATAATCACCCCT GGCTT
GAATTTAA GCA GAT CT CA CTACGA CTACT GCCA GT GT GGGT GGT GAATT GT TT CT GT GT CCA GAAAAT
GTTT CCTT CA CCA CAAACATTT CT CA GTT GT T CTT TTTATGATTAATT CATTT CCTTT
GT GCCGGTAT CT CTCGT GATT CT CTAAAGAAA GAAAATTCACAAACTATTATTA CA GT GTT CCTTT CA C
CTGATGTATCCCGACAAAGGTA CCTGTGGTTAGGCTACCGGACCCGTGGT CGTGAATTGTGGGTCCCG
GGGTT CGAGCCTCAACT CGGTCCGATGT T GCGT CCTTGGGGAAAGGCGTTTGAAGTTTCAAAAATGT C










Gene Bank Accession Number: 158906123
Reference: (Zhang et al., 2008)

Prothoracicostatic peptide 2
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
CAAATGTCCGTT T GGGGTAAAAGAGAT GAAGAT GGCACTTAGATAAGCGGTGGAAACAAAT GTCTGTT
T GGGGAAA GA GGGA T GGA GAT GGCAACTTA GA TAAACGGT GGAA GCAAATGT CCGTT T GGGGGAA GA
GAGACGAGGATGGCAACTTAGATAAACGGTGGAAACAAATGTCTGTTTGGGGAAAGAGA GACGATGA
CATA GGGGA C TTA GATAAAAGAT GGAAACAAATGTCGGTTT GGGGAAAACGCAAT GGGGCT GAT GTT
GGT GGT GATAATTT GGA CAA GA GGT GGAAACAAATGT CT GT GT GGGGCAA GA GGT TACAA GA CCT GG
ATTCAAACAAAAAGT GGGCT CAAAT GT CAA GCT GGGGGAA GCGG GGT TTGT GGGA GAT GTAA GCGA
CGT CGATA GCTTA GTTAACAA GA GAT GGAA GCA GATA GCCGCA T GGGGCAAACGA CT GA GCCA GGA C
GACGA TCTCCAGAAAAAGTGGAAACAAATGCCTGTTTGGGGGAAGA GA GGCGATACCAACGA CGTCG
ACGATAA GA GAT GGC GA GAAAT GGCT GTTT GGGGGAA GA GAAA CT CGGAAAATAAC GA CAAC CGAA
ACATCGTAAACGAAGGGA GCGA CGAAAACACT GCGGA CAAGCGATGGAAACAGATGTCAGTAT GGG
GGAAAAGGGATGCC GGGA CGGCTCCAGCCGCC GTCATGGAAGCA CCATCTCCA GTGTCATCAT CATCA
TCATCCT CATCATCATCACCAACAT CTT CCTTATCGTCCCCCTCATTATCAGCCT CAGCCGCA GAAGCA
GCA GCT GCT GCA GGGGC GCCGC GCGT GGA CAA GA GGT GGCCC GA TA GCT CCGT CT GGGGCAA GA GAA
CGTACA GGCA CATCA GGAACT GGCGA GA CAT GGA CAT GT GGGGCAA GA GGCCCA GCT GGT CGA GGA C
AGGCTT CACCAGCTGGGGAAAACGCT CT GGGGCCGCT GTGGA CCTGGCTCA GTTCAAAGACTATCTCC
TAAACAGCT CCCA GCAACA GCAA CT GCAACA GCA GCA GCA GCCGTT GATA GGT GA GT CGT CAACATA
ATCAATAAAAATGAAAAATAAAATGACGAGGATGAATAAG

Putative pheromone-2+
Gene Bank Accession Number: n/a
Reference: n/a
Partial Clone:
T GGCAAT GGTTTAAAA GGCA GTTAAATCGCAT CTTTT GGTT GA GACATATTTATTA GTTTT CT GA TT CA
TGTAATATCAGTAAAACTATATAGAGA GAGAGGATGGGAAAAGAAAGA GGGAGA GAGA GA GGGGGG
GGGGAGAAA GAGAGAGAGA GAGA GA GAGA GAGAGA GAGA GAGAGA GAGAGA GAGA GAGAGA GAG
AGA GAGAGAAGTAGA GACAGA GAGA CGGAAA GGGGGA GATATATGGAGA GAAAGGGGGATA GTGA
AATTGT CA CTATT GGAATTA GGTT CTTTATA CTATT GT CAAACGCAT T GTA CGTT CTTTT GT GGAAAT GC
GTTAATTATATTAAAAGTAAGGTATCATTTCTAAATAGCGATTGTCAATCCTGAGCATGCATATATTAT
TTTACCTATCTCAACA GAGAAGT TCTCGACGA CCGCCTTCCCCACCACCATCACCCCACGCCGC GT CAA
ACCA GT GA CCACA GA CCT CCCCA GCT CC CCACCGGAA GCGACCA CCAACAT CT GCGACTACCT GT GCT
CCGT GCA GCT GGGT GGT GAT GCCT GCCA GT GCA GTAACCCCA GT CT GCCCGGGAA GAA GTA GTCGGCG
GTTCGAACCCCGCCCGAAACGATGCTCTCACCACAGAATCGTCGGCGCACCTAGCTAGACATCTAGAA
CGTACCGCTGTTTGAACGACTTGTCCCGACACAAGGGGC GCTGTCTGTTCCAAAGACGTAGAGCTGAG
ACTTCCGGTTGTT GTTTTATGTCGTGAATGTATCCAATACAAGACGGA GCATCTTTCGCAAACGTTTGA
CTTTT GTT T GA GGT CT CT GTTT T GAAT CCAAT GGCTTT TTAAAGA CTATATACAAA GT CTT CACATAATT
TATTCTTCCATACGTTT T GCTT T GTCGGTTGTTGATACTTTATACGGCCTGTCCGGGTGCGTTTTGGAAG
T GGCT GTT TTTT GTT GTT GTTTTTT TT GGT GCTTATTTT CCCT CTT CATAAAAGA GA GTAA GGGCAA CTA
TTTT GT T GCA CTTATT GTT GT GTT TATT GTT T CATTTT GCT GGGGAT GTTTT CCT CTTTT GGGAAATT GGG
AACAACTGTGCTTTCGCACCTTCCTGCTTTTAATTTCATTTCTTTTCATTTCCTTCATTGATGTTGGTTTT
AAATAAAAGCA CATCCAATATCGCCCGGTTT GA GCT CAT CTTTT GT CTTA CA GATTTA CCTAA GTTTT G
AACTCT CT TTTTTTATA GGT CAATACTT GGT CA GT CT GT GCCA GCGTATATAAATCTTT GA CAACCTATA
CCAATACGTATCA CTATTAACCCGTCTACCCTAAA GCTACA CA GTTT TATCT GA CTTTT GT GA CCGTTTT
GAGTTACTAAGACCAAGAGGCAGAGACAAATGTTCTATCAGCTCTTTTGAAATCGTAGACAATTCGAG
CCGTATCATGATATTCTAAGAACTGCACGTGAAAAAGCTTGACGTGTTGTGAACTCAAACTTTCAGCG









FMRFamide 5

The partial sequence and clone for FMRFamide 5 was obtained (not submitted) using

terminal primers 5'-GGTGGATCAAGCCTTGGAGCT-3' and 5'-CACTCAGGTTATTCAACACGTCAAC-

3'. The resulting cDNA fragment was 698 bp.

Allatotropin-OR precursor

The partial sequence and clone for Allatotropin-OR precursor was obtained (not submitted)

using terminal primers 5'-CAAGTGGTGATTCGGCCGC-3' and 5'-GTCAATATAGTTCCCTCTCGTGG-

3'. The resulting cDNA fragment was 746 bp.

Second

The partial sequence and clone for Second was obtained (not submitted) using terminal

primers 5'-GCTAATGAGCGATTTCTGCGAG-3' and 5'-CGTCCTTCTCCGAAAGGCCTAAGCC-3'. The

resulting cDNA fragment was 712 bp.



Neurosecretory Predictions

Preditions were performed by Jinnie Sloan, Mathew Citarella, Drs. A. Kohn and L. Moroz.

Software scripts were written by M. C itarella.

Homology search using unbiased shotgun approach

A cross species master list of secreted signaling molecules submitted to NCBI was created

by manual word search. Search terms included any combination of the following words: signal,

peptide, growth factor, hormone, and neuropeptide. Any results containing the word "receptor"

were eliminated.

All sequences submitted to PeptideDB (www.peptides.be, May 2009), a public resource

for bioactive peptides that includes cytokines and growth factors, peptide hormones,

antimicrobial peptides, toxins and venom peptides, and antifreeze proteins, were manually









SFY 1-like peptide ......................................................................................... .. ........ ......36
SFY3-like peptide ................................................. .. ...................... ... 36
FIRFamide related neuropeptide precursor .............................................................36
M ajor royalj elly protein (M RJ) .....................................................................36
F M R F am ide 5 ...................................................... 37
A llatotrop in-O R precursor........... .................. .......... ............... ............... 37
S e c o n d ..............................................................................3 7
N eurosecretory Predictions ...................... ...................... ................................ 37
Homology search using unbiased shotgun approach ................. ...... ............37
Genomic approach....................... .. .. ............................. ... .. 38
C classical Secretory Prediction* ........................................ .......................... 38
Non-classical Secretory peptide Prediction* ................................. .................39
Cross-Species Analysis of Predicted Proteins ........................ .................... 39
Annotation of Predicted Proteins................................................... .... ........... .40

3 MAPPING EXPRESSION OF NEUROPEPTIDE TRANSCRIPTS..................................50

In tro d u c tio n ....................................................................................................... ............. 5 0
M ytilus Inhibitory Peptides .......................................... ................... ............... 50
F ulicin ....... ............................................................. 5 1
R results ........................... ... ......... ...... ..... ................................... 5 1
Cloning ofAplysia Fulicin precursor mRNA ...............................................51
Localization ofFulicin in the CNS ofAplysia ........................... ............... 52
C o-localization of M IP and F ulicin ....................................................................... ... ...52
Isolation ofFulicin in the CN S ofAplysia ................................................. ............... 53
MIP and Fulicin share 5' UTR and coding region ......................... ........................53
TxFrag2: Structure and Function.............................. ................. ............... 53
Discussion .................. ........................................................54
Colocalization of Fulicin and M IP ............................................................................ 54
TxFrag2: M odulator of Expression? .......................................................... ............... 55
M materials and M methods .......................... ...................... .. .. .... ........ ........ 56
Animals .................... ................... ....... .... ...............56
Cloning of full-length cDNA encoding Fulicin.............................................................56
Sequence analysis and A lignm ents ............................................................................... 56
In-situ hybridization of Fulicin and M IP in Aplysia ....................................... .......... 57
Im a g in g ................... ......................................................... ................ 5 8

4 GENE EXPRESSION PROFILING FOR INDIVIDUAL NEURONS ...............................65

In tro d u c tio n ............................................................................................................................. 6 5
A advantages ofAplysia ........................... .......... ................. .. ........ .... 65
Limitations of Sequencing Technology............................................ ...................... 66
Results and Discussion .................. .............. ................ ............ .. ....... 67
Sensory N eurons ...................................................................... ........ 67
Motor Neurons.............................................. 67
Intern e u ro n s .......................................................................................6 8
D isc u s sio n ................... ...................6...................9..........


6









GLOSSARY OF TERMS


* BLAST: finds regions of local similarity between sequences. This program can be used to
compare nucleotide or protein sequences to a sequence database in order to determine the
functional and evolutionary relationship between sequences. The statistical significance
calculated between sequences can be used as a guide to compare sequences and identify
members of gene families.

* Exon: the nucleic acid sequene within a gene that is present in the mature form of an RNA
molecule.

* In situ hybridization: used to localize a specific DNA or RNA sequence in a specific
tissue or cells by using labeled complementary DNA or RNA. Transfrag

* Intron: a region of DNA within a gene that is not translated into protein. These regions
are included in the transcribed pre-mRNA and removed by splicing to produce a mature
RNA.

* Neuropeptide: small protein-like molecules that can be secreted by neurons to
communicate with each other. Neuropeptides can act on neighboring neurons as
anterograde or retrograde messengers or the neuron secreting the neuropeptide (orthograde
messengers) through cell surface receptors to modulate or mediate neuronal
communication. Typically neuropeptides alter the communication of a neuronby
increasing or decreasing its excitability. Neuropeptides are assembled by ribosomes
attached to the ER then transferred to the Golgi Apparatus to be packaged into vesicles and
transported to synaptic terminals. Some neuropeptides are secreted directly into the blood
stream and are referred to as neurohormones. Secreted neuropeptides can have long lasting
effects from seconds to days.

* Prepropeptide: the inactive precursor of a peptide that requires posttranslational
modifications to become the active peptide molecule. The prepropeptide of neuropeptides
is characterized by a signal peptide that targets the protein to the secretary pathway for
posttranslational modifications including the cleavage of the signal peptide in the ER.

* Signal Peptide: a short (3-60 amino acid) sequence made up of a positively charged
sequence found on the N-terminal region, a hydrophobic region and a polar uncharged C-
terminal region (cleavage site) that targets the transportation of a protein. In the case of
secretary signaling peptides, the signal peptide targets the preprotein to the ER for further
posttranslational modifications.

* Transcript: an RNA molecule produced from a gene.

* Transmembrane Domain: a short span of amino acids (15-35) composed of mostly
hydrophobic regions separated by polar connecting loops that form stable secondary
structures in membranes.










Prediction:
GGCTCGACAA GTA GACCTGCTTCAATCCCA GAGGTGAATACTGA GACGAATAAGCATGCAGCT CTTAA
TACTTCTTGCTGCCGTT GTGGGTGTAT CCCT GGCCGCCCCAAGCCCTGATGTTTGCTCCAACGT CAACG
CTA CGGCTATTAA GT GTAT GA CA GACAA CGGAATT CCT CT TCAA GAT GT GCTCT CTAT GCA GAA GGCT
GCTTTGT CTGGGAACATTGCAGCAATGAACCTGACCCGCT TCTGTGGCGACAAAAAAGATGGCTACCT
GAAA GGTGTGGAGTGTGTAATGAACT CCGTGGCTGTGTGTATCCCATCGTTCAAACAAACATTAGGAG
ACTTGGACTT CAAAAAAATGCTGGATGT GCAATGTAC C GACAAAAACATCAAGTACAACTGCCTGGGC
CT GAT GCAACT GCCT GCTAATAAAGCT GTT GT CCA GA CCT GT GT CCA GAAA GCTTT T GA GAAT CT GAA
GGATTT GTCTTCAGACAAGA GA GTTTGTGA GGCGACCAAGA GCGCCTACGTGTGCGA GGATGCCATTG
TGAGGTCATGTGATGTGCACACCGCTGATGTGTACAAACAGTTCATTGACCCTTTCATGAAGAGGGAC
TGCGATAACTTTGTCCT CGGGTGAATGTGACAAAGTGT CCAGGACAACCGGGGTATCAAAGGATAAAA
AAGAAACTCGCAAC

Whitnin precursor (SPTR)
Gene Bank Accession Number: 56200044
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGGAACT CCAA GCCTGTAACATTTTTGCCCTTTTCGTT GTGGTT GTGACGCTCTCA GTTGCCA GCAGT
CTCCCTGCCAGCCGGACGGACGATGTGCTGCAGGAGGCCTCGGGTCTGGCTTTGAACAAACGGCCAAA
GTACATGGACACCCGACGGGACCTTGACTTAAAGACGTCGCTCATCAGCATTCAAGAGCTTG
TGGACGAAAACCGCCTGAACCCCGCCTTGTTGCCAGAGGAAGATGCCCCCAAGCCCGTTGAGAAGCG
AATGCGCTACAT GGGAAT CTGCATGAAGAAACAGTACAACAACTTCATCCCCTTCCCCTGCCTCAGGA
GTGGTCGCTAA
Protein:
MELQACNIFALF VVVVTLSVASSLPASRTDDVLQEASGLALNKRPKYMDTRRDLDVFKDLVLISIQELVD
ENRLNPALLPEEDAPKPVEKRMRYMGICMKKQYNNFIPFPCLRSGR

Signaling Molecules

Wnt-2 protein precursor
Gene Bank Accession Number: 46981372
Reference:
Brown, B., Kohn, A.B et al. (2007). Unpublished.
CDS:
ATGA GATCATCA CCAGGCTATCCACTCTTCATTTT GGAAGTGGTCCTACT CCTA GGGCTTGTT GCTAAG
GAA GTTT CT GCAT CT T GGT GGT TTATCA GCCA GCTAAACATCCAT GCCCT GGGCCCT GGT GT GGT GT GT
GACAACATCCCCGGT CTCGTGGGCAGACAGCGCA GGCTAT GT CGTCATCACCCTGACGTCATGGT GCC
GCT GA GCGAA GGCGTA CGCCT CGGA GT GGAA GA GT GCCA GT TCCATTT CC GGAA CCA GA GGT GGAAC
TGTA GCA CGCT GGA GA GGGATA CCT C CTT GTTT GGGAA GGT CAT GCTAA GA GGCA GT CGC GAA GCT GC
CTTTGTGTACAGCATCT CTTCCGCTGGC GTGGTCCA CGCCATCACCCGTTCTTGCA GTCGTGGCCAGCT
CAT GCA CT GCGCCT GCGA CCCCA CCAAAAGA GGAT CT GGCA GA GA CAA GGGA GGT GCCTTT GA CT GG
GGA GGAT GTT CA GATAAT GTT CGCTACGGCA GTACTTT CT CCA GGAT GTT CAT CGA CGCTAA GGAACG
CAA GCA GA GA GA CGGGA GGGCTAT GAT GAA CTTACA CAACAACA GA GCCGGCCGAA GGGC GGT GAA
GAA GTTCCGCAAACTGGA GTGCAAGT GTCACGGT GTGAGTGGATCCTGTACCATCCGCA CGTGCTGGC
TGGCCATGCAGGAGTTCCGTCTGGTGGGGCAGCACCTCAAAACACGCTACAACGGGGCCACGCAGGT
CATGAT GAAGCAAAACGGCGCAAGTCTCATTGT GGCAGATAAGAA CCACAAGCGTCCTACGCGCTCTG
ACCTCGTCTACTTAGAATCTTCACCCGA CTACTGCGT T GAGAATT CGGAAGTTGGATCGCTCGGGACTA
CA GGGC GCA CGT GCAA CAAATCTT CTAT GGGCA CCGA CGGTT GT GA CAT CAT GT GTT GT GGGCGGGGC
TACCACACACGCGTGGTGAAGCAACATTACAAGTGTGAGTGCAAGTTCCACTGGTGCTGCTTTGTACA
CTGCAAGGATTGTCAAAGGTGGGTGGAGCTACACACCTGCAAAGGCCCCGCCCCTCAGGGTCTCATTG
GCTCCTGA









other intergenic sRNAs (IRNAs), TxFrag2 includes the 5' boundary of the protein-coding gene

but excludes most of the other exons of the coding region. Previous work on IRNAs suggests

they are involved in regulation of gene expression (Davis et al., 2006; Martianov et al., 2007).

These studies suggest that IRNA transfrags serve as precursors to functional sRNAs via sense

and antisense transcription of the IRNA (Kapranov et al., 2007).

To see if TxFrag2 is transcribed in the sense or antisense direction, 454 and SOLiD

transcripts that aligned to the 480 nt TxFrag2 region were counted and compared to the

quantities of sense and antisense MIP and Fulicin (Figure 3-5B). This showed that TxFrag 2 is

transcribed in both the sense and antisense direction where as the coding region of MIP and

Fulicin are made only in the sense direction.

Discussion

Colocalization of Fulicin and MIP

This is a unique example of two neuropeptides showing exclusive co-localization

throughout the CNS ofAplysia. After several in situ experiments, we have found that transcripts

for both MIP and Fulicin consistently colocalize to specific neurons of the CNS ofAplysia,

predominately those neurons in the abdominal ganglia responsible for the gill and siphon

withdraw.

Analysis of the protein sequence of MIP and F ulicin reveal that structurally, they share a

conserved C-terminal. Further analysis of the nucleotide coding region of the sequences shows

that this results from a conserved 5' UTR region that extends through the first exon of both

genes. Preliminary analysis using recently released genomic information fromAplysia

californica further supports that MIP and Fulicin share the same splice sites and exon regions

covering the TxFrag2 region until after the signal sequence.









produces an electropherogram image along with a gel-like image of the sample representing peak

ratios of the 28s/18s RNA contained in the sample. Additionally RNA concentration was

measured spectrophotometrically by using the GeneSpec III systemTM (Mirai Bio).

454 Library Construction

Library construction was completed by Dr. Kohn and Jinni e Sloan. The protocol for

transcription analysis was developed by Drs. A. Kohn, Y. Panhin and L Moroz.

3' end amplified cDNA library construction for 454 s equencing:

This technique targets the 3' end of the transcripts being expressed. Amplified cDNA was

generated using the Marathon cDNA amplification kit (Cat # 634913, BD Biosciences, Clontech,

Mountain View CA). The first strand synthesis utilized the AMV Reverse Transcriptase and an

oligo(dT) primer, Trsa (Matz, 2002). After second strand synthesis and clean-up following the

Marathon kit protocol, the entire sample ofcDNA was fractionated by digestion with 20 units of

Alu 1 and NEBbuffer 2 (Cat # RO 137SO, New England BioLabs, Ipswich, MA) for 1 hour at

370C. The enzyme was heat inactivated at 650C for 20 min. The double stranded adaptor was

made with A adaptors then added to the ligation mixture at a final concentration of 1 IM along

with the digested cDNA, 2 Units T4 DNA ligase and 5 x ligase buffer (Cat # 634913, BD

Biosciences, Clontech, Mountain View CA). The ligation was performed at 160C overnight.

The cDNA was purified using DNAclear (Cat#1756, Ambion Inc, Austin, TX) and eluted in 16

1l of RNAase-free water. Amplification of the cDNA was performed using 16 ld of the purified

cDNA, Advantage 2 buffer, dNTP and taq polymerase (Cat # 639201, BD Biosciences,

Clontech, Mountain View CA). The primers A per B per added to the amplification were at a

final concentration of 0.05 IM with B adaptor at a final concentration of 0.01 pM. This full

length B adaptor was modified to complement with the Trsa primer along with the B adaptor on

the beads and was added to ensure the eventual attachment of the cDNA on the beads. The PCR










Pedal peptide 2+
Gene Bank Accession Number: 94434888
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGGC CAAA GTC GGA GT GA GCA GT CT CT GCCTT GT CCTTAT CCTT CT CT GC T CT GCCGT GA GCGGA GGT
GAA GTT CCA GAT GGA GT TCTCAA GAAAAAATCACAATTT GT CCAA GCCT CCT CCGT GA GT GA GA CA GC
CGA GGG GCA GGT GAT GGT T CT CT CA GTAAACGACAT GT GGGTT CCATCGGAA GTT CTTTTAT CAAAC
GACCT GT CGATT CTATT GGAA GCT CTTTTAT CAAACGACCA GT CGAT TCCAT CGGAA GCT CTT TCAT CA
AACGACCA GT CGATT CCAT CGGAA GCT CTTT CAT CAAACGGCCA GT CGATT CCATT GGAA GT T CGTT CA
TCAAACGACCA GT CGACTCCATT GGAA GTT CCTT CATCAAACGGCCA GTT GACT CCATT GGAA GTT CCT
TCAT CAAACGGCCA GTT GACT CCATT GGAA GTT CTTT CAT CAAACGGCCA GT CGATT CCATT GGAA GCT
CTTT CATCAAACGGCCA GTCGA TTCCATT GGAA GCTCTTT CAT CAAACGACCCGTCGATTCTATAGGAA
GCT CTTT CAT CAAACGGCCA GT CGATT CCATT GGAA GT TCGTT CAT CAAACGGCCA GT CGA TT CTATT G
GAA GTT CGTT CATCAAACGGCCA GTCGATT CCATT GGAA GTT CGTT CAT CAAACGGCCA GT CGATT CTA
TT GGAA GTT CTTT CAT CAAACGGCCA GT CGATT CTAT CGGAA GT T CTTTTAT CAAACGGCCA GTT GATT
CCATA GGAA GCT CCT TCAT CAA GCGACCT GTT GAT TCAAT CGGAA GCT CCTTTAT CAA GCGA CCGGTT G
ACT CTATCGGAA GCT CTTT CAT CAAACGT GGC GTT GA TT CAATT GGAA GTT CTTT CATTAAAAGACCTA
TT GACA CTATT GGCA GTT CGT TTATAAAGCGCGGGGT GGATT CGATT GGTA GTT CTTT CAT CAAACGGC
CGGT GGA CA GTATT GGTA GCA GCT TCAT CAAAAAGA GAA GATT CGGGGGC GAAAAT GAATTTAT GAA
GA GACCAAT CGATA CTATT GGAA GCA GTTT CAT CAAAAAGAAT GT CGA CT CTAT CGGTA GCA GCTTTA
TCAAACGGCCCATTGACACGATTGGAA GCAGTTTTATCAAGAAGAATATGGACCA GGCAGCGTTTATT
AAGA GGCCTATT GA CACTATCGGAA GTA GTTT CAT CAA GAAAAACATTGATT CAATT GGCA GTA GCTT
TATAAAGCGA CC CGTT GACA GTAT CGGAA GCA GTTT CAT CAA GAAAAACATTGATT CTATT GGCA GTA
GCTT CATAAAGCGGCC GATA GA CA CCATT GGAA GCA GTTT CATTAA GAAAAGT TAT GGA CAAAACGA
AATGAT GAA GCGACCT GT GGA CT CGA TCGGCA GCA GCTT CAT CAAACGA CCGATA GA CACCAT CGGG
AGCTCT TTCATCAAGCGA GGGGCTTTCGTTTA CGGGGGA CGCCATTTCA GGAAAAGGCCAAAACCCAC
TT GGCA CA GGTT CAT GAA GA GGCCAATCGA CACCATA GGAA GTA GTTT TATCAA GAA GCGA GAT GT GT
T GGA GGA GGA GGC GGC GA T GGA CGGCA CA CA GGGCAAATTA GGCGCGA GT GATT CCGAA GGT CA GA
GCAGA CGAAAGCGTAGCACGAGCTTCCACGTGTT GGACTCACTT CCCCCTGA GA CGTGTGATCAACA GC
CTGTACA CGTCAGATCTTGATGGGTCACGCGACGAGCCTGCCCAT GAGGGCCA GCTCACGTACGGCGA
CGTA GTGGAGCT CCTCCGATTCTTGGACAGGAACGAGTCTGT CATCAGTGATGAGAA GTTCGGA GA CA
TTCTGGACAAGCCATACCTGGGCTCCGGTCA GATGGGGAGGTAA
Protein:
MAKVGVS SLCLVLILLC SA VSGGEVPDGVLKKKSQF VQA SSVSETAEGA GDGSLSKRHVGSIGSSFIKRP
VDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGS SFIKRPVDSIGSSF
IKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSI
GSSFIKRPVDSIGS SFIKRPVDSIGSSFIKRPVDSIGS SFIKRPVDSIGSSFIKRGVD SIGS SFIKRPIDTIGS SFIKRG
VDSIGS SFIKRPVDSIGSSFIKKRRF GGENEFMKRPIDTIGS SFIKKNVDSIGSSFIKRPIDTIGSSFIKKNMDQAA
FIKRPIDTIGS SFIKKNIDSIGSSFIKRPVDSIGS SFIKKNID SIGS SFIKRPIDTIGS SFIKKSYGQNEMMKRPVDSI
GSSFIKRPIDTIGSSFIKRGAF VYGGRHFRKRPKPTWHRFMKRPIDTIGS SFIKKRDVLEEEAAMDGT QGKLG
ASDSEGQ SRRKRSTSFHVLDSLPLRRVINSLYTSDLDGSRDEPAHEGQLT YGDVVELLRFLDRNESVI SDEK
FGDILDKPYLGSGQMGR

Pedal peptide 4+
Gene Bank Accession Number: 94434899
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGAAT CAAAACTATTT GT CCAAAAGA CCGCT GGATT CCATAT CT GGTA GCAAATTT CACGGAAT GAA
TCAAAACTATTTGT CAAAACGA CCGC T GGATTCCATATCTAGCGGGTTCAACGGAATGA GACAGAACT










TGAGA GGGAT GAGGTA GACGAATT CCCTGATGATTCAGGAA CAGAGGATTT GGA CAAAAGGGC GGGA
TTT GCGCCTT GGGCGGGCAAAAGA GA CGGA GA CGGT GTCGT GGATAAAAGA CCA GCATTT CAT GCGT
GGT CA GGGAAAA GAACA CCACATA GCGCT GTA GAT GCA CA GAAAAGA CCCGC GTTT CAT GCAT GGT C
GGGCAAACGTTCAACGTACCCCTACAAAAACAATGAAAAACGACCC GCATTCCATGCCTGGTCGGGC
AAAAGATCAGAAAAACGGCCTGCATTTAGCGCTTGGAATGGAAAACAAAAAAAAGTACGGCTGCG

LFRFamide precursor*
Gene Bank Accession Number: EU886298
Reference: n/a
CDS:
AAGGTGCGAGCGTCCAGGGTCCAGCTGTCCCAGAATTCCCTGTGTGAGGTCCAGGACAGCACAGCTTA
GCCCTGCCCCGCCTGCCGC GCA GTGTGTT CNCCCCATCGTCT GACAGGCCTCAGTTCGAAACCTCGTCC
CTGAGCAAGCGGAAGACAAGAGAATGGAACCCAGA CCCATCACGCTCAGCCTGGCTGTTCTACTCGC
AGCTCTCTGCACAACCTGT GCCACCGGCT CGGCCAAATTAGTCAGCTCAAGCGAAGACAATACCAACA
GCCACACTGACGGCCTCACCCGGGACCACCAGCCACAGAAGCGGTCAGCGGCGGCCATCTTGTCCCCA
GCCGAT GA CGCGCT GTT GGT CA GA GC GGA CGA GGGA GA GGA GGA GGGA GT GGGA GGAA GCTATCCA
GGGATCCCCCTCTCTCTCCCAGGAACCGCCGACAAGAGAAGCACACTCTTCAGGTTCGGCAAACGAGG
CGCA GGGACCCT CTT CCGGTT CGGGAA GCGA GGCT CT CT CTCT CCGGTT CGGGAAAA GGGGCGGGGCTC
TCTTCCGGTTCGGCAAAA GGGGCGGGGCTTTGTTCCGGTTCGGCAA GAGAGGC GTGGCTT CGGGGTCG
GGGGCGTTCCCGCTGGCTGACGACTCCGACGTGAAGAGGACGCTCTTCCGCTTCGGCAAACGATCCAA
CCTTGA CCTTCTCAGGGACGTCTTGT CGCAGTA CCAGCTCA GCGCCTACCCCGACGCCTACCCCGA CGC
CTACCCCGACGCCTA CATCGAT GACGTA GACAGCAA GCGA GGGGTAGA CGGGTTT CA CTGGGGAGAT
GA C CAATAAGC GAACT GT GT GT GAATA GCCGA GGA CA GGGT GA CCAATAAACTAACT GT GT GGT GA T
AAGCT GA GA CA GGGGT GA CCAATCAACTAA CATT GGGGGGA CATTAACATT GT GATTA GCGAA GAT G
AAGGCGACGAATAAACGAACTTTGTA CACTTTCAGCAAAGA GTGAGACGACGAATAAAATAAGGAAA
TTTCA CAA GTT CAAA GAT GGA GA T GA C GAATAAACAAACATATTTCA CA GGGGCCAAAA
Protein:
MEPRPITLSLA VLLAALCTT CAT GSAKLVS SSEDNTNSHTDGLTRDHQPQKRSAAAILSPADDALLVRADE
GEEEGVGGSYPGIPLSLPGTADKRSTLFRFGKRGAGTLFRFGKRGSLFRFGKRGGALFRFGKRGGALFRFGK
RGVASGSGAFPLADDSDVKRTLFRFGKRSNLDLLRDVLSQYQLSAYPDAYPDAYPDAYIDDVDSKRGVDG
FHW GDDQ

Major royal jelly protein*
Gene Bank Accession Number: n/a
Reference: n/a
Partial Clone:
GGCTC GA GATTA CGT CA CACCA GAT GGAT CGGAA GTCCGGTACGCGTATATTT CT GATACT GGCGATG
CGAAT CT GGTGGTATTCGATTT TCA GACGAA CACGTCAT GGGCTTT CAA GGACA CGACAATGACACCG
GCA CCAAACGGTAT CTT GA CCATTAAT GAT GT CAACTACA CCAT GGA CTTT CCTAT CAACGGAATCGG
AATGT CT CCCAATTT CGATTA CGTCTACTACTGTGT TATTGGCAACAATAACCT GTA CCAGATT CCCAC
GTCA GTCCTCCGAAACAAAACCGCAGA CTTTAAGGAATATGTTA GGAAT GTT GGAGA CAAAGGTGGG
TCCTCTGACGCAATTGTTGTTGGGAAGAAAAATCTGTACTTCGGAGTAATGCAAGAAAATGCTGTTTC
CGA GT GGCAACGCA GCAAA GACAT CAA GT CCCAAAA T GT CT CCGAA GGCGA GGT CAA CAT GGCCA CC
ATACAAGAGCT GACCTCTAACGACGTGACCATGCAATGGCCGGACGGCTTCACTCTGGACGATAAAGG
CTA CCT CT GGTT CA CCACA GAA CGT CT GCA GCT CTA CCT GAAT GA CAA GAT GGA CTT CT CT GGGGGT C
AGGGCTCCAATTTCCGT GT CTTCCGGATGTTCGTT GGT GACGTCAGCTACTTGA CAAAACCCACGA GTG
GAAA GAA GA GT T GGT GGGA GGAATA GCCTT CT GAAA GATTAAAATAGTAT CAACGAAATA GAT GA GA
TTATATTCTAATA GTTTA GTAA GCGT

MIP-related peptide precursor
Gene Bank Accession Number: 8886135
Reference:
Fujisawa, Y., Furukawa, Y. et al. (1999). The Aplysia mytilus inhibitory peptide-related












A Buccal g.

Cerebral g.
7 Pleural g.
\ Pedal g.

/ nominal
JB Abdominal g


Head ganglia


Parapodium


Mantle and
Visceral Hump


Figure 1-2. Organization of the Aplysia central nervous system. A) Diagram showing the central
ganglia that make up the central nervous system ofAplysia. B) Representations ofAplysia
showing the areas, color coded as in (A), innervated by specific ganglia. Some ganglia
(pleural/pedal) have multiple functions innervating the same regions of the body as indicated by
the gradient coloring, however there is some specificity in innervating. [Modified from Moroz,
L.L., Edwards, J.R., Puthanveettil, S.V., Kohn, A.B., Ha, T., Heyland, A., Knudsen, B., Sahni,
A., Yu, F., Liu, L., et al. (2006). Neuronal transcriptome of Aplysia: neuronal compartments and
circuitry. Cell 127, 1453-1467.]


iccal mass







-Intestine









Li, L., Floyd, P. D.et al. (2001). Cerebrin prohormone processing distribution and action in
Aplysia californica. J Neurochem. 77(6), 1569-80.

Clionin precursor+
Gene Bank Accession Number: FJ214338
PubMed ID Reference: n/a not released
CDS:
ATGA CGTCATCCAT GATCCTCGCT GTCCTGGCCATCTCCCTGTCGTGCCTGCTGA CCGCTGTGA CGTCA
TCACCACTTGGCCCAGTCCAGCCGGCCATATCCCTGTCCAAGATGGAACCCGATCACGCCTGTTTGTTC
ATGT GCAACAT CT GTTT CCCGGAT CT GGA GGA CACGGGT CT GCT CCT GGA CT GCA GCAA CAAAGT GT G
TGGTCCCATCATGGC GGGCATGTGTGCTAT GGAGAAGCTCGTCTGGCTGGGCCACAACTGCCGTCAGT
ACGA CAT GGT GCA GAAAAT GT GGGCT CCGCA CGGC GCT CT CTAA
Protein:
MTSSMILA VLAISLSCLLTA VTSSPLGPVQPAISLSKMEPDHA CLFMCNICFPDLEDTGLLLDCSNKVCGPIM
AGM CAMEKLVWLGHNCRQYDMVQKMWAPHGAL

ELH precursor [Contains: Beta-bag cell peptide (Beta-BCP)
Gene Bank Accession Number: 119268
Reference:
Scheller, R. H., Jackson, J. F. et al. (1983). A single gene encodes multiple neuropeptides
mediating a stereotyped behavior. Cell. 32(1), 7-22.

Enticin precursor
Gene Bank Accession Number: 74814556
Reference:
Cummins, S. F., Nichols, A. E. et al. (2004). Characterization ofAplysia enticin and temptin, two
novel water-borne protein pheromones that act in concert with attraction to stimulate mate
attraction. J BiolChem. 279(24), 25614-22.

Feeding circuit activating peptide precursor
Gene Bank Accession Number: 22947345
Reference:
Sweedler, J. V., Li, L. et al. (2002). Identification and characterization of the feeding circuit
activating peptides, a novel neuropeptide family ofAplysia. J Neurosci. 22(17), 7797-808.

FMRFamide neuropeptide precursor
Gene Bank Accession Number: 84551
Reference:
Taussig, R. and Scheller, R. H. (1986). The Aplysia FMRFamide gene encodes sequences related
to mammalian brain peptides. DNA. 5(6): 453-61.

FUR
Gene Bank Accession Number: 453657
Reference:
Chun, J. Y., Korner, J. et al. (1994). The function and differential sorting of a family ofAplysia
prohormone processing enzymes. Neuron. 12(4), 831-44.

Glycoprotein hormone beta subunit (GPB5) +









Feeding circuit activating petide precursor 3 (FCAP-3)

The partial sequence and clone for FCAP-3 was obtained (not submitted) using terminal

primers 5'-AACAGGCGCAGGCTCAGA-3' and 5'-ACACGTGGATCGCCGCCGCC -3'. The

resulting cDNA fragment was 367 bp.

SN4

The partial sequence and clone for SN4 was obtained (not submitted) using terminal

primers 5'-ATGCAGTGAGGGATGCTGTGT-3' and 5'-GGTCAAGGCCAATTTCCCGTG -

3'. The resulting cDNA fragment was 755 bp.

SFY1-like peptide

The partial sequence and clone for SFY1-like peptide was obtained (not submitted) using

terminal primers 5'-GTGGAAGTACGCAGAGAGG-3' and 5'-CATCCCTCTTGTAGAAGCTGGA-3'.

The resulting cDNA fragment was 1198 bp.

SFY3-like peptide

The partial sequence and clone for SFY3-like peptide was obtained (not submitted) using

terminal primers 5'-GTATCAAGACCGACGACCATG-3' and 5'-GTGAGCTCATCCCGCGGTTG-3'.

The resulting cDNA fragment was 729 bp.

FIRFamide related neuropeptide precursor

The partial sequence and clone for FIRFamide related neuropeptides precursor was

obtained (not submitted) using terminal primers 5'- CGTCATCGCTGGTGCTGTCAC-3' and 5'-

CCTCGACAAGGCTTCTCCTTCACC-3'. The resulting cDNA fragment was 2032 bp.

Major royal jelly protein (MRJ)

The partial sequence and clone for MRJ protein was obtained (not submitted) using

terminal primers 5'-CGGAAGTCCGGTACGCGTATATTTC -3' and 5'-

CTTTCAGAAGGCTATTCCTCCCACC -3'. The resulting cDNA fragment was 774 bp.









In-situ hybridization of Fulicin and MIP in Aplys ia

Full-length cDNA from Fulicin and MIP was cloned and used for the preparation of in situ

probes. For co-localized expression studies, clones were made for Fulicin and MIP from unique

regions starting after shared 5' UTR and signal sequence. The antisense probe was generated by

digestion ofcDNA from Fulcin and MIP with Not I (New England Biolabs), then transcription

with T3 polymerase from the DIG (digoxigen) RNA labeling kit (Roche Diagnostics). The

control sense probe was produced by the same protocol but used Pmel (New England Biolabs)

to digest the cDNA and T7 polymerase for transcription. The DIG-labeled antisense probes were

hybridized in whole-mount CNS preparations, and the neurons containing the probe-target

duplex were localized and visualized with alkaline phosphatase-conjugated anti-DIG antibody

fragments (Boehriger Mannheim). The detailed in situ hybridization protocol has been described

(Jezzini et al., 2005; Jezzini and Moroz, 2004; Walters et al., 2004).

Expression ofFulicin was investigated in central ganglia of four experimental CNS

preparations and two control experiments. Control in situ hybridization experiments with full-

length "sense" probes revealed no specific and selective staining in the CNS under identical

conditions and labeling protocols for either probe.

Co-localization using two-color in-situ hybridization was performed as previously

described (Jezzini et al., 2005). Briefly, unique regions ofMIP and Fulicin were cloned and

sequenced. Two probes were then made using different NTP labeling mixes. The MIP probe

was made using fluorescein-12-UTPs and the Fulicin probe was DIG-labeled. First the

fluorescein-MIP probe was hybridized in whole-mount CNS preparations, and the neurons

containing the probe-target duplex were localized and visualized with Fast Red substrate. After

the first development is stopped, a second hybridization is preformed using the DIG-Fulicin









There is currently one additional publication indicating that the preproprotein of two

neuropeptides, brandykinin and temporin in frog, share a conserved 5' UTR and signal sequence

(Suzuki et al., 2007). In this paper the authors suggest that the sequence similarity may suggest a

linked evolutionary history involving exon shuffling.

TxFrag2: Modulator of Expression?

With the completion of several genome projects, including the Human Genome Project,

the once accepted view that of noncoding RNAs (ncRNAs) as "junk" is rapidly shifting to accept

non-protein-coding transcripts are crucial for cellular function. Recent reports suggest that only

2% of the human genome codes for translated proteins (System, 2008) while nearly half of the

genome is transcribed and expressed as RNA without any messenger (mRNA), transfer (tRNA),

or ribosomal (rRNA) functions (Szell et al., 2008). Understanding the role of these ncRNAs

represents a new realm of understanding genomic data, and the accumulating data suggests

ncRNAs may play a role in organism complexity, specificity, cell regulatory machinery, and

regulation of these ncRNAs has been implicated in several human diseases (Szell et al., 2008).

Work on the human genome has demonstrated that the transcriptome does not exist

exclusively of protein-coding transcripts but also regulatory genomic elements and non-protein-

coding transcripts (Szell et al., 2008). This project, termed ENCODE (the Encylopedia of DNA

Elements), along with unbiased tiling-array data has identified a new set of transcripts (TxFrags)

that are made from fragments of defined genomic regions. Cross species genome sequencing has

shown that increasing biological complexity is correlated with increasing number of non-protein-

coding DNA sequences (Taft et al., 2007) and suggsts that differences between species may rely

upon non-coding genes rather than protein-coding genes (Pollard et al., 2006).

While the function of TxFrag2 remains to be determined, expression ofboth sense and

antisense transcripts suggest that it may serve in a regulator mechanism in these cells. It is our









probe and the neurons are localized and visualized with alkaline phosphatase-conjugated anti-

DIG antibody fragments (Boehriger Mannheim).

Imaging

Images were captured with a Nikon Digital Sight DS-5M digital camera mounted on an

upright Olympus SZX12 microscope. Figures were prepared using Adobe Photoshop.











Results and Discussion

Identification of Predicted Secreted Signal Molecules

Homology results

Through cross species homology search, 109 putative neuropeptides were identified from

the Aplysia CNS transcriptome, 88 predicted to be secreted signaling molecules (Object 2-2),

and 21 predicted to be involved in the secretary pathway though not necessarily secreted (Object

2-3).

While homology searches can provide a wealth of information when initially annotating

transcriptomes, it limits annotation to only sequences with homology to known sequences.

Indeed when annotating the >203,000 transcripts of the Aplysia transcriptome (Moroz et al,

2006), only 43,672 sequences can be annotated, showing the enormous complexity of the

neuronaltranscriptome (Figure 2-2, insert). Furthermore, preliminary results from cross-

species transcriptome analysis suggst that many neuropeptides are quickly evolving even

between closely related species (Figure 2-2). Due to these limitations, we suspected that many

SSMs ofthe Aplysia CNS could not be identified through homology searches, and decided to

employ bioinformatics approaches for a more comprehensive list of SSMs using genomic scale

searches.

Prediction results

To identify novel and unannotated SSMs, publicly available software was used to complete

genome-scale profiling of all SSMs. These software programs rely upon conserved protein

motifs to predict prepropeptides. Signal peptide prediction software identifies conserved protein

motifs required for processing and directing proteins to secretary pathways (Figure 2-3).

Accurate predictions rely upon the use of full length peptide sequences. Because there is









CHAPTER 2
IDENTIFICATION OF NEUROSECRETORY PRODUCTS IN GASTROPOD MOLLUSCS

Introduction

Gastropod molluscs have served as powerful model organisms for cellular and system

neuroscience for more than 60 years. The central nervous system (CNS) of these models

consists of a simplified network of relatively large identified neurons, allowing unprecedented

opportunities to study the principles of organization of neural circuits as well as learning and

memory mechanisms. However, a major limitation to identifying the neurosecretory products of

neurons of the molluscan models has been the lack of genomic information.

To overcome these limitations, we have sequenced >980,000 ESTs/cDNAs from the CNS

of eight molluscan species (Gastropod s: Aplysia californica, Pleurobranchaea californica,

Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea stagnalis, and Cephalopods:

Octopus vulgaris, Nautilus pompilius) (Figure 2-1). These sequences were assembled and cross-

annotated using the extensive transcriptome and genomic information from Aplysia californica

(Moroz et al., 2006). This comparative approach allowed identification of both evolutionary

conserved neuronal genes and numerous novel genes including neuropeptides, prohormones and

other predicted secretary products. It is estimated that there are >320,000 putative unique gene

products (including non-coding and small RNAs) present in our comparative neurogenomic

database, which likely correspond to >50-60% of the total number of genes expressed in the

nervous systems of these molluscs.

A selected list of genes identified in that study represents major group of transcripts

implicated in the control of neural excitability, synaptic functions and plasticity, receptors,

adhesion molecules, developmental genes, and homologs of genes involved in neurological

disorders, etc. Specifically, we looked for putative neurosecretory products that may function as









BD Biosciences, Clontech, Mountain View CA). This ligation was performed at 160C overnight.

Again the cDNA was purified using DNAclear (Cat#1756, Ambion Inc, Austin, TX) and eluted

in 16 ld of RNAase-free water. Amplification of the cDNA was performed using 16 [l of the

purified cDNA, Advantage 2 buffer, dNTP and taq polymerase (Cat # 639201, BD Biosciences,

Clontech, Mountain View CA). The primers added to the amplification were at a final

concentration of 0.05 IM and the sequences were A per and B per. The PCR amplification

protocol consisted of 17 cycles of 95C for 30 sec, 65C for 30 sec, 720C for 1 min. Preparation

and sequencing of the Amplicon product used the GS emPCR Kit II (Amplicon A and Paired

End) (Cat # 04 891 384 001 Roche Applied Science) GS Sequencing Kit (70x75) (Cat # 04 853

342 001 Roche Applied Science)

Primers for 3' and 5' libraries are listed in Table 2-4.

Cloning of selected Signaling Molecules

Amplified cDNA libraries were constructed from the CNS ofAplysia californica, as

described elsewhere (Matz, 2002; Moroz et al., 2006). Full-length cDNA sequences for six

sequences and the partial sequence of an additional nine sequences were obtained. The full-

length copy of coding sequences were amplified from appropriate CNS cDNA libraries and

cloned into pC R4-TOPO (Invitrogen). For each sequence, three clones were isolated and

sequenced by SeqWright (Houston, TX).

Hypothetical protein 2

The full length sequence for Hypothetical protein 2 was identified based on BLAST results

against the National Center for Biotechnology Information (NCBI) as Aplysia californica

hypothetical protein previously cloned (Cummins et al., 2004) (Genebank accession number

AAN83922). Using terminal primers 5'-CTCTGAACCGTCGCGAACTGTGT-3' and 5'-











Predicted:
GA GA GAAATACAATCCTT CT CAANT CGCA GGA GA CGGGA GAAACA GAT TTT GTT GT GT GATAACGT CA
GATTATCTCCTCGAGGGAAACGCGCGCTGAGAAATAGTAAATAGTGAGCCGAGGAATTTATCTATCGT
TTTGTGCTTCTTCGCGCAGTCTTGTAATTGAAGAGTGGGCCTCCTTCCAACTTTTCTTCCCTTCTTCTGTT
TGACAT CACTGA CCGCAA GT CGA GT GTT CT CT GGT GT T GTGT GTT CTTATAATT GT GGAA CTA CT GCA
GAGGATTTGTTGTATGGACACTTCGGATTTTTCA GACGTTTGGAAATAATGCGCGA GTGTTTTGACT GA
TTTCT GTA CA GTA CCT GGAAAATAAACTACGCACGTT CT GT GATT CTTACGGTACA CTT GAAATTACT G
ACAGACAGTTTTCCATTTCCTGACGCGACCCCGTTATAGGATGGGGCAACCGCCTACTCAAGCGGGCT
GCT CTA GCAACT CGT GT GT CTAT GAAT GCGT CCGT GA GT CT GTT CGT GA GT CT GTACAT CGT GGGT CAA
GTCACATAAAGGTGGTCAACAGCTGAGGAGGTCTAGAGAGGACGAAACCAGAGACAGCTGCCAGCCG
CCCATAACAT GCA GAA GAT GA CT CA GAT GCT GGT GTATAAAGT GCT CAT CTT CGT CAT GGTATATCGCT
GCA GT GGAAA GCCTT CGGGA GGCC CGT GT GTTTTA GGCGGA GA GCA GTACAA CGT T GGA GA GA CGT G
GCAT C CTAAAGA GTTTCTT CCACCGCCAACTCCTGTGTCCACT GCA CGTGCCTA GA GGGC GGTCAGAT
AAATTGTACCGGCGTGGACTGTCCTTCTCCAGAGTGCGAGGTGCCCAGATTTATCCACGGACAGTGTT
GTCCTACATGTGATGTCCGGGGGGACGA GGCCGGAAGTCCCGAGAGCA GACATGCCCGGATGTGATTTC
CAT GGCGA CCACTACGA GGA CGGT GA CATATTT CCGT CCAACAA GA CGGCTTA CAAACCCAA GCAT GA
CAACCA GT GT GT GTT GT GCGGGT GTTATA GA GGT GA GGT CAT CT GT CA CCT GAA GA CTT GT CT GCCCA
GCCCCAGGTGTCGGCGGGTAGTCCGGGTGGATGACGACTGCTGCCTACAGTGTGAAGAGGAGTCCAG
CATA GACGAATATTT CAT GT CTAT CGGGT CA GA CAACATAAACCATACA CT GGA CGA CGAA GA CT GT C
TGTCA GCGACAGGACGCCGTAAGAACGGCA GTA CGTGGAAGCCA GTGGTGGGC GAGTA CGGAGA GAT
GCATTGTATCGTCTGCT CCTGTTTAGAAGGTCA GGT CGACTGCAAGCGTCTGACCTGCCCTGACGT CAC
AACCTT GACCTGCGGACACCCAAGGCCA CGACA GGACGGGTGCTGTAA GGAAT GCCCTGAGA GCGAC
AGACAGAGGGACAACA G

Wnt-16 precursor
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
TTTTTTTCCCCCGTCTACTTAACATCATTTATGCAGCTGGTGACAGTAACGAAAGCACGACATTCCGCT
TTTCGAAAGCATGGGC GTGTACCTACACAAAGCCACGATGGGTA GAAATAGCGCA CTGAATGT CCATG
CTGAA GTTCTCAGATTCTTGAACA GGACTCA GGAGTCCACAATCCTCTGGGATT T GATCACGCCT GTCA
GAGTCCCAAATACTGCCAGAAGTGATTACACCGGTCAAATGACATGTGGTATTAATTGTTTTTTTAAAA
GTAAACAT GAA GGGA CTTT C GTAT CTAAAGT GAA GTA CATTTT GGAAAAGT C CAACT T CATA GGAAAA
GTT CCCAAAAATCATGA CA GA GATT CCCAAAACAAAGGT GGAACCA GAATTT CTAAAATGAA GAAAA
TAATTGTCTCTGAGGTGTACTCATGTGCAGTTTTCAGACTGGACCAAGAAGGCTGCACTCCGAAAGAG
AAGGCTTCACCGA GTATTTAAATGGGAATAAGCCA CACTTT GGTGATCA GCTCGTCGAAAGA GCCAAA
TTCCT CTT T GT CGGT GA CTTTTAATTAAAATTTT GCATCTA CATTTAATA GT GGGGTAT TATT GGTATA G
ATCTA GTATAA GCA GGGT GGT CCCAT GCCCCTTTTT GGGT GA CCA CT GAAT GCCCTTAATAATTTTCTT
TTCAAAATCA GTA CGTT GT CAAAA GT CCA GCAT TT CTA GCA GAAAAAGGT CA GATTTTT GT GA CA CAT
TCAACAA GGGCT GGT CAT GT CAT GCT GA GAATATTT CTA CACTAAAACAT GCAACGCCT GA CAAAT GA
AATACAACAC CACTA GT CTTTAATTTTT CAAACATAAAGA CTTT GTA GAA GCA GCT CCGT CGGTA CAA
ACTGATTCACGTCAA GTGTTTAA GTATGCTCTTGCTAGGATTCATCATTTCCTATCA CATCGTATT GCA
AACTCCTGCTTTCGATTATACAATAAAAGCA CAGGAT GTTCACAA GGGCCA CT GAAAA GTACTTCAGC
CAA GAGAATT GGCGTTGAT GGTAGGCTAATATATTGT TCTCACAGA GCAATACTGGCATCGCTCGAAA
AATGGTTTCCTTCAGT GTAAAATAGTGTTACCAAGCTTATCCTCGGATACATTGTTTGA GAGGTTGTGA
GGGAACGTTTCAATTTCGGCTTTAGAATGAGTTACAACTCCGTGGTCAGCAGTTTTCAAAGCTGTTGTT
CGGGGA CTT CAT GCA CCACCAATATCTT CCATAAATTCAATT GGA CCT GCATA CATTTAA GATCA CGCA
CAGCT CTCCTGAGTTAAATGATCGACCATCGGTGATCCATGCCAGA CTCAGTGAAAGGTTTCCT CAAG
AAGAGACAGACCGCATGCGCTTTAAAACTTTTGT CGGAAACTCGGCGGCGCAAGGTGCTCCATTATGT
AAATGACA CTT CACCA CAT GT CCT GT CA CGGTATACT T CTAATCA CAAGT GCA CACT CT TAAAGT CA GG
AAGGAATTCAGTCGCAGTGACAGAATTCCTTCTTCTCGGACAACGAGTTTCAATGTGCACGCCACGCC
ATTTCTT GTTT CT CT T GGA TTATAATATATACACAAAATCCAACA CTTTT CGATT GA CA GT GA CGCA GG
T GCT GT CACTT GCA GGTAT GAAT GTCAA CCAAA GTTT CA CAA GT CTT GCA CT CAACGTAACAA CACCA










TCTTAT CAAA GAAAAGCTTT GA CT CTATT TCCGGCA GCGGGCT GCA CGGGAT GGGCCA GAACTTT CT C
GACGGGAAGAGAACATTTGA CTCAATCGGA GGGGGTGACTTACAAGGAATGGGCCAAAATTTTCTGA
CAT CAAAAAGA GGT TT GA CCCAATTTCAA CA GGCTCTCT GCA CGGAAT GCGT CAAAACTTTTT CGGA
CACAAAAGAAGTTTTGA CTCAATAA GCA GAAATCGGTTA GGAAATTTACGTCAAAATTTTTT GA GTAA
ACGAGCAGACGAGAGCGCGGATGAGTGGAGTGTTAATGACGCTTCACCCGAGAAAAGATTCGATTCG
ATCAGCACTAGCGCTCTCAATGGCATGGGCCAAAATTTCTTCGACAAAAAGTACTTTGACAATTTTCCA
GAA GGTGCTGTATCGAAGA GGTTCGA CCCCATTTCTTCAT CGGGGA CCATAAGCGGATTACAACAGAA
TTTCCTTAAGAAAAAAGACTCT GACTCCA GGTCGGCGGGCGGGATGGGAGGAGTCCGCCTGAGTTTT C
CACGACAGACAGGAAGTGTGGACGGCATGACGTCATCTAAACGCCAGTTTGACTCCATCTCTGGGGGC
AGCGGCATCGGC GGTCTGGGTCAGAACTTCCTGAACAGGCGA CAGTTTGATT CGATAAACGAGGGGG
ATATAGGCGGGATGGAGCAAAACTTTCTCAGTGGGAAACGATTCGATACCATTTCCAGTGGTAAAATG
GCAGGATTCAATCAGAATTTTCTTGGCAAGCGACAGTTTGATTCCATTTCTACTGGGGAGATGAGCGG
GAT GGA CCAAAACTTT CT GGGAAAACGCGGCTT CGATT CCATCAACA GCGA CT CT CGCATTTCCGGAA
TGGGGCAAAA GTATTT GGT TCGT CGTA GCT CCA GCCT CGAA CCT CTA CCCT CA GCA GAA GT GA GCGA G
CAGCCCCATGACGTCTACAAACGCAGCCCAGAGGATGTGACGTCTCTGGAAGCCAGTCACGACTCGGC
TGTTGTCACCGGC GATACTTCAGAACCGAT CGGACCTGAGCATCGTTTTATAAAACGCAGCTTCGATTC
TATTTCCAGATTTTCAGGAGGTATGGCGGGCATGAGTCAGAACTATCTGTCTCGCAGAGCGTCTAACA
CT CGT GCT CT GGA CT CGAACT CCCAACCGT CGT CGT CAT CA GCAA GCTACA CTA GCCGT GGCA CTAAC
GACGTGACTTCACTCGCCGGAGTAGACAACGATCACGAACTCTGGCGCATCCTGGACAAGAAAAACTT
GGA CTCAATCTCCA GATTGGGC GCTTATTTCCGGCTTT CATCA GAACTTCTTA GGCTTGCCGT CTGCCA
AACGCGCGGGCCAGGGC GC GCGA GACTCCA GCCAACATCACCCCGCCACCGAT CCTCTCTTCAATCGA
T GGGATTA CCAAA GAT CGGGGT CCGCA GATTT CT CA GA GATTAA
Protein
MNQNYLSKRPLDSISGSKFHGMNQNYLSKRPLDSIS SGFNGMRQNFLSKKSFDSISGSGLHGM GQNFLDG
KRTFDSIGGGDLQ GM GQNFLTSKRGFDPIST GSLHGMRQNFF GHKRSFDSISRNRLGNLRQNFLSKRADE
SADEWSVNDASPEKRFDSIST SALNGMGQNFFDKKYFDNFPEGA VSKRFDPI SS SGTISGLQQNFLKKKD
SDSRSA GGMGGVRLSFPRQT GS VDGMT SSKRQFDSI SGGSGIGGL GQNFLNRRQFDSINEGDIGGMEQNF
LSGKRFDTISSGKMAGFNQNFLGKRQFDSI ST GEM SGMDQNFLGKRGFDSINSDSRISGMGQKYLVRRS S
SLEPLPSAEVSEQPHDVYKRSPEDVTSLEASHDSA VVT GDTSEPIGPEHRFIKRSFDSI SRF SGGMA GMS
QNYLSRRASNTRALDSNSQPS SS SASYT SRGTNDVTSLAGVDNDHELWRILDKKNLDSISRLGA YFRLSS
ELLRLA VCQTRGPGRARLQPTSPRHRS SLQSMGLPKIGVRRFLRD

Pleurin+
Gene Bank Accession Number: 56200040
Reference:
Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome ofAplysia: neuronal
compartments and circuitry. Cell 127(7), 1453-67.
CDS:
ATGCA GTCTCACGT GAGCAGCAGCCTACTGGCCCT GTGCGTCCTGACGGGCAGCGCAT GCGCAATGTT
CTACACCAAA GGCAGCGACAGCGA CTACCCCCGGATCGGGCGTCGGA GCTTCTACACAACCGGAAAC
GGAAACCATTACCCGCGAATA GGA CGA CGGGATT CCCCT GGC GCA GCT GCT GGCA CGGA CATT CTTTA
CCCGAACACGGCTTCAGGGTTT TCCGATTTCTCGGCAGGC GCTCCGGCGGTGAAGAGAGGGATTTTCA
CGCA GT CGGC GTACGGT T CTTA CCCGA GGGTA GGA CGAA GGA GT CA GGCGGGA GAT GAT GGTT CCCA
GGAA GGT CTT CA GA CT GCGA GAT CTAA GATTTT CCA GAA GATTA CGGGGCT GGA CGGGCC GGT GGAA
GAA GAGCTCGGGGATGGATCGGCC GAGAGATTGGAA GCTGACAACCAGATT GGGCTTGA GCTCATGT
TCCT CGCCTTT GA CCTA GAT GGT GAT CAA GCTTT GT CAAAATCA GA GTT CCA GT CCGGGAT GGAAAAG
TTCCGA CGACACAACCCACTTT GCTAA
Protein:
MQSHVSSSLLALCVLT GSA CAMF YTKGSDSDYPRIGRRSF YT T GNGNHYPRIGRRDSPGAAA GTDILYPN
TASGF SDFSAGAPA VKRGIFTQSAYGSYPRVGRRSQA GDDGSQEGLQTARSKIFQKITGLDGPVEEEL GD
GSAERLEADNQIGLELMFLAFDLDGDQALSKSEFQ SGMEKFRRHNPLC

Preprogo nadotropin-releasing hormone-like protein









signaling molecules since these are important for modulation of the central and peripheral

nervous system in bot h vertebrates and invertebrates are involved in neuron communication,

identity and development. Most neurosecretory signaling molecules appear to be neuron-

specific, making them ideal candidates for markers for neuronal identification and to understand

the genomic basis for neuronal identity.

Previous work using released genomes to screen for secreted signaling molecules and

neuropeptides precursors. Several models including Danio rerio (Klee, 2008), Drosophila

(Wegener and Gorbashov, 2008), Arabidopsis thaliana (Emanuelsson et al., 2000), Apis

mellifera (Hummon et al., 2006), Tribolium castaneum (Amare and Sweedler, 2007) and Homo

sapiens (Emanuelsson et al., 2000) have been investigated. These results have been useful for

understanding organism-wide expression of SSMs; however, none of these predictions have

aimed at understanding neuron-specific expression of SSMs due to limitations in cell-specific

mapping in these models. The large and easily identifiable neurons ofAplysia will allow the

current work to go beyond the scope of these investigations to look at what SSMs are responsible

for learning and memory, neuron identity and plasticity in single neurons.

At the start of this project, 31 non redundant signaling peptides had been found by

traditional cloning techniques and submitted to NCBI for Aplysia californica (Object 2-2). This

represents over 20 years of work by numerous labs searching for individual proteins. Here, a

comprehensive list was created using both genomic and transcriptomic screening to identify all

non redundant transcripts predicted to encode secretary signaling peptides expressed in the CNS

ofAplysia californica. It is shown that screening genomic and transcriptomic data for transcripts

encoding proteins of interest represents a faster, more comprehensive way to identify putative

SSMs important for neuronal processes.









LIST OF OBJECTS


Object page

2-1 Intron/Exon Boundaries of selected neurosecretory genes........................ ...............77

2-2 Identified neurosecretory products likely to be signaling molecules.............................82

2-3 Controversial predicted neurosecretory products. ....................................................127

2-4 Predicted Lottia secreted signaling proteins. ...................................... ............... 149

2-5 Controversial predicted Lottia secreted signaling proteins ................ ......... ..........155

2-6 Predicted signaling molecules found in Lymnaea stagnalis. ........................................170

2-7 Predicted secreted signaling molecules found in Pleurobranchaea californica..............181

2-8 Predicted secreted signaling molecules found in Tritonia diomedea. ..........................189

2-9 Predicted secreted signaling molecules found in Melibe leonine ..................................195

2-10 Predicted secreted signaling molecules found in Clione limacine..............................199

2-11 Predicted secreted signaling molecules found in Octopus vulgaris ..............................201

2-12 Predicted secreted signaling molecules found in Nautilus pompilius. ..........................209









CHAPTER 1
INTRODUCTION

Introduction

One major goal of neuroscience is to understand the molecular mechanisms underlying

learning and memory. Despite several advances in our understanding of these mechanisms, the

complexity of the mammalian brain poses many technical challenges to studying mechanisms of

synaptic plasticity and learning. The small neuron size of mammalian brains makes isolation of

individual neurons difficult and the overall complexity of neuronal networks affecting behavior

poses problems for correlating neuronal function to specific behaviors. For this reason, many

researchers turn to simpler model organisms, such as invertebrates, for studying learning and

memory.

Aplysia californica: a model for learning and memory

Aplysia californica (Figure 1-1) is an important model for studying mechanisms of

learning and memory because it has a simple nervous system that consists of 20,000 relatively

large and identifiable neurons (Figure 1-1) (Kandel, 1979). The nervous system ofAplysia

consists of a system often connected ganglia with specialized functions (Figure 1-2). The

simple and quantifiable behavior, the gill and siphon withdraw reflex, has been extensively

studied over the last 40 years providing keys to the cellular understanding of learning and

memory (Kandel, 1976; Kandel, 2001; McPhie, 2006). Although about 200 neurons identified in

the central ganglia can participate in the gill and siphon withdraw reflex, the cellular circuitry

that modifies this reflex can be simplified to a network of monosynaptic connections between

three neuron types: a presynaptic sensory neuron, a post synaptic motor neuron and a modulatory

interneuron ((Kandel, 1976; Kandel, 2001). When the application of serotonin (5-HT) or nitric

oxide (NO) are applied locally to substitute for the action of the facilitory interneuron (Antonov










TCCGCAAA GA CAAGA GA GCACT GAA GGT GGA GCT CAA GGA GCA GGA GCTA GCTCAT GAA G
ATGTCATCAAGAACCTGAAAAAGA GAAATGATGAAAACATGACCTCCCTTCGCAACAGCT
TTGAGCGCCAAGCCAAA GAAATCGA GTCAAAATATGAGAAGAA GATGAGGGCTCTTCGTG
ATGA GCTAGACTT GAGAA GAAAGACAGAAATTCATGAGA TTGAGGA GAGGAAGAACGGCC
AGAT CAACA CCTTATGAA GAATCA CGA GAAGGCGTT CAGCGA TATTAAGAATTATTACA
ATGA CATTACATTGAACAACCCTGCCCTTATCAACACATT GAA GGGACAAAGTTGAAAAA
AAAAAAAAAAGGACCGGCGGGCCCGGGGTGGACCCCCGGCTTAAACCCCAGGGGC GGCCG
CCGCCGGGCGACCATATGGAAAACTCCCAACGGCGTGGAGGCAAACTTGGGTATTCCAAA
GGGGCCCCAAAAACCTGGGGGAACCGGTCAAGGCGTCTTTT TGGCGGGGAAAANTTTNTT
TTAGAAAACCCCCCCCAACCCCCCGGGGCCAGGGGGGGGGGGGAAAAGGGAGCCCCCGCC
CAAATTGGCGCAAAAAGGGGGAAGAATAAAATATTGGGGGAAAAACTTTTAAAAAAAAA
AAAAAAAAAAAAAAA

Secretory signaling peptides/proteins found by homology search to Lottia genomic
screening


Strong Annotation

Lottia Protein Model: jgi_Lotgil_119019_e_gwl.30.87.1
NR Annotation: PREDICTED: similar to seleno protein N, 1 isoform 2 precursor [Canis familiaris]
NRE value: 2.00E-85
Ac Annotation: Seleno protein Nprecursor_[0.0]_ [6]_APL all 052305.8412.C1
Ac E Value: 7.00E-69
Sequence:
MVLFTLLCCLFIIGRHDPTQIPEEIVINIGDDGVLLFQQHDRDNDGYLSIQEFEPLVYRLLEINVSGPVYDVPIS
TDDEMITLKSYFIPIVKESMSKDLND S VSIGLLRTMNSLHGLEKW QNVNLQWMNFGA SHFSGFLPKDVDSI
MLGSSYFIINVEKGLFNAALS SNRYYPPKVTTNESIIVHRLLTLFHPRPF VVSRFPAQSSVA CVRA YNDKYLD
IVFRIHAEFQLNEPPYHPFWFTPAQFTGNLIISKDGKHIQYFNLYVPNNKRLNIDMEWLNGPNESENMEVDI
GFMPLMQLNSTQSSVPVKNLEEYELLEPLPERPQHKTEGENIEWMATIDLEDAKSSLEKALYPFKKVPYYN
FTEAFKKAEDNKKLVHSILLWGALDDQSC*

Lottia Protein Model: jgi_Lotgil_121665_e_gwl.37.7.1
NR Annotation: hedgehog [Patella vulgata]
NRE value: 1.00E-171
Ac Annotation: Sonichedgehog protein precursor (SHH) (HHG-1) [Contains: Sonic hedgehog protei_[3.65288E-
39]_[1]_L7ALL-ET 3 V2MC01DELVF_253
Ac E Value: 3.00E-32
Sequence:
MKLFF SFQSVCSWTFIFLFIT SLTHACGPGRGSGSRRRPRKRTPLVFKQHVPNVSENTLGA SGISDGKIRRNS
EKFKNLVKNENPDIVFKNEEGDGSDYLMSRRCQDKLNSLAVSVMNNWKDVRLRVTEAWDDSPNSHAKD
SLHYE GRA VDITT SDRDRSKYGMLARLA VEA GFDW VYYESRGHIHCSVKSDSSVAIKIGGCFPPT GSVQTL
HGWKTM GQLT VGDKVLSINSA GQLEYSPVIAFIDRNDLEFEKYLTLHTEDDTDITLTSKHLIYA SGTNSSNF
ESYDVVYADDIMEGDHVLIT SSEKGAISPTRVVTI SEKTLQGVYAPLT VNGNIVVDGVVWSCYA VVSDANL
AHA VFAPMRGLHYLSQYVPWFLHST QQENAPQNGVHW YAKMLYNIGSTFLNEKTLYIA*

Lottia Protein Model: jgi_Lotgil_159314_fgenesh2_pg.C sca 20000054
NR Annotation: Buccalin precursor [Contains: Buccalin-D; Buccalin-E; Buccalin-F; Buccalin-G; Buccalin-H;
Buccalin-A; Buccalin-I; Buccalin-J; Buccalin-K; Buccalin-L; Buccalin-B (BUCb); Buccalin-M; Buccalin gene-
predicted acidic peptide A (BGPAP A); Buccalin-N; Buccalin-O; Buccalin-P; Buccalin-Q; Buccalin-R; Buccalin-C;
Buccalin-S; Buccalin gene-predicted acidic peptide B (BGPAP B)] gb|AAB27696.21 buccalin precursor [Aplysia
califomica]


































Figure 1-1. The Aplysia model system.. A) Image ofAplysia californica. (Image courtesy of
(Rudman, 2003). B) The abdominal ganglia from Aplysia, showing large, easily identifiable
neurons. C) The gill and siphon withdraw reflex can be simplified in cell culture to two neurons,
a sensory cell (i.e. SN) and a motor neuron (i.e. L7) and still exhibit many of the same behavioral
and cellular responses seen in mammalian conditioning including long-term synaptic plasticity
and memory. [ Image A courtesy of Rudman, W.B. (2003). Ink glands (Syndney, Sea Slug
Forum). Images B and C courtesy of Lovell andMoroz, unpublished.]









ABSTRACT OF THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE


IDENTIFICATION OF NEUROSECRETORY MOLECULES IN APLYSIA CALIFORNICA
AND RELATED MOLLUSCS: GENOMIC APPROACHES

By

Jinnie Amber Sloan

August 2009

Chair: Leonid L. Moroz
Major: Medical Sciences

Several major questions related to the molecular underpinnings of neuronal identity and

function such as, "What make s a neuron a neuron? What is the genomic basis of unique neuronal

phenotypes? How different is the transcriptional profile of one neuron from another? Can

molecular techniques be used to determine neuronal identity?" remain at the center of research in

neuroscience. As a first step to answering these questions, and providing a list of molecular

markers to identify specific neurons types, we have attempted to identify and quantify nearly all

transcripts likely to be uniquely expressed in different neuronal classes (such as neuron-specific

secretary products) and play a crucial role in neuron identity and function. This includes

transcripts that encode neuropeptides, prohormones and related secretary signal peptides.

Molluscs have served as powerful model organisms for cellular and system neuroscience

for more than 40 years (McPhie and Miller, M., 2006; Kandel, 1970). Their nervous systems

consist of simplified networks of large identified neurons, allowing unprecedented opportunities

to study the principles of organization of neural circuits as well as learning and memory

mechanisms. As our major experimental models, we chose Aplysia californica and related

species, sea slugs belonging to the class of Gastropod Mollusca and species belonging to










Feeding circuit peptide 2+
Gene Bank Accession Number: n/a
Reference: n/a
Partial Clone:
CT GGA CGGTAT CGCAA GCT C GTT GAT CAAACGGCAA GA CGT GCT GGGT T CT GA CGT GGC CAT T GGGA G
CACGGGGAACAGCGGGCCGGACAAACGATACCTTGACCACT TGGGTA GTTCACTGGTCAAGAAATCT
GATGGACAA GACAAACGATACCT TGA CCACTTGGGTAGTTCGCTGGTCAA GAAATCT GAT GGACAAG
ACAAACGATACCTTGACCACTT GGGCA GTTCT CTGGTTAA GAAAT GGGACGA GGGTGACCAGGAAAA
GA GATATTTGGA CCACTTAGGCA GCT CACTAGT GAGAAAATCA GATA GTCA GGACAA GAGGTATTTGG
ATCATC T GGGGA GT T CTTT GGT CAAAAAGT CA GAT GGT CAA GAAAAAAGATATTTA GACCATTT GGGT
AGT TCA CTGGTAAAAAAGT CTGACGGTATAGACCA GGAGAAGAGATATTTAGACCATTTGGGAAGTTC
TTT GGT GAAAAAATCT GATACT GA GGA GAAAA GATATTTA GACCATTT GGGAA GTT CATT GGT GAAAA
AGT CA GAT GGT CAA GAAAAAAGATATTTA GA CCATTT GGGAA GTT CTTT GGT CAAAAAGT CT GA CGGT
ATAGACCAAGAGAAGAGATATTTAGACCATTTGGGAAGTTCCT T GGTGAAAAAATCTGATACTGAGGA
GAAAA GATATTTA GACCATTT GGGAA GT TCTTT GGTAAA GAAATCT GATA CT GA GGA GAAA CGATATT
TAGACCATTTGGGAAGTTCTTTGGTGAAAATCTGATAGAGGA GAATCTAATATTTA GACCATTTG
GGAA GTT CTTT GGT CAAAAAGT CT GA TACT GAA GA GAAAAGATATTTA GA CCATTT GGGAA GTT CGTT
GGT GAAAAAATCT GATACT GA GGA GAAAAGATATTTA GAT CATTT GGGAA GTT CGTT GGT CAAAAAGT
CT GA CA GT CAAGAAAAAAGATATTTA GA CCATTT GGGTA GTT CA CT GGTTAAAAAATCTAAC GGCATA
GACCAAGAAAAAAGATATTTAGATCATCTAGGCA GTTCT TTGGTCAAAAAGTCAGACA GTGAAGAAA
AAAGATATTTAGACCATTTGGGCAGT TCGC T GGT TAAAAAATCTGACAGAGAAGAAAAAAGATATTTA
GACCATCTGGGAA GTTCGTTGGTGAAAAAATCGGAGA GCCCGGA GTTGGCAA GATTTGAGCAGCGGA
TGTTTACA GACTACAACAACGACCTTGTTGCTATGGAGAAAGACTT CCTGGAGAAGAACAGAGAACTC
CAGCA GTCGA GCCCGGAT GTGACGTCACAGGGGAACAAACGTTATCTGGACAGGA TCGGCA GCTCGC
T GGT GAA GAA GGAA G

FFELamide/FMRFamide -3
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Predicted:
GGCT CGA GA GGACCCT TTTACCCT TT CGGTA GA GGC GA CCCATTTAT CA GATTT GGTA GA GGCGT CCCC
TTCATTCGATTT GGTCGAGGCGATCCATTCATCCGATTTGGCAAAAGAGGAGCAGACAGCGAAGAAGG
TGTCGGTGAAGGA GAAGCCTTGTCGA GGACAGCCAGATCGACAGAAACGACACAGGAACGAGCCAAC
TCT CGT CAAATACGCA GT GCA GA CGCCAACAAACGAT TCA CGA GGTT CGGCCGCT CCGCCA CT TCCGC
TTTT GT GA CCCA GCA GGA GGCT GA GGT CAA GGA CGCA GT GA GGAAAA GGT CA GT CGA GAA CTT GGA C
AACGA GGT CAAACGA GCGAT GT GGA GCAACAACCAACA GAT GA CGCGA TT GT CCAAAAGA GGT CA CC
CA GA GTT CAT GCGAT TT GGGCGGA GT CAA GGA GA CGA GA CA GA GGACGACGAAATT GGCGGA GAT GA
CGT CATCA GTTT GGGGGAA GGT CTA GCA CA GA GGGT GC GCCGGGT GTA CGAA GA CA GGA GCT CA CT G
CCACGATTT GGGAA GA GGCA GGA GGT GGA GGA GGA GGA GGA GGA GGACTTT GGGAA GGAAAA GA GA
CA GAA GTACAT GCGTTTT GGAA GACGGA GT GAT CCA GA GCA GCA GGC GT CA GCGGAA GT GGGC GGGG
CGGAA GT CCGA GACA GT GT GCT CT CT GCCA T GTA GT CACGT GCT CACT GGGA GGAAA GGAT GCA CGCA
CAACA CGCT CTT T GGACGTATTATT GCA GCCGGCA GCA CAACATTTTTA

Fifth (short peptide fragments obtained from MS data)
Gene Bank Accession Number: n/a
Reference: n/a
Comment: Predicted only, not cloned
Partial:
CCACGTACGGGCGTTAGCGCCAACTTCCTGGCCCCAGGCAGACTCGAAACATGGGAGATAATAGAAA
AATCCATTT GGAAAATACT GCTA GTT GT CT CCCATCCT CT CTTT CCGT TAAACCCTATT CCGT GAA GGC
ATAGT GGGA GAATT GT CAAAATTCTACA GT TT GTCAATATACT GAT CCT GAAATCAACCCT CTA GGCCC
TTA GTT CGGTTT CTTTTACA GT CTTT CTAT CCCTTTT CTAT CATA GA GTCT CCCATTT CA CT GAT GGGCA









Digital Expression Profiling

Digital Expression Profiles (DEP) are made by taking the entire set of data from a

sequencing project before assembly. In this dataset, each sequence read represents the

expression of a single transcript in the cell or tissue the library was made from, such that there is

a 1 to 1 ratio of sequence read to transcript.

From this data set, any sequence of interest can be aligned using BLAST (le-04) and by

counting the number of reads generated from the 454 sequencing that have significant BLAST,

the number of times that transcript was sequenced from the library can be determined. To

determine the frequency of expression, these counts are normalized to the total number of

sequences in the sequencing project. By normalizing the counts, the frequency of expression

across different sequencing projects can be compared.












Secretory Product DEP In situ

SN MN IN SN MN IN

Sensorin + + +

PTSP-like + +

Capsulin + + +

LFRFamide + + +

SF Y3 +

7B2 s.g. + + +

Heatshock + + +

MIP + +

FMRFamide + + +

R15 + +

Delta-like +

Ependymin + +

Putative Pheremone 2 + +

Theromacin +

Fulicin +

Myomodulin + +

Insulin-like + +

Buccalin + + +


Table 4-1. Validation of DEP using in situ hybridization. Comparison of secretary products
predicted to expressed in Sensory (SN), Motor (MN) or Inter (IN) neurons by DEP compared to
expression confirmed by in situ hybridization.










Table 2-3. Genomic organization of neurosecretory genes. Preliminary analysis of the genomic
organization of selected neurosecretory products shows that all genes have a similar
intron/exon organization, with an average of 3-5 exons. (See Supplemental A for
detailed information.)
Neuropeptide Number Number
Introns Exons
Atrial gland-specific antigen precursor 9 10
Feeding circuit activating peptide precursor 1 2
FMRFamide neuropeptide precursor 2 3
L11 neuropeptide precursor 2 3
Buccalin precursor 3 4
Conopressin 3 4
Fulicin-like neuropeptide precursor 4 5
MIP-related peptide precursor 4 5
Neurotoxin-like- 1 3 4
Pleurin 2 3
Whitnin precursor 2 3









responsible for regulation are more highly variable. This may suggest one means for species

specificity at the neuronal level. Using recently released genomic data fromAplysia californica,

the intron/exon boundaries of neuropeptides were analyzed to determine if sequence

conservation and variability was due to the genomic arrangement of these genes. Preliminary

analysis of 5 conserved, divergent and moderately conserved neuropeptides does not reveal any

clear differences between the genomic organization of each type of neuropeptide.

Perhaps the most important finding is the large number of sequences with shared identity

found in bothAplysia and Lottia. These sequences represent a portion of the Aplysia secretome

that has be en effectively predicted without having to perform expensive genomic sequencing or

repeat the computationally expensive process of secretome prediction. This is a major step in

bringing genomic-scale proteomics to a species without a sequenced genome.

Methods and Materials

Animals and Tissue Collections

Specimens ofAplysia californica weighing 150-280 g were collected in the wild by

Marinus Scientific (Long Beach, CA). Specimens ofPleurobranchaea californica, Clione

limacina, Tritonia diomedea, Melibe leonina, and Lymnaea stagnalis were obtained from the

Friday Harbor Labs, University of Washington. Octopus vulagris was obtained from Italy and

Nautilus pompilius from the Phillipines. Prior to dissection of gastropods, animals were

anesthetized by injecting a volume of isotonic MgCl2 (337mM) equivalent to 50%-60% of their

weight. Dissection ofCepholopods and Gastropod Mollusca was performed by Dr. Moroz.

Total RNA from whole CNS was extracted using RNAqueousTM (Ambion, Austin, TX) kit.

RNA isolation was performed by Dr. A Kohn, Jinnie Sloan, and Yelena Bobkova. Before

further processing of the RNA, quality was checked using a 2100 BioanalyzerTM (Agilent

Technologies). Small aliquots of extracted RNA were loaded on a 6000 Nano Lab chip that









AGGCAACAGTGGAATCGAAGCTCTC-3', our lab cloned the full length sequence of

Hypothetical protein 2, resulting in a 788 bp fragment.

LFRFamide precursor

The full length sequence and clone for LFRFamide was obtained (Genebank accession

number EU886298) using terminal primers 5'-GGCCTCAGTTCGAAACCTCG-3' and 5'-

ATTGGTCACCCTGTCCTCGG-3'. The resulting cDNA fragment was 1074 bp.

Myomodulin-like Neuropeptide Precursor3

The full length sequence and clone for Myomodulin-like Neuropeptide Precursor 3 was

obtained (Genebank accession number EU934739) using terminal primers 5'-

CCTGAGTCCAGCCTAAGCGGTAAGT-3' and 5'-GTAC TGTGTAGGACGTAGGAAGCAG

-3'. The resulting cDNA fragment was 1690 bp.

Conopressin

The full length sequence and clone for Conopressin was obtained (Genebank accession

number FJ172359) using terminal primers 5'-CCAACTACAGGATGTCTCACTC-3' and 5'-

GACGTTGAGTGGACACTGTGA -3'. The resulting cDNA fragment was 1983 bp.

Ependymin-related protein 2

The full length sequence and clone for Ependymin-related protein 2 was obtained (not

submitted) using terminal primers 5'-CTGGTATCAGAGCCACTCACCTC-3' and 5'-

TGGTTGAATGTATACGTGCATGTAC -3'. The resulting cDNA fragment was 873 bp.

Betsin

The full length sequence and clone for Betsin was obtained (not submitted) using terminal

primers 5'-GTAACTTTCGTCCCTCCTGCCA-3' and 5'-CTTCTACTTGACCCACTCGGACC

-3'. The resulting cDNA fragment was 538 bp.












Intron 1 2 3 4


Exonl1 2 3 4 5


MIP-related peptide precursor
Gene Bank Accession Number: 8886135
Intron/Exon Boundaries: coding region: 668-2873 nt
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Nucleotide
Number Sequence Length location
1 CCCTTGGGGT TGACCATAGA gt atgttgtt 1456
2 gttgtttc ag ATTCCAAAAA AGTGGTTCAG 144 gt aggttgct 457-600
3 tctcgtt ag GTTACCACAG AATTGTACFG 33 gt gagtggaa 601-633
4 cttattcc ag ATTTTrTCFG TCACGTGCAG 107 gt gagtagga 634-740
5 ttccatgc ag ATACGCAAAG AGGTAGCTTT 2139 gt ccatccag 741-2879



Intron 1 2 3 4


Exon 2 3 4 5




Neurotoxin-like-1+
Gene Bank Accession Number: 71148939
Intron/Exon Boundaries: Coding Region: 10-423 nt
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Nucleotide
Number Sequence Length location
1 GCTGAGCA AG TCACAATCAC CAGCGTGAAC 70 GT AAGTCTTA -10-60
2 TCCCCC AG GCAAAGGTGG GCAACGAAAG 130 GT GAGGCGTG 61-190
3 GATATTGC AG GGACGTGCCA TTCGTTCCAG 129 GT CAGTACAC 191-319
4 CTTACGTC AG GATTCTTTTC CATAGATGAC 129 GT CAACACCT 320448



Intron 1 2 3


Exon 2 3 4




Pleurin+
Gene Bank Accession Number: 56200040
Intron/Exon Boundaries: Coding Region: 189-755
Exon Intron 3' Exon 5' Sequence Exon 3' Sequence Exon Intron 5' Sequence Placement in
Number Sequence Length Sequence
1 TTGTCACCAG AAGTGAGTGA CTCACCAACA 155 GTAAGCCATC 2-156
2 TATCAAACAG TAAACCGCTC CTTGAGCTCA 482 GTGGCCGAAT 176-657
3 TGTTTCACAG GTGATCAAGC CTTTGATGAC 94 GTCATCTGAG 682-776










































Figure 4-1. Semi-intact preparation ofAplysia abdominal for identifying neurons of the gill and
siphon withdraw reflex. The nerves connecting the abdo minal ganglia to the gill and siphon
remain intact. By removing the sheath surrounding the neurons, cells can be impaled and
recorded from to determine cell identity. For example, L7 can be identified by impaling cells in
the vicinity of L7 until a cell is found that when stimulated, results in the contraction of the gill
and siphon.




























*. .connectives- ~2 Pleural ganglia



P5 Pee P5
P5


P4 -- Pleuroabdominal P6
connectives
P6 / P7

P8 CP8
P9 -R13 P9
P9
O0 R2
A6 Abdominal
ganglia

A5 A2
A2
A4

Figure 3-2. Schematic overview of the distribution ofFulicin-like transcript in the CNS of
Aplysia. Each circle represents a single cell showing staining for the Fulicin-like transcript.
Positions of identified cells MCC, R2, and LP1 are indicated, (viewed from caudal surface).
Most significant staining was seen in the Abdominal and Pleural ganglia.




Full Text

PAGE 1

1 IDENTIFICATION OF NEUROSECRETORY MOLECULES IN APLYSIA CALIFORNICA AND RELATED MOLLUSCS: GENOMIC APPROACHES By JINNIE AMBER SLOAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLME NT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Jinnie Amber Sloan

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3 To my mother Lisa and my family for all their support and love

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4 ACKNOWLEDGMENTS I would lik e to thank my colleagues, collaborators and teachers at the Whitney Laboratory for Marine Biosciences. This work is a reflection of the keen insight and vision of my mentor, Leonid L. Moroz, who always strives to push science and those around him to accom plish what may seem impossible. I am grateful to my dissertation committee, Peter Anderson and Nancy Denslow. I will be forever grateful to Andrea Kohn for her continual support and guidance in my pursuits both as a scientist and as a friend. To Mathew Citarella I would like to thank for years of support and his contribution to the bioinformatics aspects of this work, none of this would have been possible without his help. Jim Netherton, Yelena Bobkova, and Rebecca Virata for their technical support. I am also grateful to Thomas Ha for teaching me in situ hybridization protocols and the semi intact preparation.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 L IST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 G LOSSARY OF TERMS ..............................................................................................................12 ABSTRACT ..................................................................................................................................13 CHAPTER 1 INTRODUCTION ..................................................................................................................16 Introduction .............................................................................................................................16 Aplysia californic a : a model for learning and memory ..........................................................16 Neuropeptides .........................................................................................................................17 2 I DENTIFICATION OF NEUROSECRETORY PRODUCTS IN GASTROPOD MOLLUSCS ...........................................................................................................................23 Introduction .............................................................................................................................23 Results and Discussion ...........................................................................................................25 Identification of Predicte d Secreted Signal Molecules ...................................................25 Homology r esults ......................................................................................................25 Prediction r esults ......................................................................................................25 Confirmation of Expression of Selected Neuronal Transcripts .......................................28 Cross Species Analysis ...................................................................................................29 Genomic Organization of Select ed Neurosecretory Products .........................................29 Conclusions .............................................................................................................................30 Methods and Materials ...........................................................................................................31 Animals and Tissue Collections ......................................................................................31 454 Library Construction ................................................................................................32 3' end amplified cDNA library construction for 454 sequencin g: ............................32 5' end target amplified cDNA library construction for 454 sequencing: .................33 Cloning of selected Signaling Molecules ........................................................................34 Hypothetical protein 2 ..............................................................................................34 LFRFamide precursor ...............................................................................................35 Myomodulin like Neuro peptide Precursor 3 ............................................................35 Conopressin ..............................................................................................................35 Ependymin related protein 2 ....................................................................................35 Betsin ........................................................................................................................35 Feeding circuit activating petide precursor 3 (FCAP 3) ..........................................36 SN4 ...........................................................................................................................36

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6 SFY1 like peptide .....................................................................................................36 SFY3 like peptide .....................................................................................................36 FIRFamide related neuropeptide precursor ..............................................................36 Major royal jelly protein (MRJ) ...............................................................................36 FMRFamide 5 ...........................................................................................................37 Allatotropin OR precursor ........................................................................................37 Second ......................................................................................................................37 Neurosecretory Predictions .............................................................................................37 Homology search using unbiased shot gun approach ................................................37 Genomic approach ....................................................................................................38 Classical Secretory Prediction* ................................................................................38 Non classical Secretory peptide Prediction* ............................................................39 Cross Species Analysis of Predicted Proteins ..........................................................39 Annotation of Predicted Pr oteins ..............................................................................40 3 M APPING EXPRESSION OF NEUROPEPTIDE TRANSCRIPTS .....................................50 Introduction .............................................................................................................................50 Mytilus Inhibitory Peptides ..............................................................................................50 Fulicin ..............................................................................................................................51 Results .....................................................................................................................................51 Cloning of Aplysia Fulicin precursor mRNA ..................................................................51 Localization of Fulicin in the CNS of Aplysia ................................................................52 Co localization of MIP and Fulicin .................................................................................52 Isolation of Fulicin in the CNS of Aplysia ......................................................................53 MIP and Fulicin share 5 UTR and coding region ..........................................................53 TxFrag2: Structure and Function .....................................................................................53 Discussion ...............................................................................................................................54 Colocalization of Fulicin and MIP ..................................................................................54 TxFrag2: Modulator of Expression? ...............................................................................55 Materials and Methods ...........................................................................................................56 Anima ls ............................................................................................................................56 Cloning of full length cDNA encoding Fulicin ...............................................................56 Sequence analysis and Alignments .................................................................................56 In situ hybridization of Fulicin and MIP in Aplysia .......................................................57 Imaging ............................................................................................................................58 4 GENE EXPRESSION PROFILING FOR INDI VIDUAL NEURONS .................................65 Introduction .............................................................................................................................65 Advantages of Aplysia .....................................................................................................65 Limit ations of Sequencing Technology ...........................................................................66 Results and Discussion ...........................................................................................................67 Sensory Neurons ..............................................................................................................67 Motor Neurons .................................................................................................................67 Interneurons .....................................................................................................................68 Discussion ...............................................................................................................................69

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7 D igital Expression Profling .............................................................................................69 APPENDIX A MODERN VIEW OF ANIMAL PHYLOGENY .................................................................220 B MOLLUSCAN CLASSES ...................................................................................................222 LIST OF REFERENCES .............................................................................................................223 BIOGRAPHICAL SKETCH .......................................................................................................229

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8 LIST OF TABLES Table page 21 Annotation of Lottia predicted secretory products against NCBIs NR database. ............45 22 Summary of Cross Species Predictions. ............................................................................46 23 Genomic Organization of Neurosecretory Genes. ............................................................48 24 Adaptors and Primers for 5 and 3 454 libraries ..............................................................49 31 Comparison of Real Time PCR of Aplysia MIP related gene and 454 sequencing. ............. 41 Validation of DEP using in situ hybridization ...................................................................76

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9 LIST OF FIGURES Figure page 11 The Aplysia model system.. ...............................................................................................20 13 Conserved motifs found in neuropeptides. ........................................................................22 21 Summary of model organism and number of ESTs/cDNAs collected. .............................41 23 Conserved Signaling Molecule Motif. ...............................................................................43 24 Workflow for Computational Prediction of SSMs. ...........................................................44 25 Comparative analysis of predicted Lottia secretory products. ...........................................47 31 Fu licin Gene and Protein Predictions ................................................................................59 32 Schematic overview of the distribution of Fulicin like transcript in the CNS of Aplysia ...............................................................................................................................60 33 Colocalized expression of MIP and Fulicin transcripts. ....................................................61 34 Alignment of the coding region of Aplysia MIP like protein against the identified Aplysia Fulicin like protein. ...............................................................................................63 41 Semi intact preparation of Aplysia abdominal for identifying neurons of the gill and siphon withdraw reflex. ......................................................................................................72 42 Digital expr ession of neurosecretory products predicted by traditional cloning. ..............73 44 Digital expression of neurosecretory products predicted by genomic approach. ..............75

PAGE 10

10 LIST OF OBJECTS Object page 21 Intron/Exon Boundaries of selected neurosecretory genes. ...............................................77 22 Identifi ed neurosecretory products likely to be signaling molecules. ................................82 23 Controversial predicted neurosecretory products. ...........................................................127 24 Pre dicted Lottia secreted signaling proteins. ...................................................................149 25 Controversial predicted Lottia secreted signaling proteins. .............................................155 26 Predicted signaling molecules found in Lymnaea stagnalis. ...........................................170 27 Predicted secreted signaling molecules found in P leurobranchaea californica. .............181 28 Predicted secreted signaling molecules found in T ritonia diomedea. .............................189 29 Predicted secreted signaling molecules found in M elibe leonine ....................................195 210 Predicted secreted signaling molecules found in C lione limacine. ..................................199 211 Predicted secreted signaling molecules found in Octopus vulgaris ................................201 212 Predicted secreted signaling molecules found in N autilus pompilius. ............................209

PAGE 11

11 LIST OF ABBREVIATIONS BLAST Basic Local Alignment Search Tool CNS Central Nervous System ER E ndoplasmic reticulum ESTs Expressed Sequence Tags DEP Digital Expression Profile IN Interneuron MN Motor Neuron NCBI National Center for Biotechnology Information ncRNA Noncoding RNA NR protein database for Blast searches, compiled by NCBI PCR Polymerase Chain Reaction RACE Rapid Amplification of cDNA Ends SN Sensory Neuron sRNA Short Noncoding RNA SSMs Secreted Signaling Molecules UTR Untranslated region Symbols Cloned by Jinnie Sloan + Cloned by the Moroz lab

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12 GLOSSARY OF TERMS BLAST : finds regions of local similarity between sequences. This program can be used to compare nucleotide or protein sequences to a sequence database in order to determine the functional and evolutionary relationship between sequences. The statistical significance calculate d between sequences can be used as a guide to compare sequences and identify members of gene families. Exon : the nucleic acid sequene within a gene that is present in the mature form of an RNA molecule. In situ hybridization : used to localize a specif ic DNA or RNA sequence in a specific tissue or cells by using labeled complementary DNA or RNA. Transfrag Intron : a region of DNA within a gene that is not translated into protein. These regions are included in the transcribed pre mRNA and removed by sp licing to produe a mature RNA. Neuropeptide : small protein like molecules that can be secreted by neurons to communicate with each other. Neuropeptides can act on neighboring neurons as anterograde or retrograde messengers or the neuron secreting the neu ropeptide (orthograde messengers) through cell surface receptors to modulate or mediate neuronal communication. Typically neuropeptides alter the communication of a neuron by increasing or decreasing its excitability. Neuropeptides are assembled by riboso mes attached to the ER then transferred to the Golgi Apparatus to be packaged into vesicles and transported to synaptic terminals. Some neuropeptides are secreted directly into the blood stream and are referred to as neurohormones. Secreted neuropeptides can have long lasting effects from seconds to days. Prepropeptide : the inactive precursor of a peptide that requires posttranslational modifications to become the active peptide molecule. The prepropeptide of neuropeptides is characterized by a signal p eptide that targets the protein to the secretory pathway for posttranslational modifications including the cleavage of the signal peptide in the ER. Signal Peptide : a short (3 60 amino acid) sequence made up of a positively charged sequen c e found on the N terminal region a hydrophobic region and a polar uncharged C terminal region (cleavage site) that targets the transportation of a protein. In the case of secretory signaling peptides, the signal peptide targets the preprotein to the ER for further pos ttranslational modifications. Transcript : an RNA molecule produced from a gene. Transmembrane Domain : a short span of amino acids (1535) composed of mostly hydrophobic regions separated by polar connecting loops that form stable secondary structures in membranes.

PAGE 13

13 ABSTRACT OF THESIS PRESENTED TO THE GRA DUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL F ULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IDENTIFICATION OF NEUROSECRETORY MOLECULES IN APLYSIA CALIFORNICA AND RELATED MOLLU SCS: GENOMIC APPROACHES By Jinnie Amber Sloan August 2009 Chair: Leonid L. Moroz Major: M edical Science s Several major questions related to the molecular underpinnings of neuronal identity and function such as, What makes a neuron a neuron? What is th e genomic basis of unique neuronal phenotypes? How different is the transcriptional profile of one neuron from another? Can molecular techniques be used to determine neuronal identity? remain at the center of research in neuroscience. As a first step to answering these questions, and providing a list of molecular markers to identify specific neurons types, we have attempted to identify and quantify nearly all transcripts likely to be uniquely expressed in different neuronal classes (such as neuron specific secretory products) and play a crucial role in neuron identity and function. This includes transcripts that encode neuropeptides, prohormones and related secretory signal peptides M olluscs have served as powerful model organisms for cellular and sys t em neuroscience for more than 4 0 years (McPhie and Miller, M., 2006; Kandel, 1970) Their nervous systems consist of simplified networks of large identified neurons, allowing unprecedented opportunities to study the principles of organization of neural ci rcuits as well as learning and memory mechanisms. As our major experimental models, we chose Aplysia californica and related species sea s lugs belonging to the class of Gastropod Mollusca and species belonging to

PAGE 14

14 cephalopod molluscs Our long term goal is to identify neurosecretory molecules of peptide nature using a combination of comparative and genomic approaches. We have chosen neurosecretory molecules as putative molecular markers because they are highly abundant in neurons and are responsible for ce ll signaling and modulation Originally, the major limitation in using Aplysia californica, as well as other molluscs, has been the lack of genomic information available for these model species. To overcome this limitation, we have aimed to bridge the ga p between genomic and non genomic models. The Moroz lab has sequenced >980,000 ESTs/cDNAs from eight key mollusan species ( Gastropods: Aplysia californica, Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea stagnalis, and Cephalopods: Octopus vulgaris, Nautilus pompilius ). These sequences were assembled and cross annotated using the extensive transcriptome and genomic information from Aplysia californica My thesis deals with comparative analysis and identification of both evolutionary conserved and novel transcripts that encode neuropeptides, prohormones and other predicted secretory products. As a part of the project, we have also employed computational approaches to predict novel signal molecules based on shared pr edicted protein motifs that are conserved across all secreted signaling proteins. Through this work, I identified a selected list of candidate transcripts predicted to encode secretory molecules across selected molluscs and identif ied putative neuropeptide s present in individual neuronal classes including motor neurons, sensory neurons, and interneurons This work also led to the identification of a set of neuropeptides that is co expressed in sensory and motor neurons. The developed molecular resources an d the ability to map gene expression has allowed me to provide a detailed study of the genomics of identified cells and provide a critical bridge between genes, circuits and behavior in the broad evolutionary context. Overall these

PAGE 15

15 identified neurosecreto ry products may be the first step to creating a comprehensive list of gene products expressed in individual neurons that can be used for molecular identification of neuron types.

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16 CHAPTER 1 INTRODUCTION Introduction One major goal of neuroscience is to und erstand the molecular mechanisms underlying learning and memory. Despite several advances in our understanding of these mechanisms, the complexity of the mammalian brain poses many technical challenges to studying mechanisms of synaptic plasticity and lea rning. The small neuron size of mammalian brains makes isolation of individual neurons difficult and the overall complexity of neuronal networks affecting behavior poses problems for correlating neuronal function to specific behaviors. For this reason, m any researchers turn to simpler model organisms, such as invertebrates, for studying learning and memory. Aplysia californica : a model for learning and memory Aplysia californica (Figure 1 1) is an important model for studying mechanisms of learning and me mory because it has a simple nervous system that consists of 20,000 relatively large and identifiable neurons (Figure 1 1) (Kandel, 1979) The nervous system of Aplysia consists of a system of ten connected ganglia with specialized functions (Figure 1 2) The simple and quantifiable behavior, the gill and siphon withdraw reflex, has been extensively studied over the last 40 years providing keys to the cellular understanding of learning and memory (Kandel, 1976; Ka ndel, 2001; McPhie, 2006) Although about 200 neurons identified in the central ganglia can participate in the gill and siphon withdraw reflex, the cellular circuitry that modifies this reflex can be simplified to a network of monosynaptic connections be tween three neuron types: a presynaptic sensory neuron, a post synaptic motor neuron and a modulatory interneuron ( (Kandel, 1976; Kandel, 2001) When the application of serotoni n (5 HT) or nitric oxide (NO) are app lied locally to substitute for the action of the facilitory interneuron (Antonov

PAGE 17

17 et al., 2007) this experimental set up can be further simplified in cell culture to consist of two neurons, a sensory cell (i.e. SN) and a motor neuron (i.e. L7) and still exhibit many of the same behavioral and cellular responses seen in mammalian conditioning including long term synaptic plasticity and memory (Figure 1 1) (Kandel, 2001; Lewin and Walters, 1999) The synaptic strength or ability for these neurons to communicate can be changed, a term called synaptic plasticity. Modification to neuronal circuits through synaptic plasticity is responsible for different types of learning. One major obstacle in understanding how changes in synaptic plasticity affects learning and behavior is the het erogeneity of neuron types. Since each neuron may be unique, it is likely that they form and maintain information in different ways. Be fore the mechanism of plastcit y can be understood, neuron types (motor vs. sensory vs. inter neurons) need to reliably identified by marker s In mammals, there is strong evidence that neurotransmitters are in part responsible for changes in synaptic strength. Here, I propose the use of neurotransmitters, neuropeptides in particular, as neuronal markers to aid research of neuronal plasticity. Neuropeptides Neuropeptides are small peptides used by neurons to communicate to one another. They are the most diverse group of neuronal secreted chemical messengers and they function as hormones, neuromodulators or neurotransmitt ers to regulate physiological processes Cellular signaling through neuropeptides allows neurons to modulate the central and peripheral nervous system in both vertebrates and invertebrates (Strand et al., 1991) Neuropeptides can also regulate intercellular signaling (Caneparo et al., 2007) disease ho s t response (Boldajipour et al., 2008; Merritt, 2007) embryonic development (Ugriumov, 2009) and organogenesis (Pickart et al., 2006) Despite a range of functional roles, all classical neuropeptides are targeted to and processed by the regulated secretory pathway via conserved protein motifs (Figure 13)

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18 Before they are posttr anslationally processed into small peptides by enzymatic cleavage, neuropeptides exist as a larger prepropeptide (Southey et al., 2008) This prepropeptide is targeted for cotranslational translocation (CTT) into the endoplasmic reticulum (ER) by an N terminal signal sequence, or signal peptide. Signal peptides are composed of three regions: a positively charged N terminal region, a hydrophobi c region, and a polar uncharged C terminal region. Only the C terminal region typically shows conserved properties due to the presence of the cleavage site (Emanuelsson et al., 2007) Only those proteins lacking a transmembrane domain will pass through the ER and eventually be secreted outside of the outer cell membrane Secondary signal sequences on neuropeptides then interact with chaperone proteins to target the neuropeptide to secretor y vesicles Not all proteins that pass through the ER are targeted to vesicles that fuse with the plasma membrane to release its contents outside of the cell; those with different secondary signal sequences will be directed to various terminal destination s including the cell membrane, the ER, the Golgi apparatus, and other organelles (Klee, 2008) Prepropeptide s are then enzymatically cleaved to release functional neuropeptides. Some prepropeptides are made of highly repetitive units, and are processed to release several similar if not identical peptides. Other prepropeptides are made of unique units. Resear ch in mollusks has revealed expression of numerous families of neuropeptides (Krajniak et al., 1989; Smit et al., 1991) as well as individual neuropeptides (Banvolgyi et al., 2000; Kuroki et al., 1990; Smit et al., 1992) Physiological studies in Aplysia californica have demonstrated that neuropeptide modulation plays a crucial role in the initiation and regulation of several behaviors including feeding, egg laying, and card io activity (Campanelli and Scheller, 1987; Scheller et al., 1983; Sossin et al., 1987; Sweedler et al., 2002) Due to the lack of genomic information on Aplysia, it is likely that most neuropeptides remain unident ified. Using

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19 the conserved structural architecture of neuropeptides we will screen ou r large collection of neuronal transcripts from Aplysia and identify putative neuropeptides. Using the large neurons of Aplysia and unbiased shotgun sequencing, we ca n approach full coverage of all transcripts expressed in individual neurons in a simple memory forming network. It is my hypothesis that by using the variability of peptide, protein, and related secretory product expression in different neuron types that I will be able to identify patterns of peptide and protein expression that would give a signature for individual cell types. Coupled with the upcoming release of the Aplysia genome, this model system provides a unique opportunity to complete genome wide annotation of individual neurons to identifiy molecular markers of neuronal identity

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20 Figure 11. The Aplysia model system .. A) Image of Aplysia californica. (Image courtesy o f (Rudman, 2003) B) The abdominal ganglia from Aplysia showing large, easily identifiable neurons. C) The gill and siphon withdraw reflex can be simplified in ce ll culture to two neurons, a sensory cell (i.e. SN) and a motor neuron (i.e. L7) and still exhibit many of the same behavioral and cellular responses seen in mammalian conditioning including long term synaptic plasticity and memory. [ Image A courtesy of Rudman, W.B. (2003). Ink gl ands (Syndney, Sea Slug Forum) Images B and C cour tesy of Lovell and Moroz, unpublished.] 1mm 1mm A B SN L7 1 + Serotonin (5 HT) 0.2mm Motor neur ons Sensory neurons Motor neurons Sensory neurons C

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21 Figure 12. Organization of the Aplysia central nervous system. A) Diagram showing the central ganglia th at make up the central nervous system of Aplysia B) Representations of Aplysia showing the areas, color coded as in (A), innervated by specific ganglia. Some ganglia (pleural/pedal) have multiple functions innerva ting the same regions of the body as ind icataed by the gradient coloring however there is some specificity in innervating [ Modified from Moroz, L.L., Edwards, J.R., Puthanveettil, S.V., Kohn, A.B., Ha, T., Heyland, A., Knudsen, B., Sahni, A., Yu, F., Liu, L., et al. (2006). Neuronal transcript ome of A plysia : neuronal compartments and circuitry. Cell 127, 14531467.] Head ganglia Buccal g. Pleural g. Pedal g. Cerebral g. Abdominal g Buccal mass Intestine Buccal mass Buccal mass Intestine Intestine Eye Parapodium Foot Parapodium Foot Mantle and Visceral Hump Mantle and Visceral Hump A. B.

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22 Figure 13. Conserved motifs found in neuropeptides. All classical neuropeptides are targeted to the secretory pathway by signal peptides, short positively charged N terminal regions found on the C terminal region of a prepropeptide (Klee, 2008; Schatz and Dobberstein, 1996) The larger prepropeptides that encode neuropeptides are also characterized by regions of intrinsic disorder, and internal repeats that usually indicate the regions where peptides will be processed from. Importantly, prepropeptides destined to be fully processed into small pepti des lack transmembrane domains (Corsi and Schekman, 1996) Signal Peptide Intrinsic Disorder Internal Repeats

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23 CHAPTER 2 IDENTIFICATION OF NE UROSECRETORY PRODUCT S IN GASTROPOD MOLLU SCS Introduction Gastropod molluscs have served as powerful model organisms for cellular and system neurosci ence for more than 60 years. The central nervous system (CNS) of these models consists of a simplified network of relatively large identified neurons, allowing unprecedented opportunities to study the principles of organization of neural circuits as well as learning and memory mechanisms. However, a major limitation to identifiying the neurosecretory products of neurons of the molluscan models has been the lack of genomic information. To overcome these limitations, we have sequenced >980, 000 ESTs/cDNAs f rom the CNS of eight mollusc an species ( Gastropods: Aplysia californica, Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea stagnalis, and Cephalopods: Octopus vulgaris, Nautilus pompilius ) ( Figure 2 1). These sequenc es were assembled and cross annotated using the extensive transcriptome and genomic information from Aplysia californica (Moroz et al., 2006) This comparative approach allowed identification of both evolutionary c onserved neuronal genes and numerous novel genes including neuropeptides, prohormones and other predicted secretory products. It is estimated that there are >320,000 putative unique gene products ( including non coding and small RNAs) present in our compara tive neurogenomic database, which likely correspond to >5060% of the total number of genes expressed in the nervous systems of these molluscs. A selected list of genes identified in that study represent s major group of transcripts implicated in the contr ol of neural excitability, synaptic functions and plasticity, receptors, adhesion molecules, developmental genes, and homologs of genes involved in neurological disorders, etc. Specifically, we looked for putative neurosecretory products that may function as

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24 signaling molecules since these are important for modulation of the central and peripheral nervous system in both vertebrates and invertebrates are involved in neuron communication, identity and development. Most neurosecretory signaling molecules app ear to be neuron specific, making them ideal candidates for markers for neuronal identification and to understand the genomic basis for neuronal identity. Previous work using released genomes to screen for secreted signaling molecules and neuropeptides pre cursors. Several models including Danio rerio (Klee, 2008) Drosophila (Wegener and Gorbashov, 2008) Arabidopsis thaliana (Emanuelsson et al., 2000) Apis mellifera (Hummon et al., 2006) Tribolium castaneum (Am are and Sweedler, 2007) and Homo sapiens (Emanuelsson et al., 2000) have been investigated. These results have been useful for understanding organism wide expression of SSMs; however, none of these predictions hav e aimed at understanding neuron specific expression of SSMs due to limitations in cell specific mapping in these models. The large and easily identifiable neurons of Aplysia will allow the current work to go beyond the scope of these investigations to look at what SSMs are responsible for learning and memory, neuron identity and plasticity in single neurons. At the start of this project, 31 non redundant signaling peptides had been found by traditional cloning techniques and submitted to NCBI for Aplysia californica ( Object 22) This represents over 20 years of work by numerous labs searching for individual proteins. Here, a comprehensive list was created using both genomic and transcriptomic screening to identify all non redundant transcripts predicted to encode secretory signaling peptides expressed in the CNS of Aplysia californica. I t is shown that screening genomic and transcriptomic data for transcripts encoding proteins of interest represents a faster, more comprehensive way to identify putative SSMs important for neuronal processes.

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25 Results and Discussion Identification of Predicted Secreted Signal Molecules Homology r esults Through cross species homology search, 109 putative neuropeptides were identified from the Aplysia CNS transcriptome, 8 8 predicted to be secreted signaling molecules ( Object 22) and 21 predicted to be involved in the secretory pathway though not necessarily secreted ( Object 23). While homology searches can provide a wealth of information when initially annotating tran scriptomes, it limits annotation to only sequences with homology to known sequences. Indeed when annotating the >203,000 transcripts of the Aplysia transcriptome (Moroz et al, 2006) only 43,672 sequences can be annotated, showing the enormous complexity of the neuronal transcriptome ( Figure 22, insert ). Furthermore, preliminary results from cross species transcriptome analysis suggest that many neuropeptides are quickly evolving even between closely related species ( Figure 22). Due to these limitation s, we suspected that many SSMs of the Aplysia CNS could not be identified through homology searches, and decided to employ bioinformatics approaches for a more comprehensive list of SSMs using genomic scale searches. Prediction r esults To identify novel an d unannotated SSMs, publicly available software was used to complete genome scale profiling of all SSMs. These software programs rely upon conserved protein moti fs to predict prepropeptides. Signal peptide prediction software identifies conserved protein motifs required for processing and directing proteins to secretory pathways ( Figure 23). Accurate predictions rely upon the use of full length peptide sequences. Because there is

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26 currently no available genome for Aplysia and the Aplysia transcriptome r epresents a collection of partial length sequences, a set of full length protein models with which we could perform a computational secretome prediction is lacking Therefore, proteins predicted from the genome of Lottia gigantea, a related marine mollus c were used for computational SSM prediction. Lottia is a limpet belonging to a basal branch of G astropoda I t was chosen as the first mollusc to have its genome completed due to its small predicted genome. The availability of the Lottia genome made it possible to predict putative secreted signaling molecules that could then be used to search the Aplysia transcriptome for secretory products and effectively bridge the gap between genomic and non genomic species. A computational flowchart was used to scree n the Lottia genome for SSMs ( Figure 24). First, a set of 23,851 full length proteins predicted by the Lottia Filtered Gene M odels was downloaded from the JGI website. The Filtered Gene Models includes gene models selected as the best representative mod el available for each gene via a second layer of bioinformatics methods including manual annotation and the use of experimental data. Protein sequences were then screen e d for the presence of a signal peptide. Signal peptides are predicted by a characteri stic N terminal sequence 15 40 amino acids that contains 25 positively charged amino acids, 715 hydrophobic amino acids, and 37 neutral (often polar) amino acids that make up the cleavage site. Through the use of TargetP 1.1 (Emanuelsson et al., 2000) 3640 were predicted by the presence of a signal peptide to be directed to the secretory pathway. 1853 of those proteins were further predicted to contain a signal sequence by SignalP 3.0 (Bendtsen et al., 2004b) Not all proteins with signal peptides are secreted, like receptor proteins. To remove possible non secreted proteins, proteins were screened for the presence of transmembrane domains b y looking for repeated hydroph obic amino acid sequences 15 35 amino acids in length separated by polar

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27 connecting loops. Following screening for transmembrane domains by TMHMM 2.0c, 1034 candidate proteins were predicted to have no transmembrane domains 859 proteins were confirmed t o have a signal sequence and no transmembrane domains by Phobius (Kall et al., 2004) (see materials and methods for further information about prediction software). All sequences were then manually screened through SMART for transmembrane domains, and protein domains to remove any possible receptors or enzymes from the list. To ensure that predicted SSMs were likely to represent SSMs actually expressed by molluscs, only those SSMs shown to be expressed in the transcriptomes of two or more of our molluscan species were kept on the list. 87 predicted SSMs were predicted to be classically secreted proteins in Lottia including 22 putative neurosecretory products likely to be signaling molecules ( Object 24) and 65 of controversial or unknown function ( Object 25). Interestingly, more proteins (937) were identified by SecretomeP (Bendtsen et al., 2004a; Bendtsen et al., 2004b) as secreted via a non classical secretory pathway compared to those predicted to be secreted via classical pathways. Nonclassica lly secreted proteins are those proteins lacking a signal peptide that are still secreted by the cell These include some growth factors, interleukins and galectins. One possible explanation for this difference is that the SecretomeP program has inherent problems for prediction with our model species. SecretomeP relies upon machine learning algorithms to predict secretion from sequence information and currently has only been trained against Gram positive bacterial, Gram negative bacterial and mammalian s equences. The lack of training for invertebrate proteins may result in false positives. In fact, closer inspection revealed that many of the secretory products predicted by SecretomeP were annotated as molecules that rarely leave the cell.

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28 Annotation o f the Lottia SSMs against NCBIs NR database reveal that 14 classically secreted peptides had no hits to the database and therefore could not be annotated ( Table 21, Object 2 5). This could be a product of the evolutionary distance between Lottia and other commonly sequenced organisms used in the NR database. If the species represented by the sequences in NR are divergent enough from Lottia the predicted proteins will not be similar enough to the database entries to provide signifi cant hits for annotati on. This would suggest that homology search alone is insufficient for cataloging a list of molecular markers for neurons in the Aplysia CNS. The fact that more than 24% of the classically secreted peptides were overlooked by homology searching suggests t hat the use of bioinformatic approaches is crucial to identify all putative SSMs. Alignment of the 87 predicted Lottia SSMs against the Aplysia CNS transcriptome reveal 73 homologous putative neurosecretory molecules in Aplysia ; 21 are predicted to be secr eted signaling molecules ( Object 22 ) 28 are likely to be involved in the secretory pathway though not necessarily signaling molecules, and 24 are of unknown function ( Object 23). Confirmation of Expression of Selected Neuronal Transcripts As expected, the Aplysia neuronal transcriptome expresses many secreted signaling molecules including classical neuropeptides, growth factors, and hormones. To ensure that SSMs found through transcriptome analysis are expressed, and to determine the full length seque nce of each SSM, 94 of the identified 194 SSMs have been cloned from Aplysia cDNAs (including those previously submitted to NCBI by other labs). For this work I have cloned 15 newly identified SSMs including six full length gene products and nine partial sequences ( sequence information can be found in Object 22 ).

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29 Cross Species Analysis Using the analysis of predicted Lottia SSMs, a crossspecies transcriptome analysis of Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, Lymnaea stagnalis Octopus vulgaris, and Nautilus pompilius re vealed homologous predicted SSMs in all related molluscs ( Table 22). For each species, the percentage of all Lottia secretory proteins for which homologs exist varies across species ( Figure 25), suggesting different evolutionary rates between species. Species with more homologous proteins may be closer common ancestors to Lottia than those with few homologs. One precaution to such conclusions however, is that the number of transcripts in the EST collection for each species varies (i.e. 273,922 in Lymnaea vs. 10,209 in Melibe ) and fewer homologs may be present simply as a function of low coverage. My analysis also shows that classically and non classically secreted proteins evolve at different ra tes across species ( Figure 25). There is a consistently higher percentage of non classically secreted proteins than classically secreted proteins found in each species suggesting non classically secreted proteins are more highly conserved. Genomic Organ ization of Selected Neurosecretory Products Our preliminary comparison of predicted neurosecretory products across species revealed they are likely subject to great evolutionary divergence. To determine if variability in evolutionary divergence may be due to differences in the genomic organization, the intron/exon boundaries of selected neurosecretory products were analyzed using the recently released Aplysia genome, available on trace archives of NCBI. Most vertebrate genes are composed of seven to eight exons, however the number of intron/exon boundaries found in the selected neuropeptides from Aplysia is relatively small, an average of four exons ( Table 23). While there is some variation in the number of exons between selected neuropeptides (most have 3, 4 or 5 exons),

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30 there does not appear to be any clear correlation between the number of exons and the conservation or divergence of the neuropeptide. ( Object 2 1). Conclusions This comparative approach allowed identification of both evolutionary conse rved neuronal genes products and numerous novel predicted secreted proteins including neuropeptides, prohormones and other predicted secretory products. Our results reveal that homologous searching of transcriptomes is the quickest way to identify functio nally relevant transcripts for neuronal processes. However, genomic approaches are more useful for identifying novel transcripts, though the computational resources and manual screening require significantly greater investment in time Part of the obsta cle to using genomic approaches is identifying functionally relevant transcripts that are likely to be expres sed at the protein level, and not merely represent untranscribed regions of the genome. To ensure that the predicted transcripts are likely to be transcribed, the sequences were screened against eight gastropod CNS transcriptomes; of 676 predicted signaling molecules, only 167 were expressed in two or more of the transcriptomes. This suggests that at least 167 of the transcripts predicted to encode a secreted signaling protein are expressed and are likely functionally important since they have been conserved across mollusc species. As an additional method to validate expression of predicted transcripts, 15 of the identified transcripts believed to encode signaling molecules were cloned from cDNAs created from isolated Aplysia californica CNSs It is likely that some of the remaining predicted transcripts may also be functionally relevant but not highly conserved across species. This is not surpr ising given that the comparison between Aplysia and Lottia neuropeptides reveals that those neuropeptides responsible for development regulation share highly conserved transcript sequences while those

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31 responsible for regulation are more highly variable. T his may suggest one means for species specificity at the neuronal level. Using recently released genomic data from Aplysia californica the intron/exon boundaries of neuropeptides were analyzed to determine if sequence conservation and variability was due to the genomic arrangement of these genes. Preliminary analysis of 5 conserved, divergent and moderately conserved neuropeptides does not reveal any clear differences between the genomic organization of each type of neuropeptide. Perhaps the most important finding is the large number of sequences with shared identity found in both Aplysia and Lottia These sequences represent a portion of the Aplysia secretome that has been effectively predicted without having to perform expensive genomic sequencing or repeat the computationally expensive process of secretome prediction. This is a major step in bringing genomic scale proteomics to a species without a sequenced genome. Methods and Materials Animals and Tissue Collections Specimens of Aplysia californi ca weighing 150280 g were collected in the wild by Marinus Scientific (Long Beach, CA). S pecimens of Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, and Lymnaea stagnalis were obtained from the Friday Harbor Labs, Univers ity of Washington. Octopus vulagris was obtained from Italy and Nautilus pompilius from the Phillipines. Prior to dissection of gastropods animals were anesthetized by injecting a volume of isotonic MgCl2 (337mM) equivalent to 50% 60% of their weight. Disse c tion of Cepholopods and Gastropod Mollusca was performed by Dr. Moroz. Total RNA from whole CNS was extracted using RNAqueousTM (Ambion, Austin, TX) kit. RNA isolation was performed by Dr. A Kohn, Jinnie Sloan, and Yelena Bobkova. Before further p rocessing of the RNA, quality was checked using a 2100 BioanalyzerTM (Agilent Technologies). Small aliquots of extracted RNA were loaded on a 6000 Nano Lab chip that

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32 produces an electropherogram image along with a gel like image of the sample representing peak ratios of the 28s/18s RNA contained in the sample. Additionally RNA concentration was measured spectrophotometrically by using the GeneSpec III systemTM454 Library Construction (Mirai Bio). Library construction was completed by Dr. Kohn and Jinnie Sloan. The protocol for transcription analysis was developed by Drs. A Kohn, Y. Panhin and L. Moroz. 3' end amplified cDNA library construction for 454 sequencing: This technique targets the 3' end of the transcripts being expressed. Amplified cDNA wa s generated using the Marathon cDNA amplification kit (Cat # 634913, BD Biosciences, Clontech, Mountain View CA). The first strand synthesis utilized the AMV Reverse Transcriptase and an oligo(dT) primer, Trsa (Matz, 2002) After second strand synthesis and clean up following the Marathon kit protocol, the entire sample of cDNA was fractionated by digestion with 20 units of Alu 1 and NEBbuff er 2 (Cat # RO137S0, New England BioLabs, Ipswich, MA) for 1 hour at 37C. The enzyme was heat inactivated at 65C for 20 min. The double stranded adaptor was made with A adaptors then added to the ligation mixture at a final concentration of 1 M along with the digested cDNA, 2 Units T4 DNA ligase and 5 x ligase buffer (Cat # 634913, BD Biosciences, Clontech, Mountain View CA). The ligation was performed at 16C overnight. The cDNA was purified using DNAclear (Cat#1756, Ambion Inc, Austin, TX) and elut ed in 16 l of RNAase free water. A mplification of the cDNA was performed using 16 l of the purified cDNA, Advantage 2 buffer, dNTP and taq polymerase (Cat # 639201, BD Biosciences, Clontech, Mountain View CA). The primers A pcr B pcr added to the ampli fication were at a final concentration of 0.05 M with B adaptor at a final concentration of 0.01 M. This full length B adaptor was modified to complement with the Trsa primer along with the B adaptor on the beads and was added to ensure the eventual att achment of the cDNA on the beads. The PCR

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33 amplification protocol consisted of two cycles of 95C for 30 sec, 50C for 30 sec, 72C for 1 min, followed by 15 cycles of 95C for 30 sec, 65C for 30 sec, 72C for 1 min. Half of this PCR product was used for asymmetrical PCR to generate single strand cDNA. An excess of 10 times A pcr primer final concentration of 0.5 M was added along with 6 more cycles of 95C for 30 sec, 65C for 30 sec, 72C for 1 min performed. Controls were performed in which the ori ginal PCR product was diluted 1:50 and 1 l used in a total volume of 20 l. Only either A pcr primer or only B pcr primer were added at a final concentration 0.05 uM and 15 cylces of of 95C for 30 sec, 65C for 30 sec, 72C for 1 min were performed. Thi s was to ensure that the PCR suppressive effect was occurring. The ssDNA was measured for size and concentration then processed for bead attachment. This shotgun library was sequenced with GS Sequencing Kit (70x75) (Cat # 04 853 342 001 Roche Applied Sci ence). 5' end target amplified cDNA library construction for 454 sequencing: This technique targets the 5' end of the transcripts being expressed and is very similar to the previous protocol with a few minor revisions. Copied cDNA, first strand synthesis and second strand synthesis were generated same as described. After second strand synthesis and clean up following the Marathon kit protocol, the entire sample of cDNA was ligated with the double stranded A adaptors at a final c oncentration of 1 M along with 2 units T4 DNA ligase and 5 x ligase buffer (Cat # 634913, BD Biosciences, Clontech, Mountain View CA). The ligation was performed at 16C overnight. This cDNA was fractionated by digestion with 20 units of Alu 1 and NEBbuffer 2 (Cat # RO137S0, New England BioLabs, Ipswich, MA) for 1 hour at 37C. The enzyme was heat inactivated at 65C for 20 min. The cDNA was purified using DNAclear (Cat#1756, Ambion Inc, Austin, TX) and eluted in 16 l of RNAase free water. A second double stranded adaptor was made with B adaptors added at a final concentration of 1 M along with the digested cDNA, 2 Units T4 DNA ligase and 5 x ligase buffer (Cat # 634913,

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34 BD Biosciences, Clontech, Mountain View CA). This ligation was performed at 16C overnight. Again the cD NA was purified using DNAclear (Cat#1756, Ambion Inc, Austin, TX) and eluted in 16 l of RNAase free water. Amplification of the cDNA was performed using 16 l of the purified cDNA, Advantage 2 buffer, dNTP and taq polymerase (Cat # 639201, BD Biosciences, Clontech, Mountain View CA). The primers added to the amplification were at a final concentration of 0.05 M and the sequences were A pcr and B pcr. The PCR amplification protocol consisted of 17 cycles of 95C for 30 sec, 65C for 30 sec, 72C for 1 mi n. Preparation and sequencing of the Amplicon product used the GS emPCR Kit II (Amplicon A and Paired End) (Cat # 04 891 384 001 Roche Applied Science) GS Sequencing Kit (70x75) (Cat # 04 853 342 001 Roche Applied Science) Primers for 3 and 5 libraries are listed in Table 2 4. Cloning of selected Signaling Molecules Amplified cDNA libraries were constructed from the CNS of Aplysia californica, as described elsewhere (Matz, 2002; Moroz et al., 2006) Full length c DNA sequences for six sequences and the partial sequence of an additional nin e sequences were obtained. The full length copy of coding sequences were amplified from appropriate CNS cDNA libraries and cloned into pCR 4 TOPO (Invitrogen). For each sequence three clones were isolated and sequenced by SeqWright (Houston, Hypothetical protein 2 TX). The full length sequence for Hypothetical protein 2 was identified based on BLAST results against the National Center for Biotechnology Information (NCBI) as Apl ysia californica hypothetical protein previously cloned (Cummins et al., 2004) (Genebank accession number AAN83922). Using terminal primers 5 CTCTGAACCGTCGCGAACTGTGT 3 and 5 -

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35 AGGCAACAGTGGAATCGAAGCTCTC 3, our lab cloned the full length sequence of Hypothetical protein 2, resulting in a 788 bp fragmen t. LFRFamide precursor The full length sequence and clone for LFRFamide was obtained (Genebank accession number EU886298) using terminal primers 5 GGCCTCAGTTCGAAACCTCG 3 and 5 ATTGGTCACCCTGTCCTCGG 3. The resulting cDNA fragment was 1074 bp. Myomodul in like Neuropeptide Precursor 3 The full length sequence and clone for Myomodulin like Neuropeptide Precursor 3 was obtained (Genebank accession number EU934739) using terminal primers 5 CCTGAGTCCAGCCTAAGCGGTAAGT 3 and 5 GTACTGTGTAGGACGTAGGAAGCAG 3. The resulting cDNA fragment was 1690 bp. Conopressin The full length sequence and clone for Conopressin was obtained (Genebank accession number FJ172359) using terminal primers 5 CCAACTACAGGATGTCTCACTC 3 and 5 GACGTTGAGTGGACACTGTGA 3. The resulting cDNA fragment was 1983 bp. Ependymin related protein 2 The full length sequence and clone for Ependymin related protein 2 was obtained (not submitted) using terminal primers 5 CTGGTATCAGAGCCACTCACCTC 3 and 5 TGGTTGAATGTATACGTGCATGTAC 3. The resulting cDNA fragment was 873 bp. Be tsin The full length sequence and clone for Betsin was obtained (not submitted) using terminal primers 5 GTAACTTTCGTCCCTCCTGCCA 3 and 5 CTTCTACTTGACCCACTCGGACC 3. The resulting cDNA fragment was 538 bp.

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36 Feeding circuit ac tivating petide precursor 3 (FCAP 3) The partial sequence and clone for FCAP 3 was obtained (not submitted) using terminal primers 5 AACAGGCGCAGGCTCAGA3 and 5 ACACGTGGATCGCCGCCGCC 3. The resulting cDNA fragment was 367 bp. SN4 The partial sequence a nd clone for SN4 was obtained (not submitted) using terminal primers 5 ATGCAGTGAGGGATGCTGTGT 3 and 5 GGTCAAGGCCAATTTCCCGTG 3. The resulting cDNA fragment was 755 bp. SFY1 like peptide The partial sequence and clone for SFY1 like peptide was obtained (not submitted) using terminal primers 5 -GTGGAAGTACGCAGAGAGG3 and 5 -CATCCCTCTTGTAGAAGCTGGA3. The resulting cDNA fragment was 1198 bp. SFY3 like peptide The partial sequence and clone for SFY3 like peptide was obtained (not submitted) using terminal primers 5 -GTATCAAGACCGACGACCATG3 and 5 -GTGAGCTCATCCCGCGGTTG3. The resulting cDNA fragment was 729 bp. FIRFamide related neuropeptide precursor The partial sequence and clone for FIRFamide related neuropeptides precursor was obtained (not submitted) using terminal primers 5 CGTCATCGCTGGTGCTGTCAC3 and 5 -CCTCGACAAGGCTTCTCCTTCACC3. The resulting cDNA fragment was 2032 bp. Major royal jelly protein (MRJ) The partial sequence and clone for MRJ protein was obtained (not submitted) using terminal primers 5 CGGAAGTCCGGTACGCGTATATTTC 3 and 5 CTTTCAGAAGGCTATTCCTCCCACC 3. The resulting cDNA fragment was 774 bp.

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37 FMRFamide 5 The partial sequence and clone for FMRFamide 5 was obtained (not submitted) using terminal primers 5GGTGGATCAAGCCTTGGAGCT3 and 5CACTCAGGTTATTCAACACGTCAAC3. The resulting cDNA fragment was 698 bp. Allatotropin OR precursor The partial sequence and clone for Allatotropin OR precursor was obtained (not submitted) using terminal primers 5 -CAAGTGGTGATTCGGCCGC3 and 5GTCAAT ATAGTTCCCTCTCGTGG3. The resulting cDNA fragment was 746 bp. Second The partial sequence and clone for Second was obtained (not submitted) using terminal primers 5 -GCTAATGAGCGATTTCTGCGAG3 and 5 -CGTCCTTCTCCGAAAGGCCTAAGCC3. The resulting cDNA fragme nt was 712 bp. Neurosecretory Predictions Preditions were performed by Jinnie Sloan, Mathew Citarella, Drs. A. Kohn and L. Moroz. Software scripts were written by M. Citarella. Homology search using unbiased shotgun approach A cross species master list o f secreted signaling molecules submitted to NCBI was created by manual word search. Search terms included any combination of the following words: signal, peptide, growth factor, hormone, and neuropeptide. Any results containing the word receptor were e liminated. All sequences submitted to PeptideDB (www.peptides.be, May 2009) a public resource for bioactive peptides that includes cytokines and growth factors, peptide hormones, antimicrobial peptides, toxins and venom peptides, and antifreeze proteins were manually

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38 downloaded and added to the above results in FASTA file format. All redundant sequences were removed and the master list was aligned with cDNA ESTs from Aplysia as described below. To ensure that all results represent SSMs, all Aplysia ho mologs were screened using SMART (Schultz et al., 1998) and any sequences with a transmembrane domains or enzyme protein family domains were removed. Genomic approach To facilitate prediction of the Lottia secretome, 23 ,851 full length protein sequences were downloaded from the Joint Genome Institute (JGI) website (www.jgi.doe.gov, May 2009) These sequences represent the best filtered protein coding gene models as determined by JGIs genomic assembly. The latest relea se of NCBIs non redundant protein database, NR, was downloaded to provide annotation for any predicted secretory products. NR contains protein sequence entries from the following databases: GenPept, Swissprot, PIR, PDF, PDB, and NCBI RefSeq. Classical S ecretory Prediction* The original data set of 23,851 full length protein sequences from the Lottia genome was analyzed with TargetP 1.1. Batches of 1000 sequences were fed to TargetP via a custom wrapper script ( Citarella, M.R. unpublished), written in Pe rl using the short output option for TargetP. Sequences were considered to be targeted to the secretory pathway if they had a SP (secretory pathway) score > .80. All other sequences were discarded from the prediction. The selected sequences were then analyzed with SignalP 3.0 for the presence of a signal sequence using both Neural Network and Hidden Mark Model methods and the short and nographics output options. Proteins were determined to have a signal sequence if SignalP returned a Y for four out of the five scores for the Neural Network method and the Hidden Markov Model method predicted that it contained a signal peptide. Transmembrane domains were then

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39 predicted using TMHMM 2.0c4Non classical Secretory peptide Prediction* using the default settings. Only sequences with no predicte d transmembrane domains or one transmembrane domain and more than four residues of that domain in the first 60 amin o acids were considered further (these proteins are retained since a single predicted transmbrane domain in the first 60 amino acids of a pro tein may be a signal sequence falsely identified as a trans membrane domain). These proteins were analyzed with the web version of Phobius using the short option. Proteins with no predicted transmembrane domains and a confirmed signal sequence were sele cted as the final predicted classically secreted proteins. 14,595 full length protein sequences that were localized to other with a score > .75 by Ta rgetP during classical secretory peptide prediction were ana lyzed with the web version of SecretomeP 2.0 in sets of 100 with the mammalian option checked. Sequences were determined to be secreted via a non classical pathway if their NN score exceeded .75 and there was no predicted signal peptide by SignalP. Unless otherwise noted, the above predictions were all performed with the standalone version of the software package mentioned on an Intel Pentium 4 with 1GB RAM running Ubuntu Linux 8.04. Cross Species Analysis of Predicted Proteins All homology searches including the final set of classically and non classically secreted proteins from Lottia were aligned with cDNA Expressed Sequence Tags (ESTs) from Aplysia californica CNS using NCBIs standalone BLAST package. The following options were set program: bl astp, e value: 1e 04, processors: 3, wordsize: default(3). The results of each BLAST were parsed with a custom Perl script, findHomologs.pl. For each predicted secretory protein, a homolog was said to be found in a species if there was a BLAST hit for t hat species with an e -

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40 value less than or equal to 1e 04. Furthermore, a single sequence from a given species could be reported as a homolog for at most one Lottia secretory protein. Annotation of Predicted Proteins To annotate the list of predicted secre tory proteins in Lottia the latest release of NCBIs NR database was downloaded and each predicted protein was BLASTed against it using NBCIs standalone package set for blastp with default settings. Annotation for a given predicted protein was determine d as the identifier of the sequence from the NR database with the lowest e value hit to the predicted protein, so long as the e value was less than or equal to 1e 04.

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41 Figure 21. Summary of model organism and number of ESTs/cDNA s collected. More than 980,000 ESTs/cDNAs were collected from the CNS of five key model species ( Pleurobranchaea californica, Clione limacina, Tritonia diomedea, Melibe leonina, and Lymnaea stagnalis ). Here, the number of ESTs for each species is shown under their name Nudibranchia Melibe leonina Tritonia diomedea Aplysia californica Cl ione limacina Gastropoda Lymnaea stagnalis Lottia gigantea Pleurobranchaea californica Acanthopleura spinosa (259,277) (131,851) (115,599) (10,809) (273,922) (220,000/2,392,545) (188,590) Melibe leonina (259,277) (131,851) (115,599) (10,809) (273,922) (220,000/2,392,545) (188,590) Melibe leonina (259,277) (131,851) (115,599) (10,809) (273,922) (220,000/2,392,545) (188,590) Melibe leonina (259,277) (131,851) (115,599) (10,809) (273,922) (220,000/2,392,545) (188,590) Opisthobranchia Polyplacophore Pulmonata Prosobranchia

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42 1.00E-116 1.00E-107 1.00E-98 1.00E-89 1.00E-80 1.00E-71 1.00E-62 1.00E-53 1.00E-44 1.00E-35 1.00E-26 1.00E-17 1.00E-08 1.00E+01 Dorsal -ventral Buccalin Pedal peptide 1 Conopressin Ependymin Temptin Prothoracicostatic Myomodulin Pleurin Whitnin Intersectin -2 insulin PTSP-like Neuropeptide Y SN 4 Enterin Tolloid 2 Orcokinin Venom protein 2 FMRFamide Fulicin-like MIP-related Neurotoxin -1 LFRFamide Neuronal Transcript Figure 22. Analysis of Evolutionary Dynamics of Neuronal Transcripts. Preliminary analysis suggests that some neuropeptides may be fast evolving molecules. The sequence conservation between Aplysia and Lottia reveals that proteins involved in development (i.e. Dorsal ventral patterning) have the highest sequence similarity (indicated by a high e value from sequence alignments), while neuropeptides involved in regulatory mechanisms (i.e. FMRFamide) have t he most variability. These likely reflect differences in biological constraints between these two neuronal processes. The fast evolutionary divergence of some neuronal genes may account for the large amount of unannotated sequences from the neuronal tran scriptome of Aplysia (insert). E value Aplysia californica 203,440 transcripts 43,672 annotated 159,768 unannotated

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43 FMRFamide Internal Repeats Intrinsic Disorder Signal Peptide Figure 23. Conserved Signaling Molecule Motif. Example of the conserved motifs of secreted signaling molecules (here the neuropeptide FMRFamide is shown). Secreted signaling molecules are targeted to the secretory pat hway by their signal peptides, and often are characterized by intrinsic disorder, internal repeats, and cysteine rich regions. They are differentiated from proteins encoding receptors by a lack of transmembrane regions.

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44 Figure 24. Workflow for Com putational Prediction of SSMs. The filtered gene models for Lottia gigantea were downloaded from JGI. After filtering the original 23,851 predicted proteins through four protein prediction software programs, 859 proteins were predicted to be classically secreted signal molecules. That is, contained signal sequences, no transmembrane domains, and were targeted for secretion outside the cell. Using BLAST alignments, 400 classically secreted homologs were identified in Aplysia ETSs.

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45 Table 2 1. Annotation of Lottia predicted secretory products against NCBIs NR database. The results of the annotation show the ~24% of the classically predicted secreted peptides cannot be mapped to a biological function due to poor annotation. This could be a product of th e evolutionary distance between Lottia and other commonly sequenced organisms. If the species represented by the sequences in NR are divergent enough from Lottia the predicted proteins will not be similar enough to the database entries to provide signifi cant hits for annotation. Data Set Number Annotated Number Unannotated Percent w ith Predicted in Annotation Percent w ith Hypothetical in Annotation Percent of Peptides with unknown or ambiguous functions Classically Secreted Peptides 83 1 4 46.53 % 5.94 % ~ 24%

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46 Table 2 2. Summary of Cross Species Predictions. Results of c ross species predictions using homology search against nonbiased shotgun transcriptomes and homology search against our Lottia genomic predictions. Results show th at more predicted neurosecretory products are found using the genomic approach perhaps suggesting a genomic approach as a more robust form of predicted signaling molecules in gastropod molluscs. It is unclear whether differences across species represents sequence divergence from the model organism used ( Lottia ) or is simply an artifact of different levels of coverage of the sequencing for each species. Species Shotgun Genomic Object Aplysia californica 90 74 2 1 to 2 3 Lottia gigantea 75 87 2 4 to 2 5 Lymnaea stagnalis 66 48 2 6 Pleurobranchaea californica 45 42 2 7 Tritonia diomedea 37 28 2 8 Mel ibe leonina 18 14 2 9 Clione limacina 9 2 10 Octopus vulgaris 31 2 11 Nautilus pompilius 44 2 12

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47 Figure 25. Comparative analysis of predicted Lottia secretory products. Th e percentage of all predicted Lottia secretory proteins for which homologs were found in each species. While some variation in expression of homologous sequences may be due to the number of ESTs collected, it may also provide insight to the evolutionary d istance between species. These data suggests SSMs between Lottia and Aplysia are the most conserved, followed by Lymnaea and Pleurobranchaea. Furthermore, classically and non classically secreted proteins appear to evolve at different rates across specie s with non classically secreted peptides being more highly conserved. Aplys ia Clione Lymnaea Melibe Pleurobranchaea Tritonia

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48 Table 2 3. Genomic organization of neurosecretory g enes. Preliminary analysis of the genomic organization of selected neurosecretory products shows that all genes have a similar in tron/exon organization, with an average of 3 5 exons. (See Supplemental A for detailed information.) Neuropeptide Number Introns Number Exons Atrial gland specific antigen prec ursor 9 10 Feeding circuit activating peptide precursor 1 2 FMRFamide neuropeptide precursor 2 3 L11 neuropeptide precursor 2 3 Buccalin precursor 3 4 Conopressin 3 4 Fulicin like neuropeptide precursor 4 5 MIP related peptide precursor 4 5 Neu rotoxin like 1 3 4 Pleurin 2 3 Whitnin precursor 2 3

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49 Table 2 4. Adaptors and Primers for 5 and 3 454 libraries Primer Name P rimer sequence Trsa 5' CGCAGTCGGTAC (T) 13 3' 3 bias library A Adaptor 5' CCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAG 3' and 5' CTGAGACAGGA 3' A pcr 5' CCATCTCATCCCTGCGTGTC 3' B pcr 5' CCTATCCCCTGTGTGCCTTG 3' B adaptor,Trsa 5' CCTATCCCCTGTGTGCCTT GCCTATCCCCGCAGTCGGTACTTTT 3'). 5 bias library A Adaptor 5' GCCTCCCTCGCGCCATCAG 3' and 5' CCTGATGGCGCGAGGG 3' B Adaptor 5' GCCTTGCCAGCCCGCTCAG 3' and 5' CTGAGCGGGCTGGCA 3' A pcr 5' GCCTCCCTCGCGCCATCAG 3' B pcr 5' GCCTTGCCAGCCCGCTCAG 3'

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50 CHAPTER 3 MAPPING EXPRESSION O F NEUROPEPTIDE TRANS CRIPTS Introduction One major fun ction of n europeptides in both vertebrates a nd invertebrates is to act as extracellular chemical messengers to modulate the communication between neurons of the central and peripheral nervous systems (Kaldany et al., 1985; Strand, 1999) Physiological studies in Aplysia californica have demonstrated peptidergic modulation plays a crucial role in the initiation of several behaviors including feeding, egg laying, and cardioregulation (Campanelli and Scheller, 1987; Scheller et al., 1983; Sossin et al., 1987; Sweedler et al., 2002) Several families of neuropeptides are expressed in molluscs (Fujiwara Sakata and Kobayashi, 1992; Price and Greenberg, 1977; Smit et al., 1991) as well as individual neuropeptides, (Banvolgyi et al., 2000; Kuroki et al., 1990; Smit et al., 1992) To support the identification of putative signaling molecules identified in the first chapter of this thesis, in situ hybridization was performed for a select number of signaling molecules to test for expression of the RNA transcript. Here, the expression of two putative neuropeptides MIP and Fulicin, is described for the CNS of Aplysia. Mytilus Inhibitory Peptides One set of identified neuropeptides in Aplysia belongs to the Mytilus inhibitory peptides (MIPs) family (Fujisawa et al., 1999) Originally isolated from t he pedal ganglia of the bivalve Mytilus edulis (Hirata et al., 1987) MIPs can inhibit tar get muscles and hyperpolarize central neurons (Kiss and Osipenko, 1997; Kissler et al., 1997; Yongsiri et al., 1989) A plysia MIP related peptides (AMRPs) are expressed in the CNS and peripheral tissues including t he digestive tract, vasculature and reproductive organs where they have a dose dependent inhibitory action on target tissues (Fujisawa et al., 1999; Hirata et al., 1987)

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51 Fulicin Previously unidentified in Aply sia, Fulicin was initially isolated from the African giant snail Achatina fulica (Ohta et al., 1991) In Achatina Fulicin has been shown to regulate female egg laying behavior, potentiate tetanic contraction of the peni s retractor muscle and modulate actions of the ganglionic neurons as well as buccal and ventricular muscles (Fujisawa et al., 2000; Ohta et al., 1991) Fulicin was shown to share the C terminal portion Phe Val NH2 with Mytilus inhibitory peptides (MIPs), and both peptides depress the phasic contraction of the ABRM muscle in Mytilus edulis (Hirata et al., 1988; Kim et al., 1991) However, Fulicin was shown to be 10,000 t imes less potent than MIPs, presumably due to the lack of Pro residue required for inhibitory activity of MIPs (Kim e t al., 1991) This chapter focuses on the mapping of these two neuropeptides exclusively to provide further insight into possible explanations for the shared C terminal region of these two protein precursor. Here we cloned, localized, and show predicted gene products of an Aplysia Fulicin related peptide (AFRP) Using two color in situ hybridization (Jezzini et al., 2006) we show co localized expression of MIP and Fulicin transcripts in the CNS of Aplysia Fina lly, we postulate that the shared region of MIP and Fulicin, TxFrag2, is a regulatory genomic element that provides a molecular mechanism to regulate expression of two ne uropeptides in a single neuron. Results Cloning of Aplysia Fulicin precursor mRNA As d escribed in Chapter 1 of this work, a putative neuropeptide Fulicin was identified during annotation of transcripts generated fr om unbiased shotgun sequencing. The identified Aplysia Fulicin like transcript was cloned resulting in a full length sequence w ith a 1059 base pair open reading frame coding for a 352 amino acid precursor ( Figure 3 1A ).

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52 The predicted Fulicin precursor protein contains a hydrophobic signal peptide and a cleavage site between Asp and Thr, which suggests this protein is targeted to the secretory pathway. Analysis of monobasic, dibasic and tribasic cleavage sites suggest that there are 13 copies of 10 different predicted amidated peptides processed from the Fulicin precursor ( Figure 31B ). Localization of Fulicin in the CNS of Aply sia Since Fulicin has not been localized to the CNS of Aplysia we first wanted to map expression of Fulicin using in situ hybridization. The Fulicin transcript showed neuron specific expression with the most intense staining in specific neurons of the abd ominal and pleural ganglia ( Figure 3 2). Interestingly, Fulicin clearly labels the L7 motor neuron in addition to other motor neurons critical to the function of the gill and siphon withdraw circuit. Expression is localized in the LP1 neur on of the pleur al ganglia, and small subsets of neurons in the cerebral, pedal and buccal ganglia, suggesting that Fulicin expression is not ubiquitous It was noted that many of the neurons positive for Fulicin expression are the same neurons known to express MIP, sugg esting that besides sequence similarity, the expression of these two neuropeptides may also be similar. Co localization of MIP and Fulicin To further investigate the possible co expression of MIP and Fulicin in the CNS of Aplysia, co localization was perfo rmed using two color in situ hybridization ( Figure 3 3). Using two color in situ hybridization labeling with specific probes we found that MIP and Fulicin were exclusively co expressed. During the staining protocol it became apparent that some cells do s tain with different intensities, suggesting that the expression level of these peptides may not be uniform in positively labeled cells, but rather expression levels are neuron specific.

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53 Isolation of Fulicin in the CNS of Aplysia 454 sequencing revealed higher expression of transcripts aligning to the coding regions of the MIP gene than real time PCR (RT PCR) experiments ( Table 31). We found that MIP showed strong sequence similarity to another gene, Fulicin, in its 5 region ( Figure 34) as supported b y previous work in Mytilus edulis showing MIP and Fulicin have a structurally similar C terminal region (Hirata et al., 1988; Kim et al., 1991) MIP and Fulicin share 5 UTR and coding region We postulate that the s hared 5 untranslated region may serve as a molecular mechanism underlying the observed colocalization of these two neuropeptides. To understand the functional role of this shared region, we extended the sequence of the coding region into the 5 UTR of e ach of these genes to check if the shared sequence continued upstream of the protein start site. Using 5 RACE, we extended the non coding region of both Fulicin and MIP to reveal a 480 nt region, called TxFrag 2, that includes the 5 UTR and the 21 amino acid signal peptide of MIP and Fulicin ( Figure 31A, blue and Figure 35). TxFrag2: Structure and Function Alignment of TxFrag2 to genomic data available on NCBI reveals that the 480 nt region is transcribed from four defined fragments of DNA separated b y three intergenic splice sites, characteristic of transgenic noncoding RNA. The fourth defined fragment contains the signal sequence shared by both MIP and Fulicin. At the end of the signal sequence there is a splice site in both MIP and Fulicin, the st art of Exon 2 marks the beginning of the uniq ue coding region of both genes. Previous work mapping short noncoding RNAs (sRNAs) indicates sRNAs cluster at the 5 and 3 of genes. Similar to these results, Transfrag2 is located at the 5 of MIP and Fulicin Like

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54 other intergenic sRNAs (IRNAs), TxFrag2 includes the 5 boundary of the protein coding gene but excludes most of the other exons of the coding region. Previous work on IRNAs suggests they are involved in regulation of gene expression (Davis et al., 2006; Martianov et al., 2007) These studies suggest that IRNA transfrags serve as precursors to functional sRNAs via sense and antisense transcription of the IRNA (Kaprano v et al., 2007) To see if TxFrag2 is transcribed in the sense or antisense direction, 454 and SOLiD transcripts that aligned to the 480 nt TxFrag2 region were counted and compared to the quantities of sense and antisense MIP and Fulicin ( Figure 35B ). This show ed that TxFrag 2 is transcribed in both the sense and antisense direction where as the coding region of MIP and Fulicin are made only in the sense direction. Discussion Colocalization of Fulicin and MIP This is a unique example of two neuropeptides showing exclusive co localization throughout the CNS of Aplysia After several in situ experiments, we have found that transcripts for both MIP and Fulicin consistently colocalize to specific neurons of the CNS of Aplysia, predominately those neurons i n the abdominal ganglia responsible for the gill and siphon withdraw. Analysis of the protein sequence of MIP and Fulicin reveal that structurally, they share a conserved C terminal. Further analysis of the nucleotide coding region of the sequences show s that this results from a conserved 5 UTR region that extends through the first exon of both genes. Preliminary analysis using recently released genomic information from Aplysia californica further supports that MIP and Fulicin share the same splice sites and exon regions covering the TxFrag2 region until after the signal sequence.

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55 There is currently one additional publication indicating that the preproprotein of two neuropeptides, brandykinin and temporin in frog, share a conserved 5 UTR and signal sequence (Suzuki et al., 2007) In this paper the authors suggest that the sequence similarity may suggest a linked evolutionary history involving exon shuffling. TxFrag2: Modulator of Expression? With the comple tion of several genome projects, including the Human Genome Project, the once accepted view that of noncoding RNAs (ncRNAs) as junk is rapidly shifting to accept non protein coding transcripts are c rucial for cellular function. Recent reports suggest th at only 2% of the human genome codes for translated proteins (System, 2008) while nearly ha lf of the genome is transcribed and expressed as RNA without any messenger (mRNA), transfer (tRNA), or ribosomal (rRNA) functions (Szell et al., 2008) Understanding the role of these ncRNAs represents a new realm of understanding genomic data, and the accumulating data suggests ncRNAs may play a role in organism complexity, specificity, cell regulatory machinery, and regulation of these ncRNAs has been implicated in several human diseases (Szell et al., 2008) Work on the human genome has demonstrated that the transcriptome does not exist exclusively of protein coding transcripts but also regulatory genomic elements and non protein coding transcripts (Szell et al., 2008) This project, termed ENCODE (the Encylopedia of DNA Elements), along with unbiased tiling array data has identified a new set of transcripts (TxFrags) that are made from fragments of defined genomic regions. Cross species genome sequencing has shown that increasing biological complexity is correlated with increasing number of non protein coding DNA sequences (Taft et al., 2007) and suggests that differences between species may rely upon non coding genes ra ther than proteincoding genes (Pollard et al., 2006) While the function of TxFrag2 remains to be determin ed, expression of both sense and antisense transcripts suggest that it may serve in a regulator mechanism in these cells. It is our

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56 hypothesis that TxFrag2 may function similar to miRNAs, regulating the expression of MIP and Fulicin by binding antisense t ranscripts to complementary upstream translation regions. However, the large size of TxFrag2, 480nt, compared to a normal miRNA, ~20nt, challenges any conclusions about the mechanism of TxFrag2 regulatio n. Overall, these data provide evidence for a possi ble molecular mechanism underlying how neurons can regulate expression of multiple neuropeptides within a single cell. Materials and Methods Animals Specimens of Aplysia californica weighing 150280 g were collected in the wild by Marinus Scientific (Lon g Beach, CA). Animals were anesthetized by injection of 50% (volume/body weight) isotonic MgCl2 (337 Cloning of full length cDNA encoding Fulicin m M ) prior to surgical removal of the central nervous system (CNS). Terminal primers were designed from two overlapping ESTs that shared high identity to mRNA for the F ulicin precursor in Achatina fulica (Genebank accession number D13986). A full length cDNA sequence called fulicin like neuropeptide pr ecursor (Genbank accession number AAW30458) was obtained using terminal primers: 5' CAATCAACCCGCAATGTGTACC 3' Sequence analysis and Alignments and 5' CTAAGAATCCGGGCACGACGC 3' from an amplified cDNA library. The amplified PCR product was 1064 bp. The initial multiple alignment was done using ClustalX ver. 1.83 (Jeanmougin et al., 1998; Thompson et al., 1997) with default parameters. All protein predictions were determined with Prosite (Gattiker et al., 2002) and SMART (Letunic et al., 2006)

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57 In situ hybridization of Fulicin and MIP in Aplysia Full length cDNA from Fulicin and MIP was clone d and used for the preparation of in situ probes. For co localized expression studies, clones were made for Fulicin and MIP from unique regions starting after shared 5 UTR and signal sequence. The antisense probe was generated by digestion of cDNA from Fulcin and MIP with Not I (New England Biolabs), then transcription with T3 polymerase from the DIG (digoxigen) RNA labeling kit (Roche Diagnostics). The control sense probe was produced by the same protocol but used Pme1 (New England Biolabs) to digest t he cDNA and T7 polymerase for transcription. The DIG labeled antisense probes were hybridized in whole mount CNS preparations, and the neurons containing the probe target duplex were localized and visualized with alkaline phosphatase conjugated anti DIG a ntibody fragments (Boehriger Mannheim). The detailed in situ hybridization protocol has been described (Jezzini et al., 2005; Jezzini and Moroz, 2004; Walters et al., 2004) Expression of Fulicin was investigated in central ganglia of four experimental CNS preparations and two control experiments. Control in situ hybridization experiments with full length "sense" probes revealed no specific and selective staining in the CNS under identical conditions and labeling p rotocols for either probe. Co localization using two color in situ hybridization was performed as previously described (Jezzini et al., 2005) Briefly, unique regions of MIP a nd Fulicin were cloned and sequenced. Two probes were then made using different NTP labeling mixes. The MIP probe was made using fl uorescein 12UTPs and the Fulicin probe was DIG labeled. First the fluo rescein MIP probe was hybridized in whole mount CNS preparations, and the neurons containing the probe target duplex were localized and visualized with Fast Red substrate. After the first development is stopped, a second hybridization is preformed using the DIG Fulicin

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58 probe and the neurons are localized and visualized with alkaline phosphatase conjugated anti DIG antibody fragments (Boehriger Mannheim). Imaging Images were captured with a Nikon Digital Sight DS 5M digital camera mounted on an upright Olympus SZX12 microscope. Figures were prepared using Adobe Photoshop.

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59 Figure 31. Fulicin Gene and Protein P redictions A ) Full length open reading frame and UTR for Aplysia Fulicin like protein. Nucleic acids and amino acids are numbered at left and predicted amidated pep tides are shown in red Monobasic, dibasic, and tribasic cleavage sites are shown in green The signal sequence is shown in orange TxFrag2, shared by both MIP and Fulicin is shown in blue B ) Predicted amino acid sequence of Fulicin. Cleaved peptides are predicted by looking for conserved cleavage sites (shown in green in 3 1, A). B. F T E F L Q W E F V P Y D F V Y D F V Y D F L P Y D F L S Y D F L P Y E F I P Y E F V Y A E F LA. 1 GCTGGTAACGAGCCCTGAATACTGGACAATACAACAGAGTTGGAGAAACAGATGTGTGTA Splice 1 61 GCATGATGAAGTAGACACCGGTCAAGAGAGTAACACCACCGGAAACTGACCATAGAATTC 121 CAAAAACTCTGTGTTCAAAATAACTAGT GGCGGACTTTCACGAAATCTGATCTCCACACG 181 TCGAAGAGCGAATACCTGCAGTGACGAGCGTCGGTTTCCCTTCAACTCATTCTTGTCATT Splice 2 Exon 1 241 TTCATCTTGAAGTGGTTCAGGTTACCACAGAAAGAAAGACAAGAATTGTACTGATTTTTT 301 CTGAGTTCTAATTCCAATCAACCCGCAATGTGT ACCCGACCTGGCCTGGCTGCTCTGCTG 1 1 M C T R P G L A A L L Exon 2 361 GTGCTGATGACGTCTTGTGCCTCCAGCTTCTCACGTGCAGAT ACCACGAAGACTGGACAC 3 1 V L M T S C A S S F S R A D T T K T G H 421 C TGAAAAGGCCAACCCGATCAAGTGACCAGTTTTTCGACAGGAGCGACACGGAACCTCTC 5 1 L K R P T R S S D Q F F D R S D T E P L 481 CAGCCCCCAAACAGTGACCAGTACCTCTCTTCATTGAAAAACAAAGGCCAGTTCAGCAAC 7 1 Q P P N S D Q Y L S S L K N K G Q F S N 541 GAGCAATATCCTGGAGAGTATTTTGACGGGTCCGGGTTCAACGCCGAGGAGGAAGAAAGG 9 1 E Q Y P G E Y F D G S G F N A E E E E R 601 CCTCTACATCAAGGGGATGGGGACAGTGTGTCACTTTTGGGAGACAGCCCTCCGAGCCCG 11 1 P L H Q G D G D S V S L L G D S P P S P 661 GCCATGTCGAAAAGATTTACAGAGTTCCTAGGTAAAAGGGTGCCTTTGGCTAACAGTGGG 13 1 A M S K R F T E F L G K R V P L A N S G 721 ATGCCTGGCCCTGTGGAAGAGCCACACAAGGAGACCGTAGAGGATAGTCATA GGTCAGAG 15 1 M P G P V E E P H K E T V E D S H R S E 781 GGGTTATCACCATCAGAAAGATCGTATAGAGACATGGCAGAAAAGCTGGCTTTACTACTC 17 1 G L S P S E R S Y R D M A E K L A L L L 841 CGCTCTGACGCCAAAAGACA GTGGGAGTTTGTCGGAAAGCGACCATACGACTTCGTAGGC 19 1 R S D A K R Q W E F V G K R P Y D F V G 901 AAAAGATACGACTTTGTAGGCAAGCGATACGACTTTGTTGGCAAAAGGTACGACTTTTTG 21 1 K R Y D F V G K R Y D F V G K R Y D F L 961 GGAAAGAGAAATCCGTACGAGTTTATTGGGAAGCGGTACGACTTTGTGGGAAAACGTCGC 23 1 G K R N P Y E F I G K R Y D F V G K R R 1021 CCCTACGACTTTCTAGGGAAAAAGAGTTACGACTTCCTCGGGAAAAGGTACGACTTTTTG 25 1 P Y D F L G K K S Y D F L G K R Y D F L 1081 GGAAAAAGGAATCCGTATGAATTCGTTGGTAAAAGAACTCCGGCTATGGTTCATGAAGGC 27 1 G K R N P Y E F V G K R T P A M V H E G 1141 GTGATCTCACACAACCTAGACGGCGAGGGTCAAAAACTGGCCATTCCATCCTCTGATGCC 29 1 V I S H N L D G E G Q K L A I P S S D A 1201 CACACCGCAGACAGAAGGTACGCCGAGTTTCTGGGCAAAAGGTCCGAAGAAAGTGGACAA 31 1 H T A D R R Y A E F L G K R S E E S G Q 1261 GCAGCTCTGACAGACAGCGCCCGTCTGGCCGCTCTA CTCAGCAACACTGGCCTGAGGAAA 33 1 A A L T D S A R L A A L L S N T G L R K 1321 CGGCTGTCCAGAATGCTTCTGAACGGTCAGCTGGCGGAACAGTACCCAGAGTTTATTGGG 35 1 R L S R M L L N G Q L A E Q Y P E F I G 1381 AAAT AGGACACGGTATTTTCTAAGAATCCGGGCACGACGCAAAACTGTTATAGAAATCAC 37 1 K 1441 TTCCACTGAGTTAATCCAAAAGTTACGAA GGTAAATGTAGTTTCTATCCTGCGAACCGCG 1501 TAGTATGCATGTTAACGACGACAACAACT GCAAATGCATAGGTTTATGTATTAATCATAT 1561 GCATTACTCAAAAGATCAAACTGAAAATT CTAGTATCATATATTATTATCTACTAACAAA 1621 TCATGCCTTACTTCTTCTTGAACTTTGTA GTTTTCATTTTTTTTAATAGACCTGTTTAAT 1681 TTGAGAAACAATCGTATATAGAATGCCAG ACTTCTCAATAAAAAGTGGGTTATAATCATA 1741 AAAAAA

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60 Figure 32. Schematic overview of the distribution of Fulicin like transcript in the CNS of Aplysia Each circle represents a sing le cell showing staining for the Fulicin like transcript. Positions of identified cells MCC, R2, and LP1 are indicated, (viewed from caudal surface). Most significant staining was seen in the Abdominal and Pleural ganglia. Cerebrobuccal connective MCC B1 B2 B5 B6 B4 SC SC C1 C2 C3 C4 C5 E Cluster E Cluster B Clus ter A Cluster Pleuroabdominal connectives Pec Pleuroabdominal connectives R3 R13 R2 A6 A5 A4 A2 BC BC LP1 Cerebropedal connective Cerebropleural connective P5 P6 P7 P9 P8 P5 P4 P6 P9 P8 Cerebrobuccal connective MCC B1 B2 B5 B6 B4 SC SC C1 C2 C3 C4 C5 E Cluster E Cluster B Cluster A Cluste r Pleuroabdominal connectives Pec Pleuroabdominal connectives R3 R13 R2 A6 A5 A4 A2 BC BC LP1 Cerebropedal connective Cerebropleural connective P5 P6 P7 P9 P8 P5 P4 P6 P9 P8 Pleural ganglia Abdominal ganglia

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61 Figure 33. Colocalized expression of MIP and Fulicin transcripts. MIP specific staining shown in red, Fulicin specific staining shown in blue. A ) and B ) the gradual co localized development of Fulicin and MIP in the A bdominal ganglia. C ) Cells in the Pleural ganglia, including LP1 show co localized expression. A B C

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62 Table 3 1. Comparison of Real Time PCR of Aplysia MIP related gene and 454 sequencing. Comparison of 454 sequence frequency to RT PCR reveal significantly higher levels of expression of the MIP gene, suggesting that another gene may be expressed that has sequence similarity to MIP. Transcript Copy Number (Average) 5Reads 5Frequency (1,010,896 total) 3Reads 3Frequency (468,723 total) MIP (coding AF454399.1) 2.12E+05 9295 9.19E -03 170 3.63E -04

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63 Signal Sequence Figure 34. Alignment of the coding region of Aplysia MIP like protein against the identified Aplysia Fulicin lik e protein B oth share complete identity at their 5 ends, including the signal sequence. It is also noted that the transcripts share repetitive regions at their 3 ends.

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64 A B Figure 35. Intergenic splicing and expression of TxFrag 2. A ) TxFrag2 is composed of 3 intergenic splice sites and 1 intron/exon boundary that is conserved in both MIP and Fulicin. B ) Transcriptional profile showing t he number of sequences in our 454 library that align to the unique region of TxFrag2, MIP and Fulicin in the sense and antisense direction. TxFrag2 is present mostly in the antisense direction, suggesting it may function in a regulatory mechanism. 454 Counts (1,500,000 total) Sense Antisense TxFrag2 6 50 MIP 9,810 325 Fulicin 1 0

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65 CHAPTER 4 GENE EXPRESSION PROF ILING FOR INDIVIDUAL NEURONS Introduction T he primary motivations for the identification of putative neurosecretory products as described here was to identify neuron specific signaling molecules that may be responsible for neuronal identity, communication and plasticity. While other labs have created comprehensive lists of putative secreted signaling molecules in other models, the complexity of the brain systems in these models has presented a challenge to identifying neuron specific neurosecretory products. Our lab has combined high throughput sequencing with the large an d easily identifiable neurons of Aplysia californica, to determine what signaling molecules are expressed in individual identified neurons. Advantages of Aplysia In order to perform gene expression profiling of a single neuronal typ e, a homogenous sample of RNA mu st be isolated from an individual neuron. In many model systems this would be difficult if not impossible due to the complexity of the brain tissue, the inability to identify and isolate single neurons and the small size of the cells However, the large and identifiable neurons of Aplysia offer a means of accomplishing this goal. The gill and siphon withdraw of Aplysia represents a simple and quantifiable behavior with a well defined network of neurons. The cellular circuitry that drives th is behavior can be simplified to two neurons of the abdominal ganglia, a sensory ce ll (SN) and a motor neuron (L7) Due to the location of these cells, a semi intact preparation can be used to confirm the identity of these cells using electrophysiology ( F igure 4 1). By using this type of arrangement our lab has identified individual motor, sensory and interneuron cells that can be used to provide insight into the expression of transcripts in a single neuron type

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66 In addition to providing the ability to identify and isolate individual neurons, the giant polyploidy neurons of Aplysia contain vast amounts of genetic material with a chromosome copy number up to 100,000n (Gillette, 1991) and RNA content estimated at up to 0.2 g in some cases This makes Aplysia an attractive model for gene expression profiling as large amounts of RNA can be made readily available for sequencing. Limitations of Sequencing Technology Over the last several decades, our understanding and application of high and low throughput methods to study gene expression has continued to grow. Each method has different sensitivities as well as advantages and disadvantages due to basic technological constraints and the absolute number of each mRNA molecule to be measured. One major obstacle to using highthroughput sequencing to measure gene e xpression is the amount of data generated by this method. For research scientists, the complexity of the transcriptome becomes overwhelming, if not impossible, to understand without the use of bioinformatics approaches. As the sequencing technology contin ues to grow, and EST coverage begins to reach genomic scales, many researchers are faced with a daunting task of sifting through generated data to find transcripts with function relevant to their research aims. To address this problem, our lab has devised a method for quantifying transcript expression and abundance, in a user friendly output, to create a digital expression profile (DEP) for each transcript. We have applied this method to individual neurons in the memory forming network of Aplysia to dete rmine what transcripts are relevant for the function and maintenance of specific cell types, including motor neurons (MN) L7 and R2, sensory neurons (SN) and MCC interneurons. Here, we applied the method to a selected list of transcripts previously pred icted by our lab to encode secreted signaling molecules to quickly screen hundreds of transcripts from individual

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67 cells involved in learning and memory in Aplysia. This method will be particularly useful for studying model systems that are lacking in geno mic information Results and Discussion F rom the DEP, preliminary analyse s of selected transcripts encoding secreted signaling molecules could be easily screened to determine differential expr ession in specific neuron types (i.e. motor (L7), sensory (SN clu ster) and interneurons (MCC)). It also allows us to identify abundant transcripts for each neuron type as indicated below. Sensory Neurons The digital expression profile using neurosecretory products predicted by traditional cloning for sensory neurons s upports previous work indicating robust expression of Sensorin A in sensory neurons (Cai et al., 2008) ( Figure 4 2). As expected from previous publications, Sensorin A appears to be the most abundant transcript encoding a secreted signaling molecule in the sens ory cell. In addition to Sensorin A, DEP indicates a low expression of other neurosecretory transcripts determined by traditional cloning including Prothoraracicostatic peptide like protein (PTSP) and Capsulin ( Figure 4 2) Further profiling using SSMs identified here ( Figure 4 3) suggests that sensory cells of the gill and siphon withdraw response may also express low levels of the predicted SSMs LFRFamide p recursor and SFY3 like peptide. DEP also suggests expression of predicted 7B2 secretory granule neuroendocrine protein which is unlikely to a secreted signaling molecule but may be involved in the processing of neuropeptides. Screening of the SSMs predicted from the Lottia genome shows low expression of heatshock, and an unknown protein product ( Fi gure 4 4). Motor Neurons O ur digital expression profile of neurosecretory products predicted by traditional cloning ( Figure 4 2) and homology search ( Figure 4 3) support previous experiments suggesting both

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68 MIP ( Figure 4 3) and FMRFamide ( Figure 4 2) are e xpressed in the L7 motor neuron. Furthermore, these DEPs and that for the neurosecretory products predicted using a genomic approach suggest the expression of several other putative neurosecretory products including Capsulin, R151 and R152, Delta like p recursor, Ependymin related protein, 7B2 secretory granduale neuroendocrine protein, LFRFamide, Putative Phermon e 2, Theromacin like, heatshock like protein, and Fulicin. However, further investigation with in situ hybridization does not support the expre ssion of most of the products predicted by DEP including Capsulin, R15 1 and R152, and Delta like precursor. While some of these predicted SSMs may not be involved in cell signaling, several, including Fulicin, LFRFamide, and Ependymin related protein 2 may prove to be key signaling molecules involved in cell to cell communication including retrograde signaling. Of particular interest to this thesis was the prediction of a Fulicin like neurosecretory product in Aplysia and DEP supporting the expression of Fulicin in L7 motor neuron ( Figure 4 4) as suggested in Chapter 3 of this thesis. Interneurons DEP reveals the expression of Capsulin, FMRFamide, Myomodulin, R151 and R152, 7B2 secretory granduale neuroendocrine protein, Ins ulin like proteins, LFRFa mide, p utative Phermone 2, Buccalin, and heatshock protein in MCC interneurons. In situ hybridization has not confirmed the expression of any MCC specific neurosecretory product, suggesting that all proteins predicted to be expressed in MCC by DEP are due to inputs from synaptic terminals of other neurons.

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69 Discussion Digital Expression Profling Although some transcripts are co expressed in different neuron types, the level of expression for each type is unique. Furthermore, the overall expression profil e of transcripts across neuron types is unique, creating a sort of laundry list of transcripts unique to cell function. This presents a potential method for identifying neuron function based exclusively on molecular data and excluding the need for physiol ogical studies. Furthermore, this information can be applied to systems like the gill and siphon withdraw network in Aplysia californica to enhance our understanding of key molecular components of a learning network. We also suspect that transcripts wit h a low frequency of expression in DEP may indicate contamination to the library by way of synaptic input from other neurons in the circuit. Indeed, by combining DEP with in situ hybridization, we have found some transcripts with low DEP expression do no t show any positive labeling using in situ hybridization methods ( Table 41). While this may at first seem a drawback to the sequencing technology, this contamination could actually be used as insight to the identification of synaptic input to specific n eurons being studied. Implications of Neuron specific Expression While the cellular mechanics underlying learning and memory of the gill and siphon withdraw network has been well defined and studied, limitations in the molecular understanding of this net work has hindered progress in understanding cellular identity and plasticity. Some signaling molecules of this system have been identified (i.e. Sensorin) while others, for example the molecules are involved in the retrograde signaling from L7 neurons to sensory neurons, remain elusive. This work presents a major contribution to the understanding of the molecular mechanisms underlying cell identity and plasticity in a learning behavior. From here it is hoped

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70 that researchers will be able to study the eff ects of identified putative neurosecretory products to determine what if any role these molecules have on cell signaling and learning and memory. It is likely th at several of these identified putative neur o secretory products may prove to be crucial to the overall understanding of this neuronal circuit. Methods Animals and Dissection Aplysia californica weighing up to 150g were obtained from the National Resource for Aplysia at the University of Miami, and Aplysia weighing 150g 400g were collected in the w ild by Marinus Scientific, Long Beach, California. Animals were anesthetized by injection of isotonic (340 mM) MgCl2Single neuron collection was performed previously by Th omas Ha. Briefly, ganglia were first incubated in 1% Protease IX (Sigma) at 34 for 45 min to soften the connective tissue of the neuronal sheath. Then ganglia were pinned to a sylgard dish in artificial seawater (ASW: 460 mM NaCl, 10nM KCl, 55mM MgCl (approximately 50% of the body weight) before removal of muscle or nervous tissue. 2, 11 mM CaCl2, Construction of 454 Libraries 10mM HEPES, pH 7.6), the cells were exposed by mechanical removal of overlying sheath with fine forceps, and the dish was flooded with 70% ethanol. After two minutes the cells were mechanically removed with fine forceps placed in 250 l 70% E thanol and stored at 20C until RNA isolation. The MCC neurons were identified visually. The identity of the L7 neurons was first confirmed by electrophysiology (see Figure 41) before fixation and collection. Libraries were constructed as previously described in this thesis, Chapter 2 methods.

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71 Digital Expression Profiling Digital Expression Profiles (DEP) are made by taking the entire set of data from a sequencing project before assembly. In this dataset, each sequence read represents the expression of a single transcript in the cell or tissue the library was made from, such that there is a 1 to 1 ratio of sequence read to transcript. From this data set, any sequence of interest can be aligned using BLAST (1e 04) and by co unting the number of reads generated from the 454 sequencing that have significant BLAST, the number of times that transcript was sequenced from the library can be determined. To determine the frequency of expression, these counts are normalized to the to tal number of sequences in the sequencing project. By normalizing the counts, the frequency of expression across different sequencing projects can be compared.

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72 Abdominal Ganglia Siphon Gill Figure 41. Semi intact preparation of Aplysia abdominal for identifying neurons of the gill and siphon withdraw reflex. The nerves connecting the abdominal ganglia to the gill and siphon remain intact. By removing the sheath surrounding the neurons, cells can be impaled and recorded from to determine cell identity. For example, L7 can be identified by impaling cells in the vicinity of L7 until a cell is found that when stimulated, results in the contraction of the gill and siphon.

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73 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 0 0.001 0.002 0.003 0.004 0.005 0.006 Frequency of Expression Neurosecretory Protein L7 Frequency SN Frequency MCC Frequency capsulin FMRFamide myomodulin PTSP like peptide R152 R151 SensorinA Fig ure 4 2. Digital expression of n eurosecretory products predicted by traditional c loning.

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74 Figure 43. Digital expression of n eurosecretory products predicted by homology search against unbiased shot gun sequencing. Predicted Neurosecretory Products Frequenc y of Expression 7B2 secretory granule neuroendorine protein Delta like precursor Ependymin related protein 2 Insulin like LFRFamide precursor MIP Putative Pheromone -2 SFY3 like peptide Theromacin

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75 Figure 44. Digital e xpression of neurosecretory products predicted by genomic approach. Reticulocalbin1 precursor Kinesin-like Insulin-like Unknown Unknown Unknown hypothetical protein Buccalin Unknown FlbAprotein BEL-2 Unknown Unknown Fulicin ependymin-related VonWillebrand factor precursor Unknown similar to orphan G protein Cell wall protein DAN4 precursor P protein Unknown Unknown Out at first protein hypothetical protein Heatshock Unknown unknown IG-H3 precursor 0 0.00005 0.0001 0.00015 0.0002 0.00025 0.0003 0.00035 0.0004 0.00045 0.0005 Frequency of Expression Predicted Neurosecretory Product L7 Frequency SN Frequency MCC Frequency

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76 Secretory P roduct DEP In situ SN MN IN SN MN IN Sensorin + + + PTSP like + + Capsulin + + + LFRFamide + + + SFY3 + 7B2 s.g. + + + Heatshock + + + MIP + + FMRFamide + + + R15 + + Delta like + Ependymin + + Putative Pheremone 2 + + Theromacin + Fulicin + Myomodulin + + Insulin like + + Buccalin + + + Table 4 1. Validation of DEP using in situ hybridization Comparison of secretory products predicted to expressed in Sensory (SN), Motor (MN) or Inter (IN) neurons by DEP compared to expression confirmed by in situ hybridization.

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77 O bject 21. Intron/Exon Boundaries of selected neurosecretory genes Preliminary analysis of neuropeptides across gastropod species Aplysia and Lottia suggests that some neuropeptides evolve at different rates. These results (see Chapter 2) suggest that some neuropeptides involved in development (i.e. Dorsal ventral patterning peptide) have a more closely conserved coding sequence than those neuropeptides involved in neuron regulation (i.e. MIP). To see if these differences are due to underlying discrepancies in the neuropeptide genomic organization, the intron/exon boundaries of 12 selected neuropeptides were exami ned. Selected neuropeptides show a relatively simple genomic organization, with an average of 35 exons. Those neuropeptides shown to be more highly divergent do not appear to have differences in their genomic organization compared to closely conserved neuropeptides. Atrial gland specific antigen precursor (AGSA) (Mollusk derived growth factor) (MDGF) Gene Bank Accession Number: 22261794 Intron /Exon Boundaries : Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intro n 5 Sequence 1 gggcaggt ag CCATGTCGTC CATCTTGGCG 209 gt acgtttaa 2 tcttgttt ag GAAAAAAGAA ATGCCTAAAG 130 gt aagggatt 3 ctccccccc ag GCGGTGCTCT CCGGACCCAT 147 gt gagtctat 4 tcattccc ag TTTGTGTTTG TCGATGACTG 67 gt aaggaggc 5 tgctgtac ag GTTGAAGAAA TTTT GCTCAG 97 gt aagtgaaa 6 ttatcatc ag GTCATCGGAC GCTGTTTGGG 114 gt gagtcaaa 7 acccctgc ag TTCTTCGAGC CTGGCCTAAG 128 gt aggctaaa 8 ttccctcc ag GTTCAAGAGC GCTGAAACCA 206 gt acgtatgc 9 ctgttaac ag ACTGGCAGGA CTCTAATCAG 158 gt ttgtagag 10 tgtcctgc ag ATCCTG GGCC CTATCCCATC 69 gt tatctcct Intron 1 2 3 4 5 6 7 8 9

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78 Feeding circuit activating peptide precursor Gene Bank Accession Number: 22947345 Intron/Exon Boundaries: Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Seq uence Placement in Sequence 1 GACCAGACAG CCTCAACTTC CCACTGCAAG 87 GTAAGAAAAC 18 69 2 TCTCGTTCAG ACACCGGAAC AAATGTAAGG 2162 GTCAGTTACA 70 2231 FMRFamide neuropeptide precursor Gene Bank Accession Number: 84551 Intron/Exon Boundaries: Coding Re gion: 1491942 nt Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Placement in Sequence 1 tcaaatct ag AGTTCTTCAA CCGACTGATC 137 gt aagttgct 20 117 2 caatctgc ag GCGCCCGTGA CTGTGCGAGA 140 gt gagta ccc 118 257 3 ggtatctg ag ATTTGGCCGG AATACAACCA 1695 gt gatatttg 258 1952 L11 neuropeptide precursor Gene Bank Accession Number: 155779 Intron/Exon Boundaries : Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Lengt h Intron 5 Sequence Placement in Sequence 1 ggaccccc ag GTCATCATGC CTGCACGTGA 117 gt tgtcgttg 6 110 2 cccacagg ag GTTTGTTTTCG CCTACAACAG 141 gt gggctata 111 251 3 ttttccac ag AGTTTTCTTA ACGAAACTAT 87 gt caatagac 385 471

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79 Buccalin precursor Gene Bank Accession Number: 404497 Intron/Exon Boundaries: Coding region: nt 2711786 Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Exon on Seq 1 tttcggca ag AAATCCTGAC ACACACGCAG 181 gt agggatct g 1 167 2 tttgtttc ag TTTACTTAAC GCCTTTATCT 86 gt aagacttg 168 253 3 cctccccc ag GTTCCCCCCC AGAGGAACAG 1516 gt gagctggc 254 1770 4 ctccctcc ag TCGTCGAAGA AGTGACCGCT 29 gt gacgtagt 1771 1800 Conopressin Gene Bank Accession Number: FJ172359 Intro n/Exon Boundaries: Coding region: nt 106684 Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Exon on Seq 1 gtttttaa ag TTTGCATAAG TTAGAGGCAA 58 gt ttcaagtg 19 72 2 tcctgcac ag AACGAAATCA ACAGCGACAG 195 gt a agaacgt 73 267 3 ttatttcc ag TGCATGGCGT TGTGATTCAG 208 gt aggtcgtc 268 475 4 ttcccccc ag AATCATGTGC GCCCAGTGAC 209/1516 gt gaggagcc 476 684 Fulicin like neuropeptide precursor+Gene Bank Accession Number: 56792350 Intron/Exon Boundaries: 3531533 Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Nucleotide location 1 GGGGATCATC TGACCATAGA gt atgttgtt 1 247 2 gttgtttc ag ATTCCAAAAA AGTGGTTCAG 144 gt aggttgct 248 391 3 tctcgttt ag G TTACCACAG AATTGTACTG 33 gt gagtggaa 392 424 4 cttattcc ag ATTTTTTCTG TCACGTGCAG 107 gt gagtagga 425 531 5 ctccatcc ag ATACCACGAA ATAGGACACG 1001 gt attttcta 532 1533

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80 MIP related peptide precursor Gene Bank Accession Number: 8886135 Intron/Exo n Boundaries: coding region: 6682873 nt Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Nucleotide location 1 CCCTTGGGGT TGACCATAGA gt atgttgtt 1 456 2 gttgtttc ag ATTCCAAAAA AGTGGTTCAG 144 gt aggttgct 457 600 3 tctcgttt ag GTTACCACAG AATTGTACTG 33 gt gagtggaa 601 633 4 cttattcc ag ATTTTTTCTG TCACGTGCAG 107 gt gagtagga 634 740 5 ttccatgc ag ATACGCAAAG AGGTAGCTTT 2139 gt ccatccag 741 2879 Neurotoxin like 1 Gene Bank Accession Nu mber: 71148939 + Intron/Exon Boundaries: Coding Region: 10423 nt Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Nucleotide location 1 GCTGAGCA AG TCACAATCAC CAGCGTGAAC 70 GT AAGTCTTA 10 60 2 TC CTCCCC AG GCAAAGGTGG GCAACGAAAG 130 GT GAGGCGTG 61 190 3 GATATTGC AG GGACGTGCCA TTCGTTCCAG 129 GT CAGTACAC 191 319 4 CTTACGTC AG GATTCTTTTC CATAGATGAC 129 GT CAACACCT 320 448 Pleurin Gene Bank Accession Number: 56200040 + Intron/Exon Boundaries: C oding Region: 189755 Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Intron 5 Sequence Placement in Sequence 1 TTGTCACCAG AAGTGAGTGA CTCACCAACA 155 GTAAGCCATC 2 156 2 TATCAAACAG TAAACCGCTC CTTGAGCTCA 482 GTGGCCG AAT 176 657 3 TGTTTCACAG GTGATCAAGC CTTTGATGAC 94 GTCATCTGAG 682 776

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81 Whitnin precursor (SPTR) Gene Bank Accession Number: 56200044 + Intron/Exon Boundaries: Exon Number Intron 3 Sequence Exon 5 Sequence Exon 3 Sequence Exon Length Int ron 5 Sequence Placement in S S equence 1 TGTGTTTCAG ATCCTTCAGC TGTGCTGCAG 115 GTCAGACACA 13 102 2 TGTCTTCCAG GAGGCCTCGG CATTCAAGAG 99 GTGGGTGGCA 103 201 3 ATCATCTCAG CTTGTGGACG GGAGTGGTAC 146 GTGCCTTGGG 202 347

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82 Object 22. Identified neurosecretory products likely to be signaling molecules Secreted Signal Molecules found prior by traditional cloning Abdominal ganglion neuropeptides L5 67 precursor Gene Bank Accession Number: 113521 Reference: Shyamala, M., Fisher, J.M. et al. (1986). A n europeptide precur sor expressed in Aplysia neuron L 5. DNA. 5 (3), 2038. Aplysianin A precursor Gene Bank Accession Number: 26419361 Reference: Cummins, S. F., Nichols, A. E. et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert with attracti n to stimulate mate attraction. J Biol Chem 279(24), 2561422. Atrial gland specific antigen precursor (AGSA) (Mollusk derived growth factor) (MDGF) Gene Bank Accession Number: 22261794 Reference: Sossin, W. S., Kreiner, T. et al. (1989). A dense core vesicle protein is restricted to the cortex of granules in the exocrine atri al gland of Aplysia california J Biol Chem 264 (28), 1693340. Attractin precursor Gene Bank Accession Number: 38257306 Reference: Fan, X., Wu, B. et al. (1997). Molecular cloning of a cDNA encoding a potential water borne pheromonal attractant rele ased during Aplysia egg laying. Brain Res Mol Brain Res 48(1), 16770. Calreticulin Gene Bank Accession Number: 262054 Refer ence: Kennedy, T. E., Kuhl, D. et al. (1992). Long term sensitization training in Aplysia leads to an increase in calreticulin, a major presy naptic calcium binding protein. Neuron 9 (6), 101324. Capsulin Gene Bank Accession Number: 31088940 Reference: Cummins, S. F., Nichols, A. E et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert with attracti n to stimulate mate attraction. J Biol Chem 279(24), 2561422. Cerebrin prohormone precursor Gene Bank Accession Number: 74843746 Reference:

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83 Li, L., Floyd, P. D. et al. (2001). Cerebrin prohormone processing, distribution and action in Aplysia californica J Neurochem 77 (6), 156980. Clionin precursor Gene Bank Acce ssion Number: FJ214338 + PubMed ID Reference: n/a not released CDS: ATGACGTCATCCATGATCCTCGCTGTCCTGGCCATCTCCCTGTCGTGCCTGCTGACCGCTGTGACGTCA TCACCACTTGGCCCAGTCCAGCCGGCCATATCCCTGTCCAAGATGGAACCCGATCACGCCTGTTTGTTC ATGTGCAACATCTGTTTCCCGGATCTGGAGGACACGGGTCTGCTCCTGGACT GCAGCAACAAAGTGTG TGGTCCCATCATGGCGGGCATGTGTGCTATGGAGAAGCTCGTCTGGCTGGGCCACAACTGCCGTCAGT ACGACATGGTGCAGAAAATGTGGGCTCCGCACGGCGCTCTCTAA Protein: MTSSMILAVLAISLSCLLTAVTSSPLGPVQPAISLSKMEPDHACLFMCNICFPDLEDTGLLLDCSNKVCGPIM AGMCAMEKLVWLGHNCRQYDMVQKMWAPHGAL ELH precurs or [Contains: Beta bag cell peptide (Beta BCP) Gene Bank Accession Number: 119268 Reference: Scheller, R. H., Jackson, J. F. et al. (1983). A single gene encodes multiple neuropeptides me diating a stereotyped behavior. Cell. 32(1), 722. Enticin precursor Gene Bank Accession Number: 74814556 Reference: Cummins, S. F., Nichols, A. E. et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert with attracti n to stimulate mate attraction. J Biol Chem 279(24), 2561422. Feeding circuit activating peptide precursor Gene Bank Accession Number: 22947345 Reference: Sweedler, J. V., Li, L. et al. (2002). Identification and characte rization of the feeding c ircuit activating peptides, a novel neuropeptide f amily of A plysia J Neurosci 22(17) 7797808. FMRFamide neuropeptide precursor Gene Bank Accession Number: 84551 Reference: Taussig, R. and Scheller R. H. (1986). The Aplysia FMRFa mide gene encodes sequences related to mammalian brain peptides. DNA 5(6): 45361. FUR Gene Bank Accession Number: 453657 Reference: Chun, J. Y., Korner, J. et al. (1994). The function and differential sorting of a family of A plysia prohormone processing enzymes. Neuron 12(4), 83144. Glycoprotein hormone beta subunit (GPB5) +

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84 Gene Bank Accession Number: AY928334 Reference: Heyland, A., Moroz L. (2005) U npublished Hypothetical protein Gene Bank Accession Number: 26892018 Refe rence: Cummins, S. F., Nicho ls, A. E. et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert w ith attractin to stimulate mate attraction. J Biol Chem 279(24), 2561422. Insulin precursor Gene Bank Accession Number: 8886137 Reference: Floyd, P. D., Li, L. et al. (1999). Insulin prohormone processing, distribution, and relation to metabolism in Aplysia californica J Neurosci 19 (18), 773241. L11 neuropeptide prec ursor Gene Bank Accession Number: 155779 Reference: T aussig, R., Kaldany, R.R. et al. (1984). A cDNA clone encod ing neuropeptides isolated from Aplysia neuron L11. Proc Natl Acad Sci U S A 81 (15), 498892. Myomodulin [Aplysia californica] Gene Bank Acce ssion Number: 400327 Reference: Lopez, V., Wickham, L. et al. (1993). Molecular cloning of myomodulin cDNA, a neuropeptide precursor gene expressed in neuron L10 of Aplysia californica. DNA Cell Biol 12(1): 53 61. Myomodulin gene 2 neuropeptide precurso r Gene Bank Accession Number: 77378085 Reference: Proekt, A., Vilim, F.S. et al. (2005). Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of Aplysia. J Neurosci. 25(42), 963748. Neuropeptide precursor 1 Gene Bank Accession Number: 20372612 Reference: Matz, M.V., Meleshkevitch, E. et al. (2001). Unpublished Neuroprepeptide Gene Bank Accession Number: 155771 Reference: Nambu, J. R., Taussig, R. et al. (1983). Gene is olation w ith cDNA probes from identified Aplysia neurons: neuropeptide modulator s of cardiovascular physiology. Cell 35 (1), 4756. PRQFVamide precursor protein

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85 Gene Bank Accession Number: 29469911 Reference: Furukawa, Y., Nakamaru, K. et al. (20 03). PRQF Vamide, a nov el pentapeptide identified from the CNS and gut of Aplysia J Neurophysiol 89(6), 311427. PTSP like peptide neurotransmitter precursor+Gene Bank Accession Number: 87045866 Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal t ranscriptome of A plysia : neuronal compartments and circuitry. Cell 127(7), 145367. Putative pheromone Gene Bank Accession Number: 115371642 Reference: Cummins, S. F., Degnan, B. M. et al. (2008). Characterization of Aplysia Alb 1, a candidat e water bor ne protein pheromone released during egg laying. Peptides 29 (2), 15261. R151 neuroactive peptide precursor Gene Bank Accession Number: 155797 Reference: Buck, L. B., Bigelow, J. M. et al. (1987). Alternative splicing in individual Aplysia n eurons ge nera tes neuropeptide diversity. Cell 51(1), 12733. R152 Neuroactive polyprotein precursor Gene Bank Accession Number: 113523 Reference: Buck, L. B., Bigelow, J. M. et al. (1987). Alternative splicing in individual Aplysia neurons generates neuropeptide dive rsity. Cell 51(1), 12733. R3 14 neuropeptide precursor Gene Bank Accession Number: 155782 Reference: Scheller, R. H., Kaldany, R. R. et al. (1984). Neuropeptides: mediators of behavior in Aplysia Science 225(4668), 13008. Sensorin A precursor Gene Ba nk Accession Number: 134428 Reference: Brunet, J. F., Shapiro, E. et al. (1991). Identification of a peptide specific for Aplysia sensory neurons by PC R based differential screening. Science 252(5007), 8569. Temptin precursor Gene Bank Accession Number: 74811743 Reference: Cummins, S. F., Nichols, A. E. et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert with attracti n to stimulate mate

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86 attraction. J Biol Chem 279(24), 2561422. To lloid2 Gene Bank Accession Number: AY485265 + Reference: Brown, B.D., Kohn, A.B. et al. (2003). Unpublished. CDS: GCGCGCCATGGAGAACACAGTGCGAAAAGTTGCTCCTGTTCGAACTGCTAACAGAATCATCAATCTGT TTTGCTTGTGTACTTGTGCCTTGGTGCTGCAGTGTGTTTTGTTCCCCGCCACTGCCGAAAACATTCCAAG ACGAACGCCCGACGACCTGTACATGAATCCATGCAAAGCTGCTGGATACTTGGATGGTATCGCTCTAA CTGAAAACGAATACGAATCGTTTGAGAGGAGGTGGCTGGCTCTCAAGAACAACATTTCCGATAGTCCT CAACAGTCAATTGTCAACCATACAGACGCAGATAGGACGCAGTCACCCAGGGCTGTTACAGGTGACG CCATGGATATGGAGAAGAGAAAACTTCGTCTACAGAGTTTGGTGGAGGAGAAAGACGAACTCACGCA GTTAATCAAAGAAGCGCGTGCAGAGCTGAAAAAAACTTGCCATGACAGGTCAGACAGCGAATGTGAC CAGGAGAAAGAAACGCTTCACAAGCTACTGTCGGAGGCGAGACGCAAAAGAAAACAGATAAAGCTC AGACTGCATCGACTTCGGAAAGAGATTGTTCAGGGAGATGAGAACGAAAGTGACGGACGTCCTCACT CCTCCTCTTCTCGGGGGAGACACAGACGTGCGACACCTGCTGATAGG TCAAAGTTGTGGGACCATGGC GTTATCCCTTATGTCATTGAATCAAATTACTCGGGTGAGAACAAGGACCTGTTCAAGCTGGCCATGAG ACACTGGGAAAACCTCACGTGCCTCGTGTTCAAGGATAAAGGTCCGGAGGATACGAATTATATTCTTT TTACCCAGACGGATTGTGGATGTTGCTCGTTCGTTGGGAAGCACGGGAGTGGAGCCCAGGTGATTTCT TTGGGCAAAGGCTGCTACTATTTCGGCACCGTCGTGCACGAGCTGGGTCACGTGGTCGGCTTCTGGCA TGAGCACAACAGGCCGGACAGGGATAAATACGTACAGATAATACGGAAGAACATTATGCCAGGCAAA GAAAGCGAGTTCAACATCTTGGACGAAGATAAAGTGGATTCCCTTGGTGAACCGTACGACTATGGCAG CATCATGCACTACTCTCGTGACAAGTTCTCCAAGCACAGCTACCTGGACACCATCCGGCCGTTCCGTCA GAGAGGCATGATCGC CCTGCCCCGCATCGGCCAAAACATACGACTCAGTGATGGGGACGTACGACAG ACCAACAAACTCTACAAGTGCCCTACTTGTGGCCGCACCGTGATGGAATCAAAGGGCACGATTAGCCC CGACTCAGGGATACGAGGCGAAAGGACGTGCCAATGGAGGATCATAGCCTCTCACGGGGAGCGGATC CAGCTGAGCCTGACCCGACTTGACCTCAGCAACTGTGACACAGACTACGTGGAGGTCAGGGATGGAC AT TTTGTCGGCTCTCTCAGTTTGGGCAAATTCTGTGGGAAGAAGATCCCCCCGCCCATGATCTCCTCCG GCACTCGCCTCTGGGTGGAGTACAAGTCAAGGGCGGCGCGCCCGGGAGCTTTCCAGGCCGCTTTTGAG GCTATATGCGGTGGTAACATGCCTGGACCCGAAGGCTTCCTCAACAGTCCCGCTTATCCGGATGAGTA TGGCAGTGATAAAGTCTGCGAGTGGGTCATCACTGTGAGAGAGGGATATCAAG TGGCTCTAGAGTTCG CCACTTTCGAGACGGAATTCGACCCTGACTGTGCCTACGACTACGTAGAGATCCGAGATGGTGACACA AAAGACTCGCCTCTTGTGGGTACATATTACGGGGGTTACTTGGACGCACCCAGTGGGACCATCTCCTC CCCGTCCTTCCCTGACCTCTACCCCCCAGACAAGAACTGTGTCTGGCACATCAGCGCGCCCAAGGGAC ACACCCTCACCGTGAACTTTACTCATATGGATCTGGA GTGGAGGGGGGATGAATGTGAATTAGACTTC GTGCGTGTGACAAACGTCGTTGGTAATAAGGAAAGACTACAAGGCCAGTACTGTGGCTTCATGGCCCC GCCCTCAATTACGTCACTTAGCAACGAGCTGAGAATAGAATTCCGGTCGGATGACACTTTGCAAAAGA CAGGATTCTCTATGGACTATGTGGCAGACGTGGACGAGTGTGCCAGCAGCAATGGCGGCTGCAAGCAT ATCTGTGAGAACACTGTGGGA AGCTTCCGCTGTTCGTGTCGTGAGGGTTTCATCCTGGCCGATGACGA GAAGAGCTGCAAGGAGGGCGGCTGCCATTACGAGGTGACAGATACCAAGGGCGTCATCCAGAGCCCA GATTACCCAAGCTTCTACCCGGCCAGGCGAGACTGTGAGTGGCATTTCACCACAGCACCAGGACACGC TGTCCGTCTCATATTCACGGATTTCCAGGTTGAGCCTCACAGGACTTGTAGGTACGATCACGTAGAGGC GTTTG ACGGTGCAAATATTCAGGCACCACAGATTGGCAAATACTGCGGCTCAGAAAAGCCTGCACCAA TCATATCTTCCGAAAACACGTTGACCTTGACCTTCCTCTCTGACACGTCTGTGCAGCGGAAAGGATTTA GAGCCCGTCATGACACTGTGTGTCAGTCGTCACCCACAGCTACGAGTGCCCCCAAGAAGATTCTGTCC CACGTCCTGTACGGCAGCAAACCCTACGACAACAGACAGAACTGCTCCTGGAACAT CCAGGCGCCCG AGGGCCAGCACGTGGAGCTCAGGTTCACAGCCTTTGAGATTGAACAACAGTCCAGATGCCTGTACGAC TACGTGGCTGTCTACGATGGGCCGACGGAGAATGACCTGGTCCTCGGCAAGTTCTGTGGGAATCAGGT CCCAAGCCCTATCGTCTCCAGTACACGCAGTCTTCTCGTTCGCTTCCGCTCTGACGATACGATCAAATC TGGAGGCTTCAGCGCCACCTATCGCATTGCAGATGACACGGACCACACAGTGGATCTGCTGAGCCAGT GAAAACAAAAAGGAGCAACGCCTACGGGGTTCAACAATTGATGAAATGTTACTGATTATATTGAAAG CAAATATGCAGTTTTCATGTTGAACTCTGGTAAATGAAAAGTCTTCCCCCCAGCAAGACTGTACTGAA CTGTTATCTATCGCCTATTATTATCACCATCCTAACAAGATCATGTATAATAATCAAATCAGAGTCTCT TAATATTAGGCCTACGTTACCCAT ATTGCACACGACCGACTTAGAGACCAGTAAATCCTGCAATTGTA

PAGE 87

87 CAAACACATGTATGTGGGTTCTGCAACAGTAGCCCCCCTTTTCCACGCACAGAAAGGGGAGGCGTCTG CGAAGTCAACAGGAACGGATGTGAAGCAT Protein: MENTVRKVAPVRTANRIINLFCLCTCALVLQCVLFPATAENIPRRTPDDLYMNPCKAAGYLDGIALTENEY ESFERRWLALKNNISDSPQQSIVNHTDADRTQSP RAVTGDAMDMEKRKLRLQSLVEEKDELTQLIKEARAE LKKTCHDRSDSECDQEKETLHKLLSEARRKRKQIKLRLHRLRKEIVQGDENESDGRPHSSSSRGRHRRATP ADRSKLWDHGVIPYVIESNYSGENKDLFKLAMRHWENLTCLVFKDKGPEDTNYILFTQTDCGCCSFVGKH GSGAQVISLGKGCYYFGTVVHELGHVVGFWHEHNRPDRDKYVQIIRKNIMPGKESEFNILDEDKVDSLGEP YDYGSIM HYSRDKFSKHSYLDTIRPFRQRGMIALPRIGQNIRLSDGDVRQTNKLYKCPTCGRTVMESKGTIS PDSGIRGERTCQWRIIASHGERIQLSLTRLDLSNCDTDYVEVRDGHFVGSLSLGKFCGKKIPPPMISSGTRLW VEYKSRAARPGAFQAAFEAICGGNMPGPEGFLNSPAYPDEYGSDKVCEWVITVREGYQVALEFATFETEFD PDCAYDYVEIRDGDTKDSPLVGTYYGGYLDAPSGTISSPSFPDLYPP DKNCVWHISAPKGHTLTVNFTHMD LEWRGDECELDFVRVTNVVGNKERLQGQYCGFMAPPSITSLSNELRIEFRSDDTLQKTGFSMDYVADVDE CASSNGGCKHICENTVGSFRCSCREGFILADDEKSCKEGGCHYEVTDTKGVIQSPDYPSFYPARRDCEWHF TTAPGHAVRLIFTDFQVEPHRTCRYDHVEAFDGANIQAPQIGKYCGSEKPAPIISSENTLTLTFLSDTSVQRK GFRARHDTVCQSSPTATS APKKILSHVLYGSKPYDNRQNCSWNIQAPEGQHVELRFTAFEIEQQSRCLYDY VAVYDGPTENDLVLGKFCGNQVPSPIVSSTRSLLVRFRSDDTIKSGGFSATYRIADDTDHTVDLLSQ& Small cardioactive peptides precursor Gene Bank Accession Number: 134314 Reference : Sossin, W.S., Kreiner, T. et al. (1989). A dens e core vesicle protein is restrictued to the cortex of Granules in the exocrine atrial gland of Aplysia californica. J Biol Chem. 264(28), 1693340. Transcripts predicted by homology search to encode secreted proteins Achatin like neuropeptide precursor+Gene Bank Accession Number: 56792348 Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of A plysia : neuronal compartments and circuitry. Cell. 127(7) 145367. CDS: ATGACGTCATCTGCTCAATGCCTTCTGCTCACAATGCTCCTAGTGGCCACCCTTGACTTCCT GTACGCA GAAGACATCGGCTTCGCGGAAACAGACTCGGAACTGACGCCCTTTGCCGGGGCAGTCGCCGACCACGT CCTGAAAGAGGGCTTCGGGAAGAGGGGCTACTTCGACAAGCGGGGCTTTTTCGACAAACGAGGTGAC GCAAGCAAACGCGGCTTCTTCGACAAGCGAGGGTTTTTCGACAAGAAGAGTTACGCCGATAGCAAAG ATGAAGAGTCGAACGCTGCTCTCTCAGACTTCGCCGAAGACAAAAGGGGTTTCTTCGGCAAGCGATAC TACGGCAGTAAGCGGGAAGAAATCAGTCCCAGGGTGTTGAATCTTCTTCTGAGAAATTATCAGATCCA CCCTAGTCCTCTCAATGGACACAACGCTTTCAGCAGGCTTCTCGCGAAGCAGGGATTTTGGGAGAACT GA Protein: MTSSAQCLLLTMLLVATLDFLYAEDIGFAETDSELTPFAGAVADHVLKEGFGKRGYFDKRGFFDKRGDAS KRGFFDKRGFFDKKSY ADSKDEESNAALSDFAEDKRGFFGKRYYGSKREEISPRVLNLLLRNYQIHPSPL NGHNAFSRLLAKQGFWEN Adipokinetic prohormone type 1 precursor Gene Bank Accession Number: n/a Reference: n/a Partial clone: CGCAGTCGGTACTTTTTTTTTTTTTGTCTGGCGTTCTTTGGCGAAAGTGTAGAGCTCTCAGGCGTATAAA GACCAAGC GATCAGCATCATCAAATCAACTCTGCTGCAGCTGAGCCAATGCTTTCACACATGTCGTCA CAGTTCTGGGCAGGTATTCAGGCAGGAAGAGAGTTTCTTTGCGGTTCGCTGTATTAAAAGCATGATAT

PAGE 88

88 CGATGATTTCTTTGTCCGCGATCTGCCAGCAGTGTGGGATTTCCTTCTCGGTGACAGTGGAGACCGCCC TCTTCCCTGTACCCCAGTCGGGCGAGAAGTGAATCTGTGCAAGGCAAGTGGACGTGCCT ATCACAAGG ACGACAAGGATCAAAAGTATGCTAGAAGATTCCATTGTGCTTGCTCTTGTTTTCGTGTGACACCAGGA GAGCTCCTTGAGCTAGCAGAGACCAGACTGTCTTGGAAACTCAGTTAGGTCTGCGGCTCCTAAGACCC CC Allatotropin 0F precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not clone d Prediction: GGCTCGAGGCAGAGCGAAGCGAGGTTTCCGCCTCAACTCTGCGTCACGGGTTGCCCATGGCTACGGCA AGCGTGGGTACGCTTCCTCTTCCGGTGCCGTCCCTTACCCAGAGTTGGCGAGAGATGTCCTAGACAACT TACGAGCAGAAGAGGAGGAAAAAGAATTGGAGTGGTCAATCATGAGCGTGGATGAGCTGGCGTCTCT ACTGCAGTCACACCCAAAGCTTGCCCGCGCCTTAGTGA AAAAGTTCGTGGATATCAACGGTGACAACT TAGTTACGGCAGAGGAACTTTTCCGGCCCCCCACGAGAGGGAACTATATTGACATCGCTCTATCTTTTA TTTTTTTATTTTTGTTTTTGTTTCTCTGTCCAAAGAACAGCTGATGGGACCTCTGGAATTTTTTCTGAGTT GATGATACTTTCGAGGTATGGATCTTTTTTCGTCTATGAATGTCTGAAAATAACATTGTCCATATTACT GTTTGGCCCCTTTGTTC TCACGACCAAGGCTTGAGATTGTTCCTCCCTGGAAAAGAAGAAATTGGACAT TCTGACTCTTCTGGAAATACGTTTTGATTACACTTTCAAATTAACAATGTGCTAATAAACTAGAACATG TCTGAGGTTCCGTTTGCAAAAGAA Allatotropin 0R precursor* Gene Bank Accession Number: n/a Reference: n/a Partial clone: GAATTCGCCCTTCAAGTGGT GATTCGGCCGCTTTTCTTTTGTACTCGCTTCTACAACATCGGGGTGCAG TTCCCAGGACAAATTCGAGCACCGAGGTCACCGACAAGACTAAAACTCGAGAACACCAGATCTACGC CTGTGACAACCCGATCGACATACACTCCAAGACAAACCAGCACACGCCCAAGACCCGCCAAAAGCCT AGTCCTCACAATGCTCAGCGCACCCTCCATAGCCCACACAGGGGTCGCCCTCCTTGTCCTGATGTGTCT TTGT CCCTTCTCACAGTCTACAGAGGCGTCATTAAGCAGAGCGAAGCGAGGTTTCCGCCTCAACTCTGC GTCACGGGTTGCCCATGGCTACGGCAAGCGTGGGTACGCTTCCTCTTCCGGTGCCGTCCCTTACCCAGA GTTGGCGAGAGATGTCCTAGACAACTTACGAGCAGAAGAGGAGGAAAAAGAATTGGAGTGGTCAATC ATGAGCGTGGATGAGCTGGCGTCTCTACTGCAGTCACACCCAAAGCTTGCCCGCGCCTTAGTGAAAAA GTTCGTGGATATCAACGGTGACAACTTAGTTACGGCAGAGGAACTTTTCCGGCCCCCCACGAGGAAGT AGCTCTTGCCGCCTCATGGAAGGGGGAGTTGCTACGCCTGTCCCACATGAGTTGGCTCAATTATCTGCC CTTTGCAGGTCTGGGGTCCACGAGAGGGAACTAAGAGGGAACTATATTGACAAGGGCGAATTC Antigen 5like protein precursor Gene Ban k Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Prediction: TTTTTTTAAGGGCTACGGTAGATGTTTATTTTCCTCAAAACTGAAACACCACCAGATAGCAGCCCGTTA CATAACCGACTTGCATCTAGGGCTAGTTTATCTTCCATTGCGCTGGGTGTCCAATCTGCCAACAGTCAC CAGTTGTCGGCCGGAACACCCGCGGAACTGT AAAAAGTTCAGTCGTCGCCGATCCACACAACCACTTT CCAGCTGATTAGCTGAAGTTTGAATAACAAGAAACCGAAGCTTCTAGAATGGCTTCTCACCCATCCAA TTGCCAGGTGGGTAGTAAGCACAGACAACCAGCGTGCCCAGGTCCCAGGTGTGGTCACAGGTAGACA CGGCACAGCCCACCTGTCTGGTGTCTCGCCACACTACCTGAGTGTAGTGACCACAGCTTCCATCAGTCA TACACCTGTAGGTCC CGTCGTTGTAAGCTCTCTCACTGACCCACATCTCTGTGGCCTGTGCGGCGACTA ACCGGGCGCTGATGTGGCGATTTGACGTAGCCATGATGTTCTCTCCCCAATACCTCCGGCTGGCCGGTG TGTGGCTCATGCGACATGTGGAGGCCAGGACGGACACTTTGTTAGCCAGGTCATCACTCCACGTCAGA

PAGE 89

89 TCCCTCAGCCCCGCGCCTTCCTGTCTCCTGGCCTGGTTGTGGGCGTCAAGAAAAGCCTGCTTGTC TCCC CAGGTCAGGGACTTGAGATGACCGCCCCAGCAACACGTGACCAGACACAGGACACTGACCAACACAC TGACCGCCGCTCTCCTCATCCCGACAGACGTGGACACAACTTTTCGTGGAGGAGGTCAGCTGATGTTTC GGCCAAGCCAGACTCGAGCCGCCTCGAGCC Appetite regulating hormone precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Prediction: CTCTGTTTTGCTTTTTAAAATGATCCTTGCAAAACTAGCAATGTTACAGGGAAATGGTTTTCTATCTTTA AATATGACATTAAATGAGATGATGACTCCAGGAAAA Astacin like protein Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CTTGAGATCAAAGAAGGACATTGCCTGCCTCTGGCCCAGGTCACGTTGTAACAGAGGGTCACGTGTCT CCAGGGTGATGCGGCCGTTCTTGGAGAAAAACGAGGAGCCGTAGTGCATGATAGATCCTACGTCATAG GGCACTCCCATGTTGTCTATGTCGCCCCATGATTCCCGTTTAAAGTTGAACTCCTCCCCTCTCTTCACGT TCTCCTCCAGGTATGTGACGTAATCATCTCGGT CCGGTCTGGACTGCTCGTGCCAGAAGCCCAGGGCGT GGCCTAGTTCATGGAGGAGGACTCCTTTCTCAAGGCAGCCTTTGGCCGTGGAGACTTCCTGGTACTTGA AGGCTTTCTCTCGACCTACATATGACCAACACCCCACGTCTTTACGGAAGTCCAG Atrial gland specific antigen precursor 2 (AGSA) (Mollusk derived growth factor) (MDGF) Gene B ank Accession Number: 155709 Reference: Sossin, W. S., Kreiner, T. et al. (1989). A dense core vesicle protein is restricted to the cortex of granules in the exocrine atrial gland of Aplysia california J Biol Chem 264 (28), 1693340.(Sossin et al., 1989) Bradykinin like neuropeptide (LUQ 1) Gene Bank Accession Number: 155765 Reference: Wickham, L. and Desgroseillers L. (1991). A bradykin in like neuropeptide precursor gene is expressed in neuron L5 of Aplysia californica DNA Cell Biol 10 (4), 24958. Buccalin precursor Gene Bank Accession Number: 404497 Reference: Miller, M. W., Beushausen, S. et al. (1993). The buccalin related neuro peptides: isolation and characterization of an Aplysia cDNA clone encoding a fa mily of peptide cotransmitters. J Neurosci 13(8) 334657. Central nervous system APGWamide Gene Bank Accession Number: 4099286 Reference: Fan, X., Croll, R.P. et al. (1997) Unpublished.

PAGE 90

90 Chordin Gene Bank Accession Number: n/a + Reference: n/a Partial Clone: AGACGGGAGAAACAGATTTTGTTGTGTGATAACGTCAGATTATCTCCTCGAGGGAAACGCGCGCTGAG AAATAGTAAATAGTGAGCCGAGGAATTTATCTATCGTTTTGTGCTTCTTCGCGCAGTCTTGTAATTGAA GAGTGGGCCTCCTTCCAACTTTTCTTCCCTTC TTCTGTTTGACATCACTGACCGCAAGTCGAGTGTTCTC TGGTGTTGTGTGTTTCTTATAATTGTGGAACTACTGCAGAGGATTTGTTGTATGGACACTTCGGATTTTT CAGACGTTTGGAAATAATGCGCGAGTGTTTTGACTGATTTCTGTACAGTACCTGGAAAATAAACTACG CACGTTCTGTGATTCTTACGGTACACTTGAAATTACTGACAGACAGTTTTCCATTTCCTGACGCGACCC CGTTATAGGAT GGGGCAACCGCCTACTCAAGCGGGCTGCTCTAGCAACTCGTGTGTCTATGAATGCGT CCGTGAGTCTGTTCGTGAGTCTGTACATCGTGGGTCAAGTCACATAAAGGTGGTCAACAGCTGAGGAG GTCTAGAGAGGACGAAACCAGAGACAGCTGCCAGCCGCCCATAACATGCAGAAGATGACTCAGATGC TGGTGTATAAAGTGCTCATCTTCGTCATGGTATATCGCTGCAGTGGAAAGCCTTCGGGAGGCCC GTGT GTTTTAGGCGGAGAGCAGTACAACGTTGGAGAGACGTGGCATCCTAAAGAGTTTTCTTCCACCGCCAA CTCCTGTGTCCACTGCACGTGCCTAGAGGGCGGTCAGATAAATTGTACCGGCGTGGACTGTCCTTCTCC AGAGTGCGAGGTGCCCAGATTTATCCACGGACAGTGTTGTCCTACATGTGATGTCCGGGGGGACGAGG CCGGAAGTCCCGAGACAGACATGCCCGGATGTGATTTCCATGGCGAC CACTACGAGGACGGTGACAT ATTTCCGTCCAACAAGACGGCTTACAAACCCAAGCATGACAACCAGTGTGTGTTGTGCGGGTGTTATA GAGGTGAGGTCATCTGTCACCTGAAGACTTGTCTGCCCAGCCCCAGGTGTCGGCGGGTAGTCCGGGTG GATGACGACTGCTGCCTACAGTGTGAAGAGGAGTCCAGCATAGACGAATATTTCATGTCTATCGGGTC AGACAACATAAACCATACACTGGACGACGAAG ACTGTCTGTCAGCGACAGGACGCCGTAAGAACGGC AGTACGTGGAAGCCAGTGGTGGGCGAGTACGGAGAGATGCATTGTATCGTCTGCTCCTGTTTAGAAGG TCAGGTCGACTGCAAGCGTCTGACCTGCCCTGACGTCACAACCTTGACCTGCAGACACCCAAGGCCAC GACAGGACGGGTGCTGTAAGGAATGCCCTGAGAGCGACAGACAGAGGGACAACAGGAAGAAGAAGA GAAAGAAGAGTAAAAAAGG Conopressin Gene Bank Accession Number: FJ172359 Reference: n/a CDS: GGCTCGAGGTTTTTAAAGTTTGCATAAGTTCTCCACTGGACTCAAAACAAAGAACTAAACGAGAGATT AGAGAACGAAATCAAAACGCTATTTACCAACTACAGGATGTCTCACTCAAGCATGTCACCACTTTCTG TTCGGACTTTCGTCTTGGTAGCAGGACTTGCCGTGATTTCGTTCTCTG TCGTTGCTGATGCCTGCTTTAT TCGGAACTGTCCTAAAGGAGGCAAGAGATCAATGGACATGCAGCTTCTTGGACAGCGACAGTGCATG GCGTGTGGTCCTGAAGGGATCGGCCAGTGTGTAGGCCCCAACATCTGCTGCAGTCCACATTTTGGTTG CCATATTGGCACACCAGAAACGGAAATATGCCAGAAAGAAAATCAGAGTACTTCCCCTTGCAGTGTCC GAGGTGAGACGTGCGGATACCGAGACTCAGG AAACTGCGTGGCTAACGGAATTTGCTGTGATTCAGA ATCATGTGCCGCAAATGACAGATGCCGACTTCGAAAGGAAACGTCTAGAATCGGCTTTGATGACACAC AGAGCTCACGCGCCGAAGTGCTCAAGTTGATACAAAAATTACTCCGCGCTAAGGAGGAAGACTGACA CTGTATCCACAAAACACGTCACAGTGTCCACTCAACGTCATTTCCGGCAAAGACAACTCTAAGTGCCC ACTGACGTGAGGAGCCA AAACGGGATTACTTTTTCTTGTGATTGTTTTCGCTTTTTATTTGTTTTTTCTG GAGTTCACTGTCAACTTGCTTTGGGACAACTACCTTTCTGTGAATAATTCGGAGAAAGACGGGAGTAT CTATTAGGACTAGGAGACAAAGAAGTACTGTATGAGTCATGTGTGCAAGGGTATTAAAATTTAAAAAA GATAAAACGTTTTTGTGGCTTGCTGCTACTCGCAATGTCAATTATTATCTTTCATCGCTCGGAGTTA AC AAAGGCTGTTGAGGGCCACCCTGTTTCTGGATGTTCTGATAAAGAAATACTCTTGACTTCAAAGACCC AAAGCAGTGGAAGCAGAATAAACAACCTTAGTTGCGAGTCTCTCGTCTAACCAAATTACGTAGGATGC CTCGGGAAAACAAGGTGTGATGAATGAGAATGGAATCCTGAGCAAAATGGTTAGAACGGTTATTGAC AGCCACAATGGCACATTGGACATAGAAGAAAGATTTATAGACAGTACTCAT GACTCAGTACGCAATAT CACTACTTTTTAACTTAGCTCATTTAACATGAAAGAAAGAGTAGAAGTGAGTTGATATAGGTGGAGGG GAGGTACACTCTTTTGAGAAAATAGGAGGCGTTAGTCACAACACGTACTTATTTTTTTAAAAGAAATA GGCACTCCTTCTTTTGTTCAGTGGGAGGGGCTAAATTGTGTTTGAGGCCCGTATGTCATCTTCTGTGGC GAGCAGTTTTTGTCTTATGATGAAAACTGTCAGG CGATAAATACTACTTGAATTTGTATCCGATGATCC

PAGE 91

91 ATGATTGTATAAAAAAACAAACAAACGAAGGGCATCTCCAAGAGTTCAGCTGTCTCATATTATGTCCT GTTGTGTTCAATACTGCCACAGATCACGAATAATGTATTTCATGTGCACTTCAGTATCCATCTTTAATT AGAAAGAAAAACTAAAACAAACAAATGGAAAGAAGATTGAGCGGTTCAGGTGTACCATATCGGCAAT GAAGTCGCTAAGAAATG TTTATGAAAGGGAGTTTAAAAACACAGAAGGATTAATCTAGAGGGTATAA AGGGGTGGGGATGGGGGTTGATATGGAAATAAAAACAAGAAAAGAAGACCAGCAAAGGATATTATG AAAAAAATGGTTAATTAACGATGTTTCGGATATCCATGTCTGAACAAGGGAAGTAAGTCTGGTAAATT GTGGCATAATCTAAAGCTTGCTTTAAAATTGAATTGCTTCCCTTTAATTAAAAATGTGTTGGTTTTTTTC CC AAAAAAA Protein: MSHSSMSPLSVRTFVLVAGLAVISFSVVADACFIRNCPKGGKRSMDMQLLGQRQCMACGPEGIGQCVGPNI CCSPHFGCHIGTPETEICQKENQSTSPCSVRGETCGYRDSGNCVANGICCDSESCAANDRCRLRKETSRIGFD DTQSSRAEVLKLIQKLLRAKEED Corticotropin lipotropin precursor Gene Bank Accession Number: n/a Ref erence: n/a Comment: Predicted only, not cloned Predicted: CTAAAAAGCAGATGTACCCTTTGAAAGCATTTTTTAAAAGCATTTTTGAACAGTGTAAGCAGTATTCAT TTAGG Delta like precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CTTG TGCTGGCTTCGAGTGTTTGAACGGAGGTTCCTGCACTGCACCTGCCGACTTCCCATCCTGCCTAT GTCCACAAGGGTATTACGGAGACAAATGTCAATTCAGACAAGTAACCAGGGACCCTTGTGCTAACTTT GAGTGTTTGAACGGAGGTTCCTGCACTGCACCTGCCGACCTCCCATCCTGCAGTTGTCCAGGAGGATA TTTCGGGGACAAATGTCAATTCGGACCAGTAATCAGGGACCCTTGTACTGGCTTC GAGTGTTTGAACG GAGGTTCCTGCACTGCACCTGCCGACTTCCCATACTGTATGTGTCCAGAAGGATATTCCGGAGACAAA TGTCAATTCGGACCAGTAATCAGGGACCCTTGTGCTAACTTCGAGTGTTTGAACGGAGGTTCCTGCACT GCACCTGCCGATCTCCCATTCTGCAGTTGTCCAAAAGGGTATTTCGGAGCCAAATGTCAATTCTCATCA AATCCCTGCGCTAACTTCAACTGTTTGAATGGAGGTT ACTGCTTTTCATTTTTCAACCGCCCATTCTGTT TGTGTCGCTTTGGATATGTGGGGAAAAAATGTCAAACAAGAATCTATTAACGATTCCTAAGACAGTGT GCTGTGCCTTTTGCGAGTGATATCAATGTGGTTGATCCCAACAAACAGAATATATGTAATATCAATAA GTGAATTCCCACTCCGCCTTTTCCAGATTTCTTATTATGTATTTCAAAAATCCTCAGCTTCCTTCTATTA TCCGTAAAGAGAAACCT ATACATGTACTGATTTCGTCAAGAAGAATGGGCTGAAAATAGCACGATGTC AAGTTGGGGTTTTTCCAAACCCATACGTGTTTAACGATAACATGATGCAAATCCAGTTGACAGGAAAC GTATTTCTATTACAATATTGTGATTTTAGAGACAATAAACTTTATTAGTGCCATCAACAAAAAAAAAGT AGGGACTGCGCTGCGTGGATACACTGCTTAATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NN NNNNNNNNTATATGATCGTCATCGGCCCCCCCCCCTTAAAAAAAAAAAAAAAAAAAAAAGNNGGG GGGGCCCCGGCCGCGCGCGGACAAAAAAAAAATAAAACCCCCCCCCGGGGGGGGGG Dorsal ventral patterning tolloid protein precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not clon ed Predicted: GGCTCGAGGCGGCTCGAGGCGGCAAAGGTTTCAACGCAGCCTACGAAGCTATCTGCGGCGGAGAGAT CACCAGAGAGGAAGGGTTCCTCACCTCCCCCAACTACCCGGATGACTACCGGCCCGACAAGGTCTGTG TGTGGAAGATCACCGTGGGTCCTGACTTCACTGTGGCCCTCAAGTTCCAGTCTTTCGAGATTGAGAACC

PAGE 92

92 ACGACGACTGTGTCTACGACTATCTTGAAGTCCGTGAT GGCCCCTCTGAAATGTCTCCCCTCATCGGCA ACTACTGCGGCTACAAGATCCCAGAGGACATCAAGTCCACCGGAAGGCACCTCTACGTCAAGTTTGTC AGTGACGGCTCCGTGCAGAAGGCGGGATTTTCTGCCACTTTTGTCAAAGAGTACAACGAGTGTGAGAA GGAGGAGCATGGCTGTGATCACGTGTGTGTCAACACCCTGGGCAGTTACCGCTGCTCCTGTAGGATCG GCTACGAGCTGCACTCTGACGGCAAGAGGTGTGAAGATGCATGCGGCGGTTACATTGACCAGGAGAA TGGCACAATTACCTCCCCTTCCTACCCGGACCTGTACCCGCCCAACAAGAACTGCGTGTGGCAGATTGT TGCCCCTGACGACCACAAGATCAACATCAACTTCACTCACTTCGACCTGGAGGGCCACAATCAAGACT GCGAGTATGATTCAGTGCGTGTGAGCAGTGGAAAGGGGAAGGAACTCAAACTACACGGCGTGTTCTG TGGCTA CACGTTTCCAGCTCCAGTGACGTCAGA ELH Atrial gland peptide A precursor (ELH 18) Gene Bank Accession Number: 119272 Reference: Mahon, A. C., Nambu, J. R. et al. (1985). Structure and expre ssion of the egg laying hormone gene family in Aplysia J Neurosci 5(7) 187280. ELH egg laying hormone related precursor [Pro 25] B Gene Bank Accession Number: 2599363 Reference: Kurosky, A., Gorham, E. L. et al. (1997). Expression and genet ic variation of the Aplysia egg laying hormone g ene family in the atrial gland. I nvert Neurosci 2 (4) 26171. Endothelinlike precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GACAATGTGTGCGATTGTGTGTCTCCTTGTGTTTTAATGCTTGTGCTCTGTGTATGTGTAA Endozepine related 1F protein precursor ( b inds GABAA receptors in the central nervous system, and it increases the mitochondrial synthesis of pregnenolone. ) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GCCGCATGGGGTGAATATCGTACAGGCCGGATAAGTTTGAGGCAGCGGTGAAAGTAATTCGTGGTTTG CCCAAGAATGGTTCTTTCCAGCCTTCCCATGAGCTTATGCTCAAATTTTACAGCTATTTCAAGCAAGCC ACAGAAGGACTCTGCACATCTCCAAAGCCAGGTTTCTGGGATTTGGTCAATCGCAAGAAATGGGAGGC GTGGACCGATTTGGGTAAAATGGAAAGCGAGGAGGCCATGCTGCTGTATGTGGATGAGTTGAAAAAA AAAAAAAGTACCGACTGCG Enterin Ge ne Bank Accession Number: 74844518 Reference: Furukawa, Y., Nakamaru, K. et al. (2001). The enterins: a novel f amily of neuropeptides isolated from the enteric nervous system and CNS of Aplysia J Neurosci 21(20), 824761. Enterin neuropeptides precursor 2 Gene Bank Accession Number: n/a

PAGE 93

93 Reference: n/a Comment: Predicted only, not cloned Partial: GGCTCGAGGGTTACTCTCATAGTTTTGTAGGTAAACGAGTACCCGGCTATTCGCACAGCTTTGTTGGTA AACGAGTTCCTGGTTACTCTCATAGTTTTGTAGGTAAACGGGTACCCGGCTATTCGCACAGCTTTGTTG GTAAACGAGTTCCTGGTTACTCT CATAGTTTTGTAGGTAAACGGACTCCTGGCTATTCGCACAGCTTTG TAGGTAAACGGGCACCTGGCTATTCACACAGCTTTGTTGGTAAGCGGACACCGGGTTATTCCCACAGT TTTGTTGGTAAACGGGCACCTGGCTATTCACACAGCTTTGTAGGAAAACGAGCACCAGGTTATTCCCA CAGTTTTGTAGGTAAACGGGCACCAGGCTATTCACACAGCTTTGTTGGTAAGCGGGTACCGGGTTATT CCCACA GTTTTGTAGGCAAACGGGCACCAGGCTATTCGCACAGCTTTGTTGTTAAGCGGGTACCGGGT TATTCCCACAGTTTTGTCGGTAAACGGGCACCAGGCTATTCCCACAGCTTTGTAGGTAAACGAGAACT AGGTGAGGATGAGATAAACTTTCTAAAAGAGGTAGACGCAGCAGACATTTCTAGACAACTTGCAGAA GAAGACGAAAAGGAAACAATGGTATCAGTTGATGATAAAGAGACACTCAGCAACGAAGAAGACGCA TCTGAAGACGATTTTGAGAAGCGGGAAAAAAA Ependymin related protein Gene Bank Accession Number: 94434843 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of A plysia : neuro nal compartments and circuitry. Cell 127(7), 145367. C DS: ATGAAGTTTCTGGTAGCCTTTTTCGTCTTGTTCGTGGCATGTGTCTACGCCAATAGGCCATGCTCCGCTC CCCATGAGCTCACTTTCCGCGCCTCCAGATGGAACTATGGCGAAACTTACTACGAGCGGTTCTTCGGA GAGTACGACAGATTCAACAAGAGGGTAGTACTCTTTGAGGAGGACTTCGAAAGCGGCACCAAAAAAC AACGGGAGCTCCTGTTCCTTCACAAAGAAGGTGTAGGCTACGACTT CAACCTGCAGAGCAAACATTGC AAGAAGTTCAAGGTCGGAGCATTCAAGCCCTTCGAGGTGCCTTCTGGCGCCACTTACGAGGGCGAATT CTCCATTGGAGGACCCACCGAGGAAGTCACAATCGACAGATGGTCGGACAAAATCAGTTCTCGTCGTG AACACTGGATTGGGGAGTTCTCACTAAAGACCTGCTACCCTATCAGCCAGTTTATGATCGAAGAGGGT AACTTCAACAAGACATCCTTCACCTTCTTCTACGACACCGTCAAGGGAATCGTCAACCCCAACGACTTC AATGTGCCCAAAGAATGCGAGAAGTCGGCGATTGAAGTCGATATGCCAATCAACGCTCGCCTTGCCAA GGCTATGTACTCTGGAACCCTGCATGACTAA Protein: MKFLVAFFVLFVACVYANRPCSAPHELTFRASRWNYGETYYERFFGEYDRFNKRVVLFEEDFESGTKKQR ELLFLHKEGVGYDFNLQSKHCKKFKVGAFKPFEVPSG ATYEGEFSIGGPTEEVTIDRWSDKISSRREHWI GEFSLKTCYPISQFMIEEGNFNKTSFTFFYDTVKGIVNPNDFNVPKECEKSAIEVDMPINARLAKAMYSG TLHD Ependymin related protein 2* Gene Bank Accession Number: n/a Reference: n/a Full Length Clone: CDS: CCTTAATACTTCTGTCAGCCCTGGTATCAGAGCCACTCACCTCTA GTCCAACGTAGCTGGTAGACGTGA GAAGATACACCATGTTGAAGCTTCTGTTTTTCGCCCTTCTGGGATTGACACTGTCTGTCGCGGACATCC CTCGTCACTGCTTCTCTCCCCCTGAACTCACGTTCAGAGCCTTCCAGTTCGACCACGAGTATACCACCT TCAACCGTTTCAAGGCCGAGTATGACGTCAGAGACCGCAGGGTGGCTTTCCTGGAGGAGGAAGTCAA AGGACCGGCACCCGGAAAACAATACCT GTGGCTCATGTTCCTGCACGAAGAGAAAGCAGGTTTTGAAT TCAACCTGAAGACCAAAAAGTGCAAGAAGTTCAATCCCGGCAAATTTCATCATTTCGGCACCCCAGAA GGTTCCAAGTTCGAGGCTGACTTCTACGTTGGCGGACCCGGGGAGTCCATAGACGCCGAGATGTGGTC TGACAGAACCGACTTCAAAAGGGAAGACTGGCTAGGCGTGTTCACCAAACGTAATTGCTACCCTATCC GCACATTTACG AGGAACACACACAACAACCACACCCTCACCACCAACATCAACGACCTCGTCGAGGG CATTGAGGACCCAGGTCTGTTTGACCCTCCTAAGGAGTGCTTACAGCAGGGTGTGCTAGAAGAAGAGA TGAGCGAGACAGCGCGCAGAATGTACAGCATCTACTCACGTACTTTGTTGTAGATCACAAAACAGGCA

PAGE 94

94 TGCACAATTATTTTGTTTTAAGTTGTTAAAATGCTTATGTCCTTACGTCGTTCTAGTATGTCTT CTTCTC GAAAATGTACATGCACGTATACATTCAACCATTTCTAATTAAAGCATGCATTCACATCTTGTAACACGT ATATGTGTAATTCTAAGTCGATG Protein: MLKLLFFALLGLTLSVADIPRHCFSPPELTFRAFQFDHEYTTFNRFKAEYDVRDRRVAFLEEEVKGPAPGKQ YLWLMFLHEEKAGFEFNLKTKKCKKFNPGKFHHFGTPEGSKFEADFYVGGPGESIDAEMWSDRTDFKRED WLGVF TKRNCYPIRTFTRNTHNNHTLTTNINDLVEGIEDPGLFDPPKECLQQGVLEEEMSETARRMYSIYSR TLL Feeding circuit activating peptide precursor 3* Gene Bank Accession Number: n/a Reference: n/a Partial Clone: TCTTTGGGGAAAAAATCTGATACTGAGGAGAAAAGATATTTAGACCATTTGGGAAGTTCTTTGGTCAA AAAGTC TGATACTGAAGAGAAAAGATATTTAGACCATTTGGGAAGTTCGTTGGTGAAAAAATCTGATA CTGAGGAGAAAAGATATTTAGATCATTTGGGAAGTTCGTTGGTCAAAAAGTCTGACAGTCAAGAAAA AAGATATTTAGACCATTTGGGTAGTTCACTGGTTAAAAAATCTGACAGAGAAGAAAAAAGATATTTAG ACCATCTGGGAAGTTCGTTGGTGAAAAAATCGGAGAGCCCGGAGTTGGCAAGATTTGAG CAGCGGAT GTTTACAGACTACAACAACGACCTTGTTGCTATGGAGAAAGACTTCCTGGAGAAGAACAGAGAACTCC AGCAGTCGAGCCCGGATGTGACGTCACAGGGGAACAAACGTTATCTGGACAGGATCGGCAGCTCGCT GGTGAAGAAGGAAGACGACGAGGATACAGAGGTGGACGAAGACGGAGTGGGGCTATTTATAGATTTG GAAAAAGATGAAGATGAGAACGACTTGAGGCAAATGGCGGGCACGA GAGCAGGTAACCACAAGAGA TATCTGGACCGTGTGGGCAGCTCGCTCATCAAGAAACAGGCGCAGGCTCAGAAACGATACCTAGACG GGATCGCAAGTTCGCTCATTAAAAAGGAAGACAGAGGCCAGAAACGATATTTAGATGGAATCGCAAG TTCACTCATAAAAAAAGAAGACAGAGGCCAAAAACGATACTTGGACGGGATCGCAAGTTCGCTCATT AAAAAGGAAGACAGGGGTCAGAAACGATACTTAGATGGCATCGCAAGTTCACTCATCAAGAAAGAGG ATGGTTTCGTTGGCGACATCCCAAACGGACAAGAAAAACGATATTTGGACGGTATCGCCTCTTCCCTG ATAAAGAAAAATGAACTAAAAAGCGAAAAGCGTTATCTGGATGGTATCGCAAGTTCCTTAATAAAGA AAGACAACGTACAAGAAGAAAAACGATACCTCGATGGCATTGCCAGCTCTTTGATTAAAAAAGAAGT CAGACCCGGACAGAAACGATAT CTAGATGGTATTGCAAGCTCTCTCATAAAAAAGGACTCGACAAAC GAGAACAAGCGATATCTGGACGGTATCGCCAGCAGCTTGATCAAGAGACAGGACTCGGTGAGCGATG AAGAAAAGCGATACTTGGACAGTATCGGCAGCTCTCTCGTGAGGAAATCGTCCTCTACGACTCCGTAC AAACGATACCTGGACCAGGTTGGGAGCTCCCTGGTGAAGAAAGGGGCATCAGAGATGGGACCATACG AGGTGATTC CCGAGGCTGTCTACGTGGGGGAGGGGATCTCTGCGGGCGGGGAGGGGCCGGTGTACGT CACCATTGACGGTCAGCTCCCCATGTTGGCCATGTCCGAGACTGACCTGCCAATGGCCAGCCCCAGGA AGCGGATGTACGAGGTGACAGATGACGTCATGACCGGAAGTGGTCACCAACAGCTGGTGGACGATAT GGCGGCGGCGATCCACGTGTCGGCCAAACGGGCAGGACTGACAGGTAGGGGCAGCGTGGTAGG GCGA TCTTGTCTTATCTTGAATGTCGTTGTTGTCATCATCATCATCATCATCATCATCATCATCATCATCATCA TCATCATCATCATCATCATCATCATCATCATCATCATTATTTAGAACCTTCTGCCTCACCACTAGCTCAC ATGTTGACCAGGTCGAGTGCCGATTGGGTAAACGTTTTGTAACGTTTTATTTACGTGGCGTTAGTGGCC TAGTGGTTAGGCTCATCATCATCATCCAGTGCCAGATCTGCGA CAGAGATCGGCGATTATGGTTTTCCA TTCATTCATTTGCTCGGATGCTCTCAAAAAAGCTACGTTTCCCTGATCTTTCCCAGGTTAGGCTACCGG ACTCGTAATCGTGAGGTTGTCGGTGGCCTAGAGGTTAGGCTACCGGACTCGTAATCGTGAGTTTGTCG GTGGTCTAGTGGTTAGGCTACCGGACTCGTAATCGGGAGTTTGTCGGTGGTCTAGTGGCTACCGGACT CGTACTCATGAGGTTGCGGATTCGG GGGTTCGAGCCGTGGGCCCGACGTTGTGTCCTTGGAAAAGGCA CTTTACACGAATTTTCCTCACTTCACCTAATTGGGAATGGGTACCCAGCTATAGACATCGAAAGGTCTT CTCAGTATGTTAATGTTTTAGCTCCTATAAATGGCTGCATGCACTGTATGCATCCCCGAAAGTTGAGGT TGTTTTAGAATGCTATCGGGTCTGCTGGGGTAATAGTATAAGTTGTGAAACGCAAAGAGATTGCTGTT GAGCGGG AATTTGCGCTATACAAATGCTATCTATTATTATTTATGATAATGGTTACGAGTTTCGATCCT ACCTGTCAGGGCCTACCCGGGCTAAAAGGGATATAAAATCCCGCTAGAAAAACAGAGATACTCGATA AACCTTCTCCCCACTCGGGAATAGAACTCCTTTCTCCTGTGTTCGACAGCGTAACGCCTAACCTCTACA CCAAAGAAGCCAGACAAAATGGTTGTATCACAGACTCTAATGTGTCTCACGTCCATTC ATTTCAGTA

PAGE 95

95 Feeding circuit peptide 2 Gene Bank Accession Number: n/a + Reference: n/a Partial Clone: CTGGACGGTATCGCAAGCTCGTTGATCAAACGGCAAGACGTGCTGGGTTCTGACGTGGCCATTGGGAG CACGGGGAACAGCGGGCCGGACAAACGATACCTTGACCACTTGGGTAGTTCACTGGTCAAGAAATCT GATGGACAAGACAAACGATACCTTGACCACTTGGGTAGTTCGCTGGTCAAGAAATCTGATGGACAAG ACAAACGATACCTTGACCACTTGGGCAGTTCTCTGGTTAAGAAATGGGACGAGGGTGACCAGGAAAA GAGATATTTGGACCACTTAGGCAGCTCACTAGTGAGAAAATCAGATAGTCAGGACAAGAGGTATTTGG ATCATCTGGGGAGTTCTTTGGTCAAAAAGTCAGATGGTCAAGAAAAAAGATATTTAGACCATTTGGGT AGTTCA CTGGTAAAAAAGTCTGACGGTATAGACCAGGAGAAGAGATATTTAGACCATTTGGGAAGTTC TTTGGTGAAAAAATCTGATACTGAGGAGAAAAGATATTTAGACCATTTGGGAAGTTCATTGGTGAAAA AGTCAGATGGTCAAGAAAAAAGATATTTAGACCATTTGGGAAGTTCTTTGGTCAAAAAGTCTGACGGT ATAGACCAAGAGAAGAGATATTTAGACCATTTGGGAAGTTCCTTGGTGAAAAAATCTG ATACTGAGGA GAAAAGATATTTAGACCATTTGGGAAGTTCTTTGGTAAAGAAATCTGATACTGAGGAGAAACGATATT TAGACCATTTGGGAAGTTCTTTGGTGAAAAAATCTGATACTGAGGAGAAAAGATATTTAGACCATTTG GGAAGTTCTTTGGTCAAAAAGTCTGATACTGAAGAGAAAAGATATTTAGACCATTTGGGAAGTTCGTT GGTGAAAAAATCTGATACTGAGGAGAAAAGATATTTAGATCATTTGGGAAGTTCGTTGGTCAAAAAGT CTGACAGTCAAGAAAAAAGATATTTAGACCATTTGGGTAGTTCACTGGTTAAAAAATCTAACGGCATA GACCAAGAAAAAAGATATTTAGATCATCTAGGCAGTTCTTTGGTCAAAAAGTCAGACAGTGAAGAAA AAAGATATTTAGACCATTTGGGCAGTTCGCTGGTTAAAAAATCTGACAGAGAAGAAAAAAGATATTTA GACCATCTGGGAAGTTCGTTGGTGAAA AAATCGGAGAGCCCGGAGTTGGCAAGATTTGAGCAGCGGA TGTTTACAGACTACAACAACGACCTTGTTGCTATGGAGAAAGACTTCCTGGAGAAGAACAGAGAACTC CAGCAGTCGAGCCCGGATGTGACGTCACAGGGGAACAAACGTTATCTGGACAGGATCGGCAGCTCGC TGGTGAAGAAGGAAG FFELamide/FMRFamide 3 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GGCTCGAGAGGACCCTTTTACCCTTTCGGTAGAGGCGACCCATTTATCAGATTTGGTAGAGGCGTCCCC TTCATTCGATTTGGTCGAGGCGATCCATTCATCCGATTTGGCAAAAGAGGAGCAGACAGCGAAGAAGG TGTCGGTGAAGGAGAAGCCTTGTCGAGGACAGCCAGATCGACAGAAACGACACAGGAACGAGCCAA C TCTCGTCAAATACGCAGTGCAGACGCCAACAAACGATTCACGAGGTTCGGCCGCTCCGCCACTTCCGC TTTTGTGACCCAGCAGGAGGCTGAGGTCAAGGACGCAGTGAGGAAAAGGTCAGTCGAGAACTTGGAC AACGAGGTCAAACGAGCGATGTGGAGCAACAACCAACAGATGACGCGATTGTCCAAAAGAGGTCACC CAGAGTTCATGCGATTTGGGCGGAGTCAAGGAGACGAGACAGAGGACGACGAA ATTGGCGGAGATGA CGTCATCAGTTTGGGGGAAGGTCTAGCACAGAGGGTGCGCCGGGTGTACGAAGACAGGAGCTCACTG CCACGATTTGGGAAGAGGCAGGAGGTGGAGGAGGAGGAGGAGGAGGACTTTGGGAAGGAAAAGAGA CAGAAGTACATGCGTTTTGGAAGACGGAGTGATCCAGAGCAGCAGGCGTCAGCGGAAGTGGGCGGGG CGGAAGTCCGAGACAGTGTGCTCTCTGCCATGTAGTCACGTGCTCACTGGGAGGAAAGGATGCACGCA CAACACGCTCTTTGGACGTATTATTGCAGCCGGCAGCACAACATTTTTA Fifth (short peptide fragments obtained from MS data) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Partial: CCACGTACGGGCGTTAGCGCCAACTTCCTGGCCCCAGGCAGACTCGAAACATGGGAGATAATAGAAA AATCCATTTGGAAAATACTGCTAGTTGTCTCCCATCCTCTCTTTCCGTTAAACCCTATTCCGTGAAGGC ATAGTGGGAGAATTGTCAAAATTCTACAGTTTGTCAATATACTGATCCTGAAATCAACCCTCTAGGCCC TTAGTTCGGTTTCTTTTACAGTCTTTCTATCCCTTTTCTATCATAGAGTCTCCCATTTCACTGATGGGCA

PAGE 96

96 GCTAAATTTTGTAA TTCTGAATTGATTTGAGGGTATTTTTTCGAACATAATATCAATATGACCACTTGG ACTCATATAAAAAATTCGTTCTCGATACGCCATGACAAAAAGCTGTAAATGAACTAAATGCAGAGTAG AAAAGAGAAGAGCTAAATTGCGATGCATGTTTCAAGTCGGAGATAAAACAAATCGAGTGCTGGTCAA CAGATCTGCCAGCCGTTATCTTAACGGTGCTACTAGGCGTTTACTGGCAACAATGAGCGTGATAGT GTT TATTGTCAGTAGTAGCGTGACGTGAGATGGGGGAGCCGATCAATGTGTTGCTTTATTAATAAGGAAGA GAAAAAAAAGAAGGGGGTGGGTCTGCGTGGGAAGGAAATTTGTGGGAGCAGGCATTTTTGTTTTCGC GCGAACCCATTGGCCAGTGTATAAATCATCGTCTCATTTAAGACAGAAGTGTCTAGAAGCAAGACCGC ATTGGTCAGATCTTCGAGGGTGTAATTACTACTGGCTTCCTCTCTTTTCT TGTAAAATGCGTCAAATTA GAAAGCCTCTGAATAAATTCCTTTCGTCCGGTTTGTGATTGCCGTTATTTGCCATTATTACCCGGGGCTT GCCGAGAAGCACGGGAGTTCGACAACGATAAAACCATCGGGCGAGCTGTTGGAGAGAGGGAAACGA ACCGCTCGGGGAACGGTCGCAGAACTGTGCGTGAGAAGAAAAAGACGAGCTCGTGGTCACTTTGTGG TCATCTTCATTGTCTGCTGACCCCCACGGAGATT TCATTCGACTCCTCAGACTCATAATAACATCTCCT GTGTCTGACAAACAGGTTAAAGGATCAGTGAAGCTTGAACGAGTGTTTTCTTACCATTTCGTCTCTAGT GACCAAGAGCTCGTAACTTGGCTCAAAGAGTAGACCGGTCTTCTCAATATATAGTATTTAAGTGACTA CAGTACAAATTTCACTTTATCTAGACAGCCTTTCCCCCAGAGACCACTGTGATAAAACTATTAGGTCAA TTTACTTCAGATCAT CTCATTGACGTTAGTCTGGCATTGAAATACTCGCTGTGCTGGATCTATTGCTCGT GTGGTTGCATTTTGGTGCTAGTATTACTCTGCGTCTACGCCACTGAAACAACAGCATGACTTGCGCCAG TTATTTGAGGACTATCCCCGCTCTCGTCGTTGCAGTCCTATGTCTGGGAAGTGCGCTGAGCTCACCGTA CAGAGACGAGGACCTGAGACCATTAATTGCGAGCTCGGCCCTTGACTCGGACACAGGTGTCTT CCTGG ACATGCGCACACGGCGTGGGAGTGGCCAACAGCTGTATTACTCACCGCATTCTTTCACCAGCAAGAGG ATGAATCAGATATCGTGCAGCTGCTGTTCCAATTCCTTCAATAGCAACTGTTGCTTTCAGTGCATGCAG AGGCTTGGAAAGAGAAGCGGGGCAAGATCAGACAACAAAAATGAGGCTCCTGAACTCCTGAGGAACG GACCAGAGCTATATTATAACGAAGGCGGCCGCAGCAGTGACCATGAC CTCCTTCCAATGCCCCTTCAA CCCGAGGTCAGCGATTGGCGAGCCAAAGGCAGAGCTTGCTCGTGCTGTCTTTATCAGATGCACCAAGA GCCGTTCGCTCAGTCTTGGCCTTGCTGTAGATTGTGCAAGAGATCCAAGCGATCTGTTGGAGCCGTGCC CGCACCAGCTGAGACCAGCAAGAAAGAAACGTGAAGAAAGTCTTGTAGTTGTAGCATTTCAGACTGA TTATAATGACTAAATACAACACGAGTAACCT CAATCAGCGCTGATTGGAATAATCAGAATACGAAGAC ATGTTCTGTATGCCTCCTCCATCATATTATGCTCGAAAATTCATCTATATTTTAGGTGGAAAGTAGAAT TGAAAGGACAGTTTA FIRFamide related neuropeptides precursor* Gene Bank Accession Number: n/a Reference: n/a Partial Clone: GAGTAATCTCCCATTTTTCTTACTGTG TGTGTGGTAGATGAGGTCTGTCTCTATCTTCAAAAAACGGTA GATCATTGTCGTGGGGGACGACTTTAAACAAATAGTCATGACGAGTCTTTCTCAGAGAGATGGTTTTA ACAGATTCACGAGAAAGTCTGAGCCAGTGGGACAGCGAGAGCGAGCGAGAGAGCGAGAGAGACAGA GAGAGAGAGAGAGAGAGAGAGAGATGGGAGAGAGAGAGAGAGAGAGATGGGAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAGACAGAGAGAGAGAGAGACAGAGAGAGAGAGAGAGACAGAGAGA GAGACAGAGAGAGAGAGAGAGAGAGAGAGAAAGACAGACAGACAGACAGTTTAGTTTTAGTAAGGT CTTAAGGTGTTCACAGTTCTGTTTAAGGTCATATGAGCACCAAGGGGTGGATGTCAACAGAGAAGTCC AAACGGGGAATCAGGCTTACTCTGCTCACCTCTCTAGCCTTAACAGCATAAAGTCTGATTTGACTATCA AAG CACCCCGGAAATAGAAATTCACCAGAGTAACAAACATACAAAACAAGAAACGGAAAAACGACG ATAGTAAGGATGAGGAAAAGAAAAGCAGCTGCAGTTCCCCCTATGTTGTTCCTGCACAGTATGAGAAT GGCATTGATAAATCAGCCCATCTATAATTCTACTCATTCTACTTTGCATAAGAACATGTTAATACCCCC CCCCCCCCCCCCCCCCTTGGTAATGGCCTAAACACAACTTCGGTCAGCAGCACTGCCCTGGTGGGATG GTCCTCTGTGCCACACCGGAACAAACACAGTTTGTGGATATTGAAAAGGAAACAAGCAGTTGCAAAC GAGAGCAGGTCGTGTTCTACGAACTGTAAAGCAAAGATTGTTCTGATGAACCAACGTGTGGACCATCA TCATCTTCATCATAATTTATAGTAATATGTATTATTATTGTTATTATTATTACTATTATTATTATTATTAT TATTATTATTATTATTATTATTATTATTATTATTATTA CTGTTGTTGTTGTTGTTGCTTTTGTTGTTATAAT AATAATACTAATTATTATTAGTATCCTTTGTATTAATATCATCATTATCAAGACAATCTCAGTATCAAA ACAATTAATGATGATAACTACTGTAAGTCGTAACGTTATATTTATTACTTGTTTTCAGAAAGTGACGCG TCAGACAAGAGAGACTACCTCCGCTTTGGCCGCAGCACTGACGGCAGTTTCCTGCGCTTTGGCCGAGA CGCTCAACAGAACGGT GACAGCACCAGCGATGACGTCAATGACGACCTAAAACGGGACCCCATTATG CGGTTTGGAAGAGGGGATGGTTTCCTCCGATTCGGCAAAGGGGATCCGTTTATCCGGTTCGGGAAAAG

PAGE 97

97 AAGAGGAGATCCGTTTATTCGAATCGGGAGAGGCGACCCCTTTATCCGTTTCGGAAAAAGAGATTCGG ATGCTAGTGGTGGACAAACAGATAAGCGGCAAGACCCTATCATCCGATTCGGAAGAGGGGATCCGTTT A TCCGATTTGGCAGAGGGGACCCCTTTATAAGGTTTGGCAGAGGGGACCCCTTTATCCGGTTTGGAAA GAGGCAGGACCCTTTTATCAGATTCGGCAGGGGAGACCCATTTATCCGATTTGGAAAGAGGCAGGACC CTTTTATCAGATTCGGTAGAGGCGACCCATTTATCAGATTTGGTAGAGGGGACCCCTTCATTCGATTTG GTCGAGGCGATCCATTCATCCGATTTGGCAAAAGAGGAGCAGACAGCGAAGA AGGTGTCGGTGAAGG AGAAGCCTTGTCGAGGTAAAAATAGAAAATAGAAAACATCCTTTATCCGTTTTCTTATAGTACGAACT ACATTTTTTTCGTCTTCAAAGATGATCTAAAAGCAGTATGGATGATACAGCTGTGCTTAGGCAATAGAC AGTAAACATAGTGCTAGTAGATTGCCAAAGTTATAACTTTCTATACTTTTGATCATGATA FMRFamide5* Gene Bank Accession Number: n/ a Reference: n/a Partial Clone: CAAGCGGGGTGGATCAAGCCTTGGAGCTAATTTCGAAGAGGGAGATGATATTGTCAATGATGGGCCTG TCAATAAACGGTTTATGAGGGGATTCCAGAACTTTGTAAACATGGCCAGAAGAAGATACTCGTATCCT CGTGAAGGCGATTCTCAAGATAAAAGGTTCATGCGAGGTCTGTCTGCCTACAAACGTGGTGCAGGAGG GGACTCGTTAGACGAGGACA TCAGCAAACGTTTTATCAAGGGGTTAGAGAGATACGCTCTGCCAGCCT CTAAGAGATTCATGCGCGGTTTACAAAACTGGCAGAAACGAAACTCCCTCGAGGGTTCGGGTGCGGAC AAGCGATTTATCCGGGGGTTACATCTGTACCATAAACGAGGAGGCGACATGTCTTCTGTGGACAAGCG CTTTATGAGAGGGTTAAATTTGTACGCAAAGAGGTATCTGGGATGGGACATACCGGATTCTAAAAATA GATT TCCTGCCTCAAGCGTTTCAGTTGAGCCTGTCCTAGAGGTGAGCCAGGAAGATGGGCCCTCGTCG GTGTGGGAGGGAGAATACGATCAGCCGAAGAGAGATTATATGTTCACATCAAGTTTGGGGAATTCCTC ACCAAAATCTGGTTCAAGGCAGTTCATGAGTTGGAGGGGCGGAGACTACGTGCTTGGCTGACATGACA AATTATCACAGCTCCTTTCAGTTGACGTGTTGAATAACCTGAGTGTAACATGCAAG ACAAAGATAGCG GTAAAACTAAGGGGAACATGATAGTGTGATAGTTTATATCATTTTTTTCCTTTCCGATAATATGTTTAC AACGTTGTTGTTTTGTTTTGTTTTCTTATTGCTATTTAACGGACTTTTGATACTGCGTTCTAACGTGTTGG TTTTTTTAAAGCAAAGCGTATATTCTAGCTTCTGGTGCTACTGAGTCAAAATCTGTTTCGCTTGTCTTGC GTTTCAAAACCTCTCAGTTTTCTGTCTCTGAGAA TTGTCCCTGGTTTTTTTGGGGGGTTTTTTTGTTGTTG TTATATAAAATATCAATCTCTTATTTCACCAGTGTTTCAGGCTCCTATCTGAAAGTTTAAACTTAATGTT ATTGCTCTCTATCTACTCTCGATCGAGCAATGTCCAAAAAACGTGAAATTCTATGGCAAACTATATATT CTAAAACAAAATTATACAATATTTGCGTGTTCGTTGTTTTCTCGTTTATCCAGCTTAATTGTGATGAATT AAAGAGGCTT CAGATTAAGTCAATTATAATGTAGGCTTTACATGTGGACTCTTTTATGCCAGATGTGAA GGTTTTAAATGTTCGTTATATTAGTATAAAG Fourth (short peptide fragments obtained from MS data) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: ACGGCATGAACATCG TTAGTGGTTTTTGACGTGCATGTGGGCGTGCCCTGGCTGCTCCAACCTCTGACC CTGTGCCGACATGCTAAAGACAACGCAAAAGTGTTTCACTGACAATGGCATTCCTTCGGAAGAGGTAA ATGCCATGATCAAGACTGTTTCAATCGGCAACGTGGCAGGAATGAACTTCACCAACTACTGTGGCGAC AGAAAAGATGCCTTCCTGAATGCCAATAAATGTGTTAGGGACGCTGTAAATACGTGTTCTCCAAAC AG TTATGATGTTTCTTTAAATTTCGATTACTCGAAATACATGGATGCACTGTGTACTGACAAAAACATCAA ACTTGAATGCGTGTCACATTCAGGATTCATAGATGCATTTTCCAAATGTATCAAGGACGAAGCTGCTC GACAATTCAATTCGACCACGGTGAGTGTATGTGATCAAGGAAAAGTCTCTTTGTCTTGCGTAAAGACA GTAGTGGGGGCGTGCGACGAGCATACTGGTGAATTCTACCATGATTTTA TGCAGCCCACAGTGGAACA AGCGTGCAAAGGGAATACTGTTCTTGGCTAGATTTGACAGCGTGCGTGGAAGGATCTGAGCACCTAAA CTGTGAAGGGAACTAACAACAGACTTTGGCATAATGAAGTTACCCAGTGGAAATCATTGAGGACGTGC ATTCTGGTTATTCGAACGCTTGAAGACGCCCTATTTTGAAGAATTATAAAAAGAACAACTTAGCTGTTC CCACATATTAGAGTTTTTTTTTCAGTTCAGAT TTCTCTATTTGAAACTTGAATGCTCAAATCCGTGTTAC TAGTTTGTGTAAATTAGGTTTGAAATGTGCCATGATATTGAGTCCGCTACGCCTTGCAAACTTTTAAAA ACATTTTTTCGGGGTCAAGGGCCGGTTTTCATACTTTAAATGTGTACCAACTGCCAAATATCATTTAAA

PAGE 98

98 AAATCTTAATGGGAGTTTTGTTTCGCAAGAAGAGATTCGTTTTGAATTTGGGAATCCAATCAATTTAGT TTGGGTTATTC TGAGCATGCAAAAAAAGTATACATAATATTTGCAGACAATTATAATGTTCAACGATT ACTCGTCTAAAGTAATCACTACTGTTCATTATTTTCTTTATTCACGCCGTCTCTCCCTAAAAAATCTGCC TCTACTTTGGAAAGTCGGGCCTTACCTTGAATAACATGACTTCATGACTTCATGACTTCATGACTTCAT GAGTTTCATTCGAAACTTACAGTTTACAAAACAAATTTTTGATATTGAAAGAGTGGTGTA TGTTGTGAA CCAACACAAAATAGGAAACAGATAGGAAAATAATCATGCTGTTTGTTGTCCCTTGCAGTTTGTTCTGCT CTCCTTGTGTCTAATGAAATTGAACGGAAAGATAAAATACTTGTATTAAAACCAAAACATTCGGA Fulicin like neuropeptide precursor+Gene Bank Accession Number: 56792350 Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGTGTACCCGACCTGGCCTGGCTGCTCTGCTGGTGCTGATGACGTCTTGTGCCTCCAGCTTCTCACGT GCAGATACCACGAAGACTGGACACCTGAAAAGGCCAACCCGATCAAGTGACCAGTTTTTCGACAGGA GCGACACGGAACCTCTCCAGCCCCCAAACAGTGACCAGTACCTCTCTTCATTGAAAAACAAAGGCCAG TTCAGCAACGAGCAATATCCTGGAGAGTATTTTGACGGGTCCGGGTTCAACGCCGAGGAGGAAGAAA GGCCTCTACATCAAGGGGATGGGGACAGTGTGTCACTTTTGGGAGACAGCCCTCCGAGCCCGGCCATG TCGAAAAGATTTACAGAGTTCCTAGGTAAAAGGGTGCCTTTGGCTAACAGTGGGATGCCTGGCCCTGT GGAAGAGCCACACAAGGAGACCGTAGAGGATAGTCATAGGTCAGAGGGGTTATCACCATCAGAAAGA TCGTATAGAGACATGGCAGAAAAGCTGGCTTTACTACTCCGCTCTGACGCCAAAAGACAGTGGGAGTT TGTCGGAAAGCGACCATACGACTTCGTAGGCAAAAGATACGACTTTGTAGGCAAGCGATACGACTTTG TTGGCAAAAGGTACGACTTTTTGGGAAAGAGAAATCCG TACGAGTTTATTGGGAAGCGGTACGACTTT GTGGGAAAACGTCGCCCCTACGACTTTCTAGGGAAAAAGAGTTACGACTTCCTCGGGAAAAGGTACG ACTTTTTGGGAAAAAGGAATCCGTATGAATTCGTTGGTAAAAGAACTCCGGCTATGGTTCATGAAGGC GTGATCTCACACAACCTAGACGGCGAGGGTCAAAAACTGGCCATTCCATCCTCTGATGCCCACACCGC AGACAGAAGGTACGCCGAGTTTC TGGGCAAAAGGTCCGAAGAAAGTGGACAAGCAGCTCTGACAGAC AGCGCCCGTCTGGCCGCTCTACTCAGCAACACTGGCCTGAGGAAACGGCTGTCCAGAATGCTTCTGAA CGGTCAGCTGGCGGAACAGTACCCAGAGTTTATTGGGAAATAG Protein: MCTRPGLAALLVLMTSCASSFSRADTTKTGHLKRPTRSSDQFFDRSDTEPLQPPNSDQYLSSLKNKGQFS NEQYPGEYFDGSGFNAEEEE RPLHQGDGDSVSLLGDSPPSPAMSKRFTEFLGKRVPLANSGMPGPVEEPH KETVEDSHRSEGLSPSERSYRDMAEKLALLLRSDAKRQWEFVGKRPYDFVGKRYDFVGKRYDFVGKRYD FLGKRNPYEFIGKRYDFVGKRRPYDFLGKKSYDFLGKRYDFLGKRNPYEFVGKRTPAMVHEGVISHNLDG EGQKLAIPSSDAHTADRRYAEFLGKRSEESGQAALTDSARLAALLSNTGLRKRLSRMLLNGQLAEQYPEFI GK FVRFamide peptide protein 7/Secretogranin 10R precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TCTAGTTCGGTACTTTTTTTTTTTTTTGACTGATAATATCGTGTTGTATTGTTATGAAATAGAATGACAT GCTGGGTTGAAATAATGCGGTTTAT CACTGGTTTATCCGTTATCCGTATCGGGGGTACGAGTTTCGGTG AGACTGTGCACGAGGGGGAGTGCCCTTCAGGATAAAAGGAATGTGTGTGCTTGGTCTTGGCCGTCCCT GATTTCTTCTGTTGTGGTAAAATAACATTGCTGGCAACCTGCCAATACGCGCTGTGGCATACCTCTTCT TGTTCAGGCTGAGGCGAATATTGAGAGGCGGCAAGTTACTGTTTCGCTTTGAGGTTTGCTTTTCTTCCA CTGTGT TCGACTCTGAAGGCTCTGCCGGAGCGTCTATAAAGTCACTCTGTTCTGGAGAGCGTTCAAACC TCCTTGCAGAACTGGGATAGTTTCTATTCTGCGGTTTGCTTTCTGTCTTGAAGTCAGCAAAAGAGCGAT CACGTTGATCAAATTGGTGTGGACGTATTTTGTTGTCTTCGTTGTCTGTTTCAGGTCTTCTAGAGGATCT CCCAAATCGAACAAACCTATGATAGTATCTTTTGTCTGCACTCGGGTTTGGTTC TTTATCTACTTGGCTT GAGCGCTCGAAACGATAGTAATTTTTTTCGCCTTCCTCTTGCTTTTCACTCGGGCTTTGGCGCCCAATGC

PAGE 99

99 GAACAAAGCTATGCCCTGGAATTGTCCTTTTCCCAAAACGCACGAAGTTGTGTTGAGTTAAGGGAAGC CGTTTTCCGACCTCCTGCATGGCTAGTCGTCTGAGATCCTCTAAATGGTGGTGTTCCAAATACTGGAAG GCGTTTTCGATGTCCTCCCACAGCTGGCCGATG TACTCCACCGTGTC Glycoprotein hormone alpha precursor, CPA2 subunit Gene Bank Accession Number: n/a + Reference: n/a Full Length Clone: CDS: GCGCCCATTCTTCCACAGAGTTTTAGCCATGACGTCATCCTGGCCGGCACCCACCGGACACAATTCAG CCTCTGATATCTCGTCGCCTCGTTCATCATTTGGCAACTGCCGGAGATA CCTGCTTGCTCTGTCTATCGT TGTCCTCTTCTGTCCTTCTGTCACTCCACAAAGACATTCATGGGAGGCCCCTGGGTGTCACTTGGTAGG GCACACTCGTACTGTGTCCATCCCTGGCTGTGTGTCCTTTGAGGTGACCACCAACGCCTGCAGAGGCTT CTGCGTCTCTTACGCCATACCATCGCCTAGTCACACCTTGGCAGTCAACCGGAACTTTGTCATTACCTC CAGGGCTGAGTGCTGTGGGATTGTGGAC ACCCATGACGTGAAAGTGTGGGTCGCATGCAGAAGTGGG TTCCAGCAGAAGACCTTCAAGTCTGCTCGCAGCTGCCAATGCTCCATCTGCAGACGATCACAGTGACC CCAGGATGACCCCTACACGGCGAAAGTCAAAT Protein: MTSSWPAPTGHNSASDISSPRSSFGNCRRYLLALSIVVLFCPSVTPQRHSWEAPGCHLVGHTRTVSIPGCVSF EVTTNACRGFCVSYAIPSPSHTLAVNRNFVITSR AECCGIVDTHDVKVWVACRSGFQQKTFKSARSCQCSIC RRSQ Granulin like 55 ( modulate the growth of cells, is a secreted glycoprotein containing multiple repeats of the granulin/epithelins motif ) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, no t cloned Predicted: TGCCCCCAAACCTGAGGTAGAGCATGTGGTGTGCCCAGGAGGAACATCTTACTGCCCTGATGGAACCA CCTGTTGTCAACTTGCTTCTGGACAATATGGCTGCTGTCCATTGCCATCAG Hyperglycemic hormones isoform A precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: AAAAAGTACCGATGCGAAAAAAAAAAAAGTACCGAAAAAAAAAAAAAAGTACCGACTGCGAAAAAA AAAAAAAGTACCGACTGCGAGCAACTAGTTCCACAAAGCTGAACATGTCTAGACCCTAGACATGTAG ACTCTTTGTGGCTGGGGAGGGGGTTAGCGTCCGCTATGCGAACTAAAAAAAAAAGTACCGACTGCGA AAAAAAAAAAGTACCGACTGCGGGAGTAACAGTT CTCACGACAGGTGGTGGCAACGAATGGCTTACG AAAAAAAAAAAAAGTACCGACTGCGNNNNNNNNNNNNNNNNNNNNNCCACGAGGCAAAAGCCTGAA AAAAAAAAAAAGTACCGACTACGAAAAAAAAAAAAAGTACCGACTGGCGAAAAAAAAAAAAAGTAC CGACTGCGGAAAAAAAAAAAAA Hypertrehalosaemic neuropeptide Gene Bank Accession Number: n/a Ref erence: n/a Comment: Predicted only, not cloned Predicted: CCACTTTTGAAAGAGGCACAGATCTTCTGTACCGACGAGCAAGCACAATGGAATCTTCTAGCATACTT TTGATCCTTGTCGTCCTTGTGATAGGCACGTCCACTTGCCTTGCACAGATTCACTTCTCGCCCGACTGG

PAGE 100

100 GGTACAGGGAAGAGGGCGGTCTCCACTGTCACCGAGAAGGAAATCCCACTCTGCTGGCAGATCGCGG ACAAAGAAATCATCGATATCATGCTTTTAATACAGCGAACCGCAAAGAAACTCTCTTCCTGCCTGAAT ACCTGCCCAGAACTGTGACGACATGTGTGAAAGCATTGGCTCAGCTGCGGCAGAGTTGATTTGATGAT GCTGATCGCTTGGTCTTTATACGCCTGAGAGCTCTACACTTTCGCCAAAGAACGCCAGAC Hypothetical protein 2 (secretory, pheromone like p eptide similar to seductin) Gene Bank Accession Number: AAN83922 Reference: Cummins, S. F., Nichols A. E. et al. (2004). Characterization of Aplysia enticin and temptin, two novel water borne protein pheromones that act in concert with attracti n to stimu late mate attraction. J Biol Chem 279(24) 2561422. Insulinlike 2 Gene Bank Accession Number: 19850964 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGGAACCACAGTCTACCCTCGATTTATGTGTGACCTTGACTCTTCTTGTTTCGACATTCGGAGTAATG ACAGCTCAGGGACAACTCGTGTGTGACAGAGACTCAAGGCCACACCCAAGAGGTATCTGCGGCTCTCG TCTGACTCGGGCCCACAACAACCTGTGCTTCCTGTTGAGTCAAACCTACCCCGAGTACTTCCCGGGCAA GAGATCGGCCTCACCAGAGCTACAGGCGTCTCAAAACAAACGT GTTCCCAACCAAAGCCTCATTGACG GATCTACAGCATCCTCTGTCTGTGCTGCCGACCTTGACCTATCTAGGGACGACTGGGCTCGCTCGCCTT CAAGAGACCTTTGGCGGGCCCAGCCCTGCCAGGACCCTGAGGGTAGAGTAAGAGTCCGCGGCGACCC AGAGGCCAGTGCCAAGCTCAATCGGTTCACGGCCATGATGCTAGACTCAATGGCAGGGAGAAGGCTG AAGAGAGGAGGAACTCAGTCCAACATGGTCTGCGACTGCTGCTACCATATGTGCTCACCTAGACAACT CGCCACTTACTGTTAG Protein: MEPQSTLDLCVTLTLLVSTFGVMTAQGQLVCDRDSRPHPRGICGSRLTRAHNNLCFLLSQTYPEYFPGKR SASPELQASQNKRVPNQSLIDGSTASSVCAADLDLSRDDWARSPSRDLWRAQPCQDPEGRVRVRGDPEAS AKLNRFTAMMLDSMAGRRLKRGGTQSNMVCDCCYHMCSPRQLATYC Insulinlike 7 Gene Bank Accession Number: 94434859 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGACAGGAAACCTCGACAGTGGACTGGCATTCACACTACTGCTGGT GACCTCGACGATGACCTTCAC ACATGGAGAGACTCGATATTGCCGACCTGGATTTTCCAGGCCTCATCCCAGAGGCTTCTGCGGCTCAG CTCTAGCACGTCTGCACGCCAACTTCTGCCTGCTACTCCGCTGGGCCTACCCAGAACACTTTCCCATGG GAAAACGATCTGCTGACCGGGCAGCCCACCATCCGCCCACAACCACTTCCTCGCCCACCATCCACGGC ATCCCACTGTCCGCTCTGGCCGATGTGTCA CTTACTGACCACGAGCAGCTCAGAACGATGCTATACCCC GACGCCTCAGGTGAGCTCAAGAAACCATTGCTGAGTGAGCATGAAATTAATGAGGTCTGGATCCAAG ACAACGCACCATCTTCTTCCCCTGCAAATCCCTTCGTTCGACGCTTGCGCAAAAGAGCTGCGCGGGGA AAACGTAGTCTTGTTTGTGACTGTTGCTACAGTGCGTGCGATGAGCGGAAACTGGCAGTGTATTGA Protein: MTGNL DSGLAFTLLLVTSTMTFTHGETRYCRPGFSRPHPRGFCGSALARLHANFCLLLRWAYPEHFPMGK RSADRAAHHPPTTTSSPTIHGIPLSALADVSLTDHEQLRTMLYPDASGELKKPLLSEHEINEVWIQDNAP SSSPANPFVRRLRKRAARGKRSLVCDCCYSACDERKLAVY

PAGE 101

101 Insulinlike Growth factor 5 Gene Bank Accession Number: n/a + Reference: n/a Partial Clone: GCGGCTCGAGGCCTGTTTGCCTGGTTCTGTCTGCTGAGCAGTCTGTACGTGTCGAGTCTGGCCGAGAGG CGCTTGTGTGGTCAACTGCTGGCCGACACACTGGACATGGTCTGTGAAGACCGAAGGTTTCAACCTCG CCAAGAGGAATGGTCGCAGTGTCGGACAGACGCACGTGAGAGTCCGTGACTTCTCACCTTTGTACAGA ATGCTCCACAAAATCGCCCCTGCTCCCCCCAGCGTG CACGGCACCGGGTCATCGACCATCAGCGACCA CAATGACGTCACAAGCTTTACTCCTCCTACTACTCGCCCTCAGCCCAAATGGGAGGGTCAAAGGTCGC CGGGAGGTCACGGCGTGAGGTCAACTCTCGTATTGTGGACGAGTGTTGTCTCCGGCCGTGCAACTTCG CCACGCTACAGAGCTACTGCGCCGATCCGGACGATCCCGTGGTGGAAATTCCAGAGGATGTACTGCGG CAGCGCCTCGGTTTCACCAC ACAGCGAACTGCAGGTCACGTGGGTCAG Insulinlike O Gene Bank Accession Number: 94434877 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGCCTGGCCCGGTGAAT AGCCTGTTTGCCTGGTTCTGTCTGCTGAGCAGTCTGTACGTGTCGAGTCTG GCCGAGAGGCGCTTGTGTGGTCAACTGCTGGCCGACACACTGGACATGGTCTGTGAGGACAGAGGCTT CAACCTCGCCAAGAGGAATGGTCGCAGTGTCGGACAGACGCACGTGAGAGTCCGTGACTTCTCACCTT TGTACAGAATGCTCCACAAAATCGCCCCTGCTCCCCCCAGCGTGCACGGCACCGGGTCATCGACCATC A GCGACCACAATGACGTCACAGGCTTGAGGAAAGACAATCTCGGAGGAGCGAGTTTAGCAGACAGCC ACGCCCCCGTTGCCTCGGCAGCTGCCTATGATGGCTCCGGGGGTAGCCGCCAGCAGAACGAGGGAGG CGCTCGGCCCGAGCACAAGCTTTACTCCTCCTACTACTCGCCCTCAGCCCAGATGGGAGGGTCAAAGG TCGCCGGGAGGTCACGGCGTGAGGTCAACTCTCGTATTGTGGACGAGTGTTGTCT CCGGCCGTGCAAC TTCGCCACGCTACAGAGCTACTGCGCCGATCCGGACGATCCCGTGGTGGAAATTCCAGAGGATGTACT GCGGCAGCGCCTCGGTTTCACCACACAGCGACCTGCAGGTCACGTGGGTCAGACCGGAAGACGGCGC CAGACGACATCGAGGCCCTCGGCGGAAATCAACACCGGTTCAGGTCATCGGCGATTGGGCTCGTTTTT CTGTTGGAGATCGGGAATCAACCTACCCGCAAACCGCTTG TAG Protein: MPGPVNSLFAWFCLLSSLYVSSLAERRLCGQLLADTLDMVCEDRGFNLAKRNGRSVGQTHVRVRDFSPLY RMLHKIAPAPPSVHGTGSSTISDHNDVTGLRKDNLGGASLADSHAPVASAAAYDGSGGSRQQNEGGARPE HKLYSSYYSPSAQMGGSKVAGRSRREVNSRIVDECCLRPCNFATLQSYCADPDDPVVEIPEDVLRQRLGF TTQRPAGHVGQTGRRRQTTSRPSAEINTGSGHRRLGSFFCWRSGINLPANRL Latarcin 4a precursor (secreted by venom glands, antimicrobial and cytolytic) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GGAGCAGCAGAAATTGAAGAGGAGGAAGAGAAGAAGAAGAAGAAGGAGGAGGAGCAGAGGTTAAG GGAGGAGGAAGAGAAAAGGAAGAAGGAAGAACAGAGGCTGAAAGAAGAGGAACGAGAAGAGGATA AAAGAAGAGCAAAGATTGAAGGAAGAGGCGGAAAAGAAAAAGAAAGAGGAGCAAAGATTGAAAGA AGAGGAGGAAAAGAAGAAAAGGGAAGAGCAACGATTGAAAGAAGAGGAAGACAAGAGAAAGAAAG AGGAACAAAGATTGAAAGAAGAGGAAGACAAGAGAAAGAAAGAAGAACAAAGAT TGAAAGAAGAA GCTGAAACGAAAAAGAAGGAAGACCAACGGTTGAAGGAAGAAGAGGAACAGAAGAAAAGGGAAGA ACAGAGATTGACAGAAGAAGAAGAACAAAAGAAAACGAAAGAAGAACAAAGATTGAAAGGAGAGG AGGAAACGAAGAAAAGGGA New neuropeptide prohormone (Betsin)*

PAGE 102

102 Gene Bank Accession Number: n/a Reference: n/a Full Leng th Clone: CDS: GAACTTTTGCACTCCCGCGGAAACTGGATCGAGGCAGAAATAGAAGCAGAGAGTCACCCAAAACCAT TCACACATGAGCGAAAACTTCCTACCAAACAAAACTATTACGTCATAGCAACTCGGCTGCGGGTATAT ATAAGCGCGGACGGAATCACGGTCAAACATAGGGCGCTACAGAATTGATTAAAGACGGACTTAAGCT TTGACTAGGTTGTAAGTGCTTGCCTCCCTCCTTGCATTC TCAACAAAAACAACTACAATAACAACGCAA ATTCTGCTTTAGACTGAAAAAAAAGTATTCACAGTATAGGAGACCCTCCGTTCCGGCGATCAACAAGA AAACGAACTTCTGAAAACGAACAAGGAAAGGCTCACGTGTTCAGACTGAGGCTACGAAACAGAAACA ACATATATACAGAGACGGAGACTGTTAGATTGAACCAGTGAATTGAAAGACCAGGAAGAAGAATATT GAGGCCCTTGCAACGAACGACTTT AGAGCGAAAGAAAAGTTTCTCTGAACTGCAAATTGTAACTTTCG TCCCTCCTGCCAAAAACACTCCTGCACAGTCATGGAACCCCAACTTCTCAGCGTCGTCATTCTCGTTCT CTGCGCATGTGCCGCGTCTGCCCTTCCGACTAACAGCAGACGAGAGGACCTGAGTCATCTGGTGACTC TGCTGGGGAAACTGAAGAACATTGAGCGAGCACGCCAGAATAGCGAGGGGTCAGCACAGGCGCATGC ACAAGAGG CATGGGAAGGTCCCAGAGTGTCCAGCTTCCCAGAAACTGAGGTCACAGCTGACGTCATA CCAGACGAAGTGTCAGCGGCCAAGGGGTATCTCGGACAGAGGCCGGCGACTGGCGTGGTAAACAAGC GACAGGGGGCGTGGTCTTACGATTACGGTCTGGGCGGTGGCAGGTTCGGGAAGAGAAGCTACGGGGA TTACGGTATCGGAGGTGGACGGTTCGGCCGTGACGTGGACCATGTAGATCTGTCGGACGCCAC CGAGA ATGAGATTACGTCATGAAGGGCGAGCAGGGTCCGAGTGGGTCAAGTAGAAGTGACGGCGTAAGAAGA AATATCTCCATCTTTTTGTGACATCACAAGACACATTTGCTGAGGAATGATGCTTGAAGAGGTCGCTTC TACTTGCAATCGGGTCGTGAATCGTTGAGAACGATGAGATAGGATTAATTAAACAGAAATGAAACCCT TAAAAAAAG Protein: MEPQLLSVVILVLCACAASALPTNSRRE DLSHLVTLLGKLKNIERARQNSEGSAQAHAQEAWEGPRVSSFP ETEVTADVIPDEVSAAKGYLGQRPATGVVNKRQGAWSYDYGLGGGRFGKRSYGDYGIGGGRFGRDVDH VDLSDATENEITS Leptin precursor 1 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: ctgaattttggtcttcc taataaaacatctggtaggaccaatacttcagaaaggagcagaatgtcccccaaatgtgcatttctaaacaggaacactttttt Leucokinins precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GGCTGGGACTCTACCACAAGCGTAGGACAGAAGGGATTTTTTCCATACGTGATC TAGCTTCCCAGTTTT AGAATCATTTCCATTGAGGAACGAATCATTTATCTTGTGGAACCAGGTCCGATTATATTATGTCAGCAG AAAATTCAAATTCATACGAAAACCTCAAAGAATTTGTGTCATTTTGAACCAGAACGGACTGACAACTT GAAATTCTGAATATTTTAAATTATTAGTTACGGCAAAAATAAATTATAGCGCAAAGAAGAAATACGGA TTTTGCATGTCAGGACTTTTAAACCCATAACAGATG GTTATGCAAAGGAATTTTATTTCAATTTGAGCA CCGCATGTGAGGAAGTTTCAAAACCTGTCAACTCAACATTTCGAAAAAAACAAATGACTTAAAAATCT TAGTACTGCGTGAAAGGAGAAAGTGAAATCATGGAGAAACCATTTTCCATATTGGGTGCTTATAAAGT GTTTTCGAGCAGAAACAGGCCCACTAAGGATAGTTGTTTCCGTTTTCCAAACTCAAATGTGTTGCGGAT AGCTACTGTCTTTCTGAT TTGTCTACTGGGTTCACAAGACTATGCAGTTTTATGCGAAGTTCAGGACTC GGCAGTCCGTGATTTGACTACATCTGTACACACTTTTACCAAAAGATCCAGTGGGCCTTTAGACTCCTT AGAAATATTGCACTTCGGGAACGACGGGAAGTTTGGAGAATTTTCTGTGAAAAGGTCGGATAACGTTG ACCACGAAAACGAAATTGTTGTTGTGAATACAGAAGTGCCAGAGTTTTCGGAAGGTGTTGTTTCTAAA AGCGATGTTCTAGAAAAACTTGCAGACGACTTTCTTGAAGGGGAGGGGACAAATATTATTCCTGAAAA

PAGE 103

103 TGAGAGGGATGAGGTAGACGAATTCCCTGATGATTCAGGAACAGAGGATTTGGACAAAAGGGCGGGA TTTGCGCCTTGGGCGGGCAAAAGAGACGGAGACGGTGTCGTGGATAAAAGACCAGCATTTCATGCGT GGTCAGGGAAAAGAACACCACATAGCGCTGTAGATGCACAGAAAAGACCCGCGT TTCATGCATGGTC GGGCAAACGTTCAACGTACCCCTACAAAAACAATGAAAAACGACCCGCATTCCATGCCTGGTCGGGC AAAAGATCAGAAAAACGGCCTGCATTTAGCGCTTGGAATGGAAAACAAAAAAAAGTACGGCTGCG LFRFamide precursor* Gene Bank Accession Number: EU886298 Reference: n/a CDS: AAGGTGCGAGCGTCCAGGGTCCAGCTGTCCC AGAATTCCCTGTGTGAGGTCCAGGACAGCACAGCTTA GCCCTGCCCCGCCTGCCGCGCAGTGTGTTCNCCCCATCGTCTGACAGGCCTCAGTTCGAAACCTCGTCC CTGAGCAAGCGGAAGACAAGAGAATGGAACCCAGACCCATCACGCTCAGCCTGGCTGTTCTACTCGC AGCTCTCTGCACAACCTGTGCCACCGGCTCGGCCAAATTAGTCAGCTCAAGCGAAGACAATACCAACA GCCACACTGACGGCCTCACCCGGGACCACCAGCCACAGAAGCGGTCAGCGGCGGCCATCTTGTCCCCA GCCGATGACGCGCTGTTGGTCAGAGCGGACGAGGGAGAGGAGGAGGGAGTGGGAGGAAGCTATCCA GGGATCCCCCTCTCTCTCCCAGGAACCGCCGACAAGAGAAGCACACTCTTCAGGTTCGGCAAACGAGG CGCAGGGACCCTCTTCCGGTTCGGGAAGCGAGGCTCTCTCTTCCGGTTCGGGAAAAGGGGCGGGGCTC T CTTCCGGTTCGGCAAAAGGGGCGGGGCTTTGTTCCGGTTCGGCAAGAGAGGCGTGGCTTCGGGGTCG GGGGCGTTCCCGCTGGCTGACGACTCCGACGTGAAGAGGACGCTCTTCCGCTTCGGCAAACGATCCAA CCTTGACCTTCTCAGGGACGTCTTGTCGCAGTACCAGCTCAGCGCCTACCCCGACGCCTACCCCGACGC CTACCCCGACGCCTACATCGATGACGTAGACAGCAAGCGAGGGGTAGACGGGTTTCACTGGGGAGAT GACCAATAAGCGAACTGTGTGTGAATAGCCGAGGACAGGGTGACCAATAAACTAACTGTGTGGTGAT AAGCTGAGACAGGGGTGACCAATCAACTAACATTGGGGGGACATTAACATTGTGATTAGCGAAGATG AAGGCGACGAATAAACGAACTTTGTACACTTTCAGCAAAGAGTGAGACGACGAATAAAATAAGGAAA TTTCACAAGTTCAAAGATGGAGATGACGAATAAACAAACATATTTCACAGGGGCCAAAA Protein: MEPRPITLSLAVLLAALCTTCATGSAKLVSSSEDNTNSHTDGLTRDHQPQKRSAAAILSPADDALLVRADE GEEEGVGGSYPGIPLSLPGTADKRSTLFRFGKRGAGTLFRFGKRGSLFRFGKRGGALFRFGKRGGALFRFGK RGVASGSGAFPLADDSDVKRTLFRFGKRSNLDLLRDVLSQYQLSAYPDAYPDAYPDAYIDDVDSKRGVDG FHWGDDQ Major royal jelly protein* Gene Bank Accession Number: n/a Reference: n/a Partial Clone: GGCTCGAGATTACGTCACACCAGATGGATCGGAAGTCCGGTACGCGTATATTTCTGATACTGGCGATG CGAATCTGGTGGTATTCGATTTTCAGACGAACACGTCATGGGCTTTCAAGGACACGACAATGACACCG GCACCAAACGGTATCTTGACCATTAATGATGTCAACTACACCATGGACTTTCCTATCAACGGAATCGG AATGTCTCCCAATTTCGATTACGTCTACTACTGTGTTATTGGCAACAATAACCTGTACCAGATTCCCAC GTCAGTCCTCCGAAACAAAACCGCAGACTTTAAGGAATATGTTAGGAATGTTGGAGACAAAGGTGGG TCCTCTGACGCAATTGTTGTTGGGAAGAAAAATCTGTACTTCGGAGTAATGCAAGAAAATGCTGTTTC CGAGTGGCAACGCAGCAAA GACATCAAGTCCCAAAATGTCTCCGAAGGCGAGGTCAACATGGCCACC ATACAAGAGCTGACCTCTAACGACGTGACCATGCAATGGCCGGACGGCTTCACTCTGGACGATAAAGG CTACCTCTGGTTCACCACAGAACGTCTGCAGCTCTACCTGAATGACAAGATGGACTTCTCTGGGGGTC AGGGCTCCAATTTCCGTGTCTTCCGGATGTTCGTTGGTGACGTCAGCTACTTGACAAAACCCACGAGTG GAA AGAAGAGTTGGTGGGAGGAATAGCCTTCTGAAAGATTAAAATAGTATCAACGAAATAGATGAGA TTATATTCTAATAGTTTAGTAAGCGT MIP related peptide precursor Gene Bank Accession Number: 8886135 Reference: Fujisawa, Y., Furukawa, Y. et al. (1999). The Aplysia myt ilus inhibitory peptide relat ed

PAGE 104

104 peptides: identification, cloning, processing, distribution, and action. J Neurosci 19(21), 961834. Myomodulin like Neuropeptides Precursor 3* Gene Bank Accession Number: EU934739 Reference: n/a CDS: AGTCAGGGAATCGTTTGCATGGCGAGACGCGGACTATAGAAGGAGATGGC AATCTGTGTTTCACATCA ACACAAACACAGGAGGAAAACCATTAAAAGATCAAACATGGCGCAAGCATAAAATGAGATGTCGCAG TACCCTATCACACATAATTGTATAGAATAGGCTTTTTGAAAAGATAAATATACCCAAACATGGCGAAG CTGGACACAAGACGTGGCAGAAAAACAACAGCAGAGAATTGTACAAAAAAGTCCTATCATCCACTTT GTGACGTCATGGGGAGTGTGTGAGACGTGAGAGATC CGCCTCCTGTAACTCGTGTTAGGTTTCAGTCA TGCGACAATGGCGACGGTCACGTTTTGCTTGAGGCTGAAAGCTTCCTAGGAACACCCGGGATTCGTTC CCTGTTTCCTTGATCTCTCTACACTAAACCACATTAGACCTGTCAGTATTCTGTTAACATATTGGAAGCT TACCATTCCTTTATTTCTTTCTAGAAAAATGAAGTATTGGTGTCAATGCGATTTTAAAATGTCCACTAA CTTTTAACCCTAACCCT CATGGTAAGAGGATATAATAATTCTATCGTTCAAAGATAACCACTTCCTCCA TGTTTTTTTAAGGGGGATGTCATATTATATGATGTGTGTCACTTTGAAAACTGTTTAAACTCCCGAAGG TATTACAGATAAATTTGACTTTTGATTGAAGATGTTGTTATTTTTTCTTTCAGAGCAGTTTGTATTTTCG CATTTCGGCAGAGGTAACCATGAACATGACCAATCTCCACTTGCATTGTACAAGTACGACTAAGAAAG AAAAATGCTCTTTATACGCCTATTGCACCGTACACGGCTCCTACGGTATACGCTGAATGTGGCAAAAT GGTTTTTGGCTCCATCATAATACAAAAAGTTAACGTCCTGAGTCTTTCTTCAATCCCCCTCTTTGGGTTA GTTGGCTACATTAAGCTGACAGACTTTCATAAGCAAAGCCACTGGAAAAAAATAAACAAACAAATCC ACCCCCCAACCCCCCCCCCCCCCAACAAAACAACAAGAACAATTTCAG TTAGGACTTGAAAGAACAA AAGGGTAATAGTGAAACATTGTTTCGAGGATTCAGGGAGTAATTCGATTTTATCGGCTTTGGTAACAG TAGCGTCGTGAGCTCTTCTTGGTTTTACAAAAAAAAAAAAAAAAAAAAAAAAAGAGGACAGGGCTCG GGATCTGGGGCACGGAGAGTGAGAGAAACACAATGTGCTTTGGGGAAAAAAGCACGTAAGTCCCACT CGTCCCCAGACACGAAGGAGCAAACAGACATCTGG ATGGGCGTGGCCACGATCTGTGGTGACATCAC GGCTGGCGGCAATTAAAACTTTGGAGACATTGACTGGACGCAAGCAAAGCTGGAACCAAATTATGTC AACTTTTCAAAGGAACTGACGAAGGTGTCCATGTTCTATCGGATCACGTTTGACTACACGTCTGCTTCC TCTACTCTGCCTTCACGTTTTATTTAATCGTCTAAGGAACGCGGGGGAAGGCAAAGTCGATTTCAAAA ACGTAACTCAAGCTTGTTAACACCTGACCCCTCTTCGTCCCCTCTCCCTGATATAATTTAGTAGCCTAG GACAGACAAAGTCAACACACATATCTCAGTGGCATTTGGAATTAAAGGGTTTTCCGAGGAGTGTCTTT ATCTCAGATCTTAAGGACAAACGACAGGAAATGTGAAACACAAAAGGGAAGCAACACACGTGCGAAA GGAAAAGATAAATATGTGAATTGAAAAATACACTGGCGAGGAGCAAAGTTCAATAATGCTCTGAAAA AGAAACGACAGCCGGGGGCTGATCTTGTTAAAAGACAAACAAACCCGAAACACAAGAAGAGAGGAG AGGAAAAGAGCTGACTAAATGACTAAAACCAAATTAAAACAAAGGTGAGAAGAGGAGAAGATTTGA CGATTTCGTTAAATAGACCAATACATTTTTCATTTTTGAAACACATTGTACGTTTTATCAGCGTGACCT AGCAGCTGACCCCACTCCCTTTACTAATCGTTTTCTTGTTGAACTTTGGCAGTTTTATTT CCGCTGAGAC ACAATAATGTTTGCGTTATCCTCCCAAAGACGGCATACATTTTCGTGCAGCAATAATATTCAAGGTTCT TTAAAAGCACAAATGAAAGAGGATATTGAGGCGTACATTTGTCTTGAAATATAAAGTCGAATTTTAGA ATGAGAAAAAGAGCGAGAGAGAGAGAGACGAGAAGAGAAAAAGCGTACAAGCGGTAAATTTTATTG ATTTTACAATGTAATAATGCTTTTCCCCAGACTCAAGAAAAAACAAACAGACAAATAACAACAAACGA ATGCTTTGACAACTAAAATAACATTTCCTGAAACTCAACAACAAAGCTAAATGCACCCCACACCCCCA AACCCCCCTTCCTTTGCAATCACAAAAAGAAAGTGACACAAAACAAAGCAAAAAGCACACGCGACAA GACAGCATTAGGAGAAAACGAGCGAAAGCATTACCAGGGAAATAATTTTGTTTCTGTTTCATCACAGA TTAACTATTTGTAACACGAGGGAAAATT TGAAAAGATTCCTTCCCTCACCTCACCCGTCCCCTTCCCGG AGCTCTCCATTTCGTCTTCATAACATGATAGCATGATAGTTAATATTATTTTTTCCCTCCTTTTTTGTCCC CACCACACACCTTATGAACATAGGTACCATTTATAACAGGAGAATCTTTGATCTCCTCTTAGACTGCAG CAGGAAAGGCAAATCGAGAAAAAAAAATCACTTTCTCCGTTTCCGTTTTTATATTTTTATACATGTACA TGTAAT TCTTTTTGTTTCAATAGCATTTCAACCCTCCTCTTTCTTAAATAAAGCACGAATAATTTTGAAA ATAACATAATAGCGCATGTTTCTTATTCGCGGGCCTTTAGTAAGTACCGTAATAGAATATAACAGCAG CGTGTATTCTTGTGAATGGTCTTTGAACCTTTATGTTTACTCAGGGAGTGTCACTCTTTCTTTACACCTT CGTATTCTACAAGACTGAAACAAACGGGATTGTTTAAAAATGCTTTATCGGGCT CTCATTATGAAACG TCAGTTCAGGATGTTCGATCTTGAGACACCAAGTCTCTACACTCCTCTTTGCACCCTCCCCCTGCCCCC

PAGE 105

105 AAATATAAAAAAAAAAATCCCCGCTTCCACTCACTCCCCCAAAACAAGAAAAGAATCTGTGGAACAC CTTCTTTCTTCAATCTGCCCCAGCTTAAAACGGCTCCACCTCCACACGTTTCATCCAACATCCCTTCAGT TTTTGGTCCATGTTCGCATCCTTCCTCCTCATTTCG ACCTCCCAAGGACATCTCCAGATCTTGAGTACGT GGAACCGAGCTCAATTTAAAATGAAGAGATGGGGGTATTGGACAAGACCAAAATAGCAGTATCGCAT GGACAAGTTCAATGAGGTGGAAAAAAAACACACCGACAGGAAGATTAAATGAGTGTCAAATCTGTGC GGCGTTACTTGTCCTTTGCGCGGCGTTCTGTTTTTTGTCAATACGTCAATGTACTAAGACCGCTTCTGAC GTTGAATTTGATGAGGTC ATTTCTTTCTGTGTCCAGGTTTTTGTGCCTATTAGGTGGGTTCCAGGGCAG GGGATGGGCGTGGTCACAAGCACGTTTATACGTAATGTGTACGTCTATAGTGTGGTGCTGCCGAAAAA ACGGTTTTATACACTTGTACTCTAAGAACAAGGGAAACAGTATTGGCAGGCTCGTTGAGGATTTTGGA AAGTTTATTAAAGTTGTGAGGATGTCTGTACATTTCGACTTATCCAGATCATCAGGTGGTATGCTGAAG TGAATTTTATCACCACAGTAATAACTCACTGAGTGTTACGTTGTGTGCGTGTAGAAATGTGAGTGTGTG TTTGTGTCGCAAGCTCTTGTATCCAGACTAGCCTACATATTGCATTATTTAGAAAGAAGCGAGCTGATA TTATGTCGACGGTAAATGTATGTTCACTAATGTGTTTACAAGCACGTTTATACGCAAACATATGTCCAT ATTCTGATACTTCTAAAGGAACCAGAAACAATTAATTTATTTGCAGGTA ACGAGGCCGCCAGATATAG CTCGGCGTGTTTCATTAAAAAGTGTTATTTTTAGAAGTTAAAAAGGGAAAACCTTGTTTCCCGCGCACC ACTCTTACAGCCTCAAGTTTCTCCCCCTATCACCTGATGCCTTTTTAAAGTTGAACTCCAGGGCGGCCG GATCTGCTGCTCGGCATCCCAAGAGATGCCATATGTCACGAGGGGATGTAGACATTACCGAAACGGCA CACACTGACTTATTGCTGCAGGAACATACCG ACTCCTCATTTCATAGAGACTATTTTAGATAATATAAA ACTGACAGGGATTAAAAGCGGTAAATAGGTTTTGAGGAACAGAGCAGGGGTTACTACTGATAACATT GCACGAATGTAGAAGACCACAAGTTATGCATTATTTTCTTTAATATAATATTTGCACTATAATACACCC TTTTCGTCCTCTAAACAGCATTGGAAGTTGTACATAATCATTTACAGACTGGATTCTGTCATGATGGAA TTCAAAATGTGGT GACAGAGCATAAGATCTTGTTTACTTGAGATGTTTGTACAAATACAAGAAAGAGC AAAGACGTGGACAGTGAGTAAAACATTGACAACATTATATGCGCTTCTTCTGTTGATGCTAATGAATT TGTAAACATCCCTCTCACCACACCCTCTTTTCACCCCCAACCAACCCATTTTCAGAGTCTTAATTGGTA ACATCAATAATCAAAAAGACGTATCGAGCAAGCGTTGGGATTCTTGTGTACTGTGTAGGACGTA GGAA GCAGTTACTTCCCTATCCGGACAAAAGAGGATTGTCCGCCCCTGGATCCCCGCCGGTACCCTGCCCTGC GCCCGAGAGCGGGCTCCACCATGACGTCAGAGTTCTTGCCGATGCGGACAAAAGCGGAGGATGGGAT TTTCCCTATTCTGACAAACGCATGCGCTCTGCTCGCCACGCTGATCGGCTTATCGTCATCAAAAAGAAC ACCGTCCCCTAGGCTTTCTTCAGACGATGATGAGTCGGAATGTGAAG ATGAAGAAGATGAGGGCGAG GATAGAGAAGACATTGAGGATGACGACCCAACGGACGACGGTTGTGCCGGTGCCCCTGGAGATGACA AAGCTTTGCCGATTCGCACGAAGTTTGAGGCCCTCTTGTCGGAAGACAGTGCACCTTCGCCATCAGCT GCGTTGTCCATGCCCGATTTCCCGATCCGGACAAAGCTTGAGGCTCGCTTCTGACCGTCGTCCGTGCCG TCTACGCTGAGATGAGGGCCGGAATTGTCCCC GGACTTTCCAATTCTGACGAAGCTGGACGCTCTCTTA TCCTCCCCGTCACCGGACTTTCCGATTCTGACAAAACTAGAGGCGCGTTTGAGCATGTCTTGGCTGTTG AAATCTGCGGGGCTGAGATCATCATAGCCGAGAGAGCTTCCAAGATCGTACCCGATGTTTTTGCCAAT ACGGACGAAACTGCTGGCTCTCTTTTCCGCAAAATCTTCGTCATCAGGCAATTCTGACTTGCCGATGCG GACAAAACTGGAA GGGTTGCGGCCAATACGAACAAAGCTCGAAGGATTTCTGCCAATTCTCACAAAA TTTGAGGGAGCCTTGCCGATTCTGACGAAGTGAGATGCGCGTTTGATGGCGTCTTTGAGCGAGGCCAG TTGGTCAACGGGGGTTTTCCCGATCCTCACAAAACTAGACGCGCGTTTGTTGTCGTACTGGTCCTCACT GGCGTCATCGTAAACCTCGTCACCTGATCTGCCAATACGGACGAAGCTGGACGGCCGTTTGCCGA TTC TGACGAAGTGAGACTTTCGTTTTTCTGCTTCGGGCTCGTCCCCATACATCCGGTTATAGTGGTCATCTA CGTAACTTCCGTAGTCAGACATCCTGCCAATTCGGATGAATCTGTGTAGAGGTAGGTTGCTTCCTCGTC CTATCCTCACAAAGCTAGACGCCTTCTTTTCCATCTCATCAGCGTAAGGGTCGTCTCTCAGATAGTCCT GCTGGCCAGCGTCGTTCTCGTCAAAGCTCCCGTCTCCTCCAACATC TTTCCCGATCCGGACAAAACTGC TAGGGCGACCGATCCGCACGAAGTTGCTCGCCCGCTTCTCTCGAGACTCAGCCGAGTCCGTGACGTCA GCCGGACTCGTGGCCTTGACCGTTGAAGCGTCGTCCTCAGCGAGGATTGCAGACGGGCAGAAAGACAT GAAGCAGACGACACACAGGAGTAACGGTATCGCCATGGCCCTGTAGGACAAGGTCGGTCGAACGGCA GCCATCTTGGTATCACTTACCGCTTAGGCT GGACTCAGGGTCTCGGAGTACTTGATTCTATGTGAGAGA TTTGACGAGATTTGGTTCTGAGGAAATACAAAACAAAAATAAAATAGAAGGATGTGTGGAGTGGTAG CTTTCGGGGAGTTAGAGTGC Protein: MAAVRPTLSYRAMAIPLLLCVVCFMSFCPSAILAEDDASTVKATSPADVTDSAESREKRASNFVRIGRPSSF VRIGKDVGGDGSFDENDAGQQDYLRDDPYADEMEKKASSFVRIGRGSN LPLHRFIRIGRMSDYGSYVDDH YNRMYGDEPEAEKRKSHFVRIGKRPSSFVRIGRSGDEVYDDASEDQYDNKRASSFVRIGKTPVDQLASLKD AIKRASHFVRIGKAPSNFVRIGRNPSSFVRIGRNPSSFVRIGKSELPDDEDFAEKRASSFVRIGKNIGYDLGSSL GYDDLSPADFNSQDMLKRASSFVRIGKSGDGEDKRASSFVRIGKSGDNSGPHLSVDGTDDGQKRASSFVRI

PAGE 106

106 GKSGMDNAADGEGALSS DKRASNFVRIGKALSSPGAPAQPSSVGSSSSMSSLSSPSSSSSSHSDSSSSEESLG DGVLFDDDKPISVASRAHAFVRIGKIPSSAFVRIGKNSDVMVEPALGRRAGYRRGSRGGQSSFVRIGK Neuropeptide CP2 precursor Gene Bank Accession Number: 18844703 Reference: Vilim, F. S., Alexeeva, V. et al. (2001). Cloning, ex pre ssion and processing of the CP2 neuropeptide precursor of Aplysia Peptides 22(12), 202738. Neuropeptide Y mRNA, complete cds Gene Bank Accession Number: 155793 Reference: Rajpara, S. M., Garcia, P. D. et al. (1992). Identification and mole cular clon ing of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons. Neuron 9(3), 50513. Neurotoxic peptide caeron like precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CGCAGTCGGTAC TTTTTTTTTTTGAAGAGTTTGCAGATATGACACTTTATTTCCGCGCTCAGTTCATCAT CTCTCGCGACGGACTCATCTAATTCCGCCATTGGTACTGTCGCTTGACGGACTCATTGAGCAGATTCTA GCGTCATGAAGAATGTCGCGTGACTGACTCGTCTAGTAAATGATGTGAGGGTTAATGTCGAAACGTTG CTCACATGCGCTCTTGACGCGACAGGTGCCCTTGAAAATAGGGAGGTTGAAGTCTTGTCTT TTACTGAC TGCCAGGTTGAAGTAACAGCATTGGTCTTTTCCACAGTCGCTGTTGCTAGTACAGGTCGTGGCGGCCTT GCGTTTGACGACCACCGGTCTCTGACAGGTGCCCATTTGACTGAAAGGCGTGTCCGAATCCTTCACAA GCTGGCACTTGAGACCTTTTTGGCAGTTGCAGGAACTGAACAAGTTGCAGGACTCTCCCTCAGCGCTG AGTTTCTGGCAAAATCCGGGGATACCGTCCAGGTTGAGCGCTC TTCTGCTGGGCGTGACGCGCATGCA GCACTCACCCTTCCCGCACTGGTCTTCACGCACACAGTTGTCACCCTGGGCCGCAAAGGCCAGACCGA GGCCCAGAGCCAAGACAGCGATAGT Neurotoxin like 1 Gene Bank Accession Number: 71148939 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome o f Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGATGAAGGTTCTCCTGTGTGTGTCTGCTCTCGTCCTCGTCAGCGTGAACGCAAAGGTGGGTGACGA GTGTTGGAACGAAAGCACGTGCGACCCTGGAGAATGTTGTTTGAAGAACGAGGGCTCTATGATCGTCA GCAAGAAGAGATCTGACTTTGGACCCATCTTGGGAGCAACGAAA GGGACGTGCCAAAACTATCGGTT GGAAGGCGACAACTGCCAGAGAGAAGACATTTGGGACGAAGTAACAATGTGTCCCTGTGAGACCGGC TTGAGGTGTGTCTCGAGGGAAGAGCCGAGATTCGTTCCAGGATTCTTTTCAAAGAGAGTTATTATAGC GCCTGCCAAACCAGGATACATTTGGCGGGAAAGGTGTGAGAGAATCGAGAGAAACCAGGATTTTTTC GTCAGCTAA Protein: MMKVLLCVSALV LVSVNAKVGDECWNESTCDPGECCLKNEGSMIVSKKRSDFGPILGATKGTCQNYRLE GDNCQREDIWDEVTMCPCETGLRCVSREEPRFVPGFFSKRVIIAPAKPGYIWRERCERIERNQDFFVS Neurotoxin like 2 Gene Bank Accession Number: 71148941 +

PAGE 107

107 Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGAAGGTTCTCCTGTGTGTGTCTGCTCTCGTCCTCGTCAGCGTGAACGCAAAGGTGGGTGACGTGTGT CAGGACGAAAGCACGTGTGACCCTGGAGAATGTTGTTTGGAGCCCGTGGACTCTTTGATCGTCAGCAA GAAGAGATTTGACTTTGGCCCCATCTTGGAAGCAAGGAACGCGACCTGCCAAAACTATCTGTTGGAAG GCGCCGACTGCGAGAGCCAAGACATTTGGGATGGAGTGACCTGTCCTTGTGAGACCGGCTCTATGTGT ATCTCAAGACAAAAGCAGAAATTCTTTTCAGGTTTCTTTGCAAGTAGAGCATTGCCGCCATCCCAACC AGGATACACTTCGCAGACTAAGTGTGAGAGAAACAACGAGCCCATACCCAGCTAA Protein: MKVLLCVSALVLVSVNAKVGDVCQD ESTCDPGECCLEPVDSLIVSKKRFDFGPILEARNATCQNYLLEGA DCESQDIWDGVTCPCETGSMCISRQKQKFFSGFFASRALPPSQPGYTSQTKCERNNEPIPS Orcokinin peptides type A precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TAACAGAGATGAGATCA GAGACAGCAGACGTGACATGACCAGGAACAGAGACAACAGATTTGAAAC GAAAGAGATCATGCGCAACAGGCGGGGCTTGGACGAGATAAGCAGAGACAACAGGCGTGACATGAA TGAGATCAGCAGAGACAACAGTCGTGATATGGACGAGATCAAAAGCAACAGACGTGAAGTGAACAAA GTAAATAACGACGAAATCCGAGACAACATGCGTGTGATGATCGGTAACAGGGACAACAGGCATGACC TGAATGA GATAAGCAGAGCAAACAGTCGTGACTTGAACGAGATCAGAGACGACAGACATGAAGTGAA CAGGGTAAGCATAGATGAGATCAGATACAACAGACGTGACATGAACGAGATTAACAGAGACAACAGG CGTGACATGGACGAGCTCAGAAACTACAGGCGTGACCGGAGCGAGATCAGAGACAACAGGCGTCACA TGAACGAGGTTAACAGAGACAACAGGCGTGACA Orexigenic neuropeptide QR FP precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CACATCGTTTGTCGGTTTGGGGGTTTGGACAGAGCGAGGTGTGTGAGGGACACAGGTGTCAGGTC Pedal peptide 1 Gene Bank Accession Number: 56200042 + Reference: Moroz, L. L., Ed wards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGATGAGACAACTGTTCGTCCTGGTCATCGTGGCCGGAGCCGTGCTCGCCGACAACTACTGGTGCCC CAAGGCCGGCGAGGCTTTCGAGTGCTTCGAGTCGTCCCCCACAGAAAGGTTTTGCCTGACC AACGGAC GCGAGACCGCTGTCATCTGCTCCAAGTGCAGGAAGAAGTTCGACTTCTGCCGTAACGACTTCGTCAAA TCCAAGAGAGCCCACACTGACTGTGGAGCCGGTTGGGAAAGCACTCCCTGCACCCACGACAACTCACA CGTGCCCGCTGTCTTCCCCGGGAAACTTTAA Protein: MMRQLFVLVIVAGAVLADNYWCPKAGEAFECFESSPTERFCLTNGRETAVICSKCRKKFDFCRNDFVKSK R AHTDCGAGWESTPCTHDNSHVPAVFPGKL

PAGE 108

108 Pedal peptide 2 Gene Bank Accession Number: 94434888 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGGCCAAAGTCGGAG TGAGCAGTCTCTGCCTTGTCCTTATCCTTCTCTGCTCTGCCGTGAGCGGAGGT GAAGTTCCAGATGGAGTTCTCAAGAAAAAATCACAATTTGTCCAAGCCTCCTCCGTGAGTGAGACAGC CGAGGGTGCAGGTGATGGTTCTCTCAGTAAACGACATGTGGGTTCCATCGGAAGTTCTTTTATCAAAC GACCTGTCGATTCTATTGGAAGCTCTTTTATCAAACGACCAGTCGATTCCATCGGAAGCTCTTTCAT CA AACGACCAGTCGATTCCATCGGAAGCTCTTTCATCAAACGGCCAGTCGATTCCATTGGAAGTTCGTTCA TCAAACGACCAGTCGACTCCATTGGAAGTTCCTTCATCAAACGGCCAGTTGACTCCATTGGAAGTTCCT TCATCAAACGGCCAGTTGACTCCATTGGAAGTTCTTTCATCAAACGGCCAGTCGATTCCATTGGAAGCT CTTTCATCAAACGGCCAGTCGATTCCATTGGAAGCTCTTTCATCAAACGACCCGTCGATTCTATAGGAA GCTCTTTCATCAAACGGCCAGTCGATTCCATTGGAAGTTCGTTCATCAAACGGCCAGTCGATTCTATTG GAAGTTCGTTCATCAAACGGCCAGTCGATTCCATTGGAAGTTCGTTCATCAAACGGCCAGTCGATTCTA TTGGAAGTTCTTTCATCAAACGGCCAGTCGATTCTATCGGAAGTTCTTTTATCAAACGGCCAGTTGATT CCATAGGAAGCTCCTTCATCAAGCGAC CTGTTGATTCAATCGGAAGCTCCTTTATCAAGCGACCGGTTG ACTCTATCGGAAGCTCTTTCATCAAACGTGGCGTTGATTCAATTGGAAGTTCTTTCATTAAAAGACCTA TTGACACTATTGGCAGTTCGTTTATAAAGCGCGGGGTGGATTCGATTGGTAGTTCTTTCATCAAACGGC CGGTGGACAGTATTGGTAGCAGCTTCATCAAAAAGAGAAGATTCGGGGGCGAAAATGAATTTATGAA GAGACCAAT CGATACTATTGGAAGCAGTTTCATCAAAAAGAATGTCGACTCTATCGGTAGCAGCTTTA TCAAACGGCCCATTGACACGATTGGAAGCAGTTTTATCAAGAAGAATATGGACCAGGCAGCGTTTATT AAGAGGCCTATTGACACTATCGGAAGTAGTTTCATCAAGAAAAACATTGATTCAATTGGCAGTAGCTT TATAAAGCGACCCGTTGACAGTATCGGAAGCAGTTTCATCAAGAAAAACATTGATTCTATT GGCAGTA GCTTCATAAAGCGGCCGATAGACACCATTGGAAGCAGTTTCATTAAGAAAAGTTATGGACAAAACGA AATGATGAAGCGACCTGTGGACTCGATCGGCAGCAGCTTCATCAAACGACCGATAGACACCATCGGG AGCTCTTTCATCAAGCGAGGGGCTTTCGTTTACGGGGGACGCCATTTCAGGAAAAGGCCAAAACCCAC TTGGCACAGGTTCATGAAGAGGCCAATCGACACCATAGGAAGTAGTT TTATCAAGAAGCGAGATGTGT TGGAGGAGGAGGCGGCGATGGACGGCACACAGGGCAAATTAGGCGCGAGTGATTCCGAAGGTCAGA GCAGACGAAAGCGTAGCACGAGCTTCCACGTGTTGGACTCACTTCCCCTGAGACGTGTGATCAACAGC CTGTACACGTCAGATCTTGATGGGTCACGCGACGAGCCTGCCCATGAGGGCCAGCTCACGTACGGCGA CGTAGTGGAGCTCCTCCGATTCTTGGACAGGAA CGAGTCTGTCATCAGTGATGAGAAGTTCGGAGACA TTCTGGACAAGCCATACCTGGGCTCCGGTCAGATGGGGAGGTAA Protein: MAKVGVSSLCLVLILLCSAVSGGEVPDGVLKKKSQFVQASSVSETAEGAGDGSLSKRHVGSIGSSFIKRP VDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSF IKRPVDSIGSSFIKR PVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSI GSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRPVDSIGSSFIKRGVDSIGSSFIKRPIDTIGSSFIKRG VDSIGSSFIKRPVDSIGSSFIKKRRFGGENEFMKRPIDTIGSSFIKKNVDSIGSSFIKRPIDTIGSSFIKKNMDQAA FIKRPIDTIGSSFIKKNIDSIGSSFIKRPVDSIGSSFIKKNIDSIGSSFIKRPIDTIGSSFIKKSYGQNEMMKRPVDSI GSSFIKRPIDTIGSSFIKRGAFVYGGRHFRKRPKPTWHRFMKRPIDTIGSSFIKKRDVLEEEAAMDGTQGKLG ASDSEGQSRRKRSTSFHVLDSLPLRRVINSLYTSDLDGSRDEPAHEGQLTYGDVVELLRFLDRNESVISDEK FGDILDKPYLGSGQMGR Pedal peptide 4+Gene Bank Accession Number: 94434899 Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGAATCAAAACTATTTGTCCAAAAGACCGCTGGATTCCATATCTGGTAGCAAATTTCACGGAATGAA TCAAAACTATTTGTCAAAACGACCG CTGGATTCCATATCTAGCGGGTTCAACGGAATGAGACAGAACT

PAGE 109

109 TCTTATCAAAGAAAAGCTTTGACTCTATTTCCGGCAGCGGGCTGCACGGGATGGGCCAGAACTTTCTC GACGGGAAGAGAACATTTGACTCAATCGGAGGGGGTGACTTACAAGGAATGGGCCAAAATTTTCTGA CATCAAAAAGAGGTTTTGACCCAATTTCAACAGGCTCTCTGCACGGAATGCGTCAAAACTTTTTCGGA CACAAAAGAAGTTTTGACTCAATAAGCAGAAATCGGTTAGGAAATTTACGTCAAAATTTTTTGAGTAA ACGAGCAGACGAGAGCGCGGATGAGTGGAGTGTTAATGACGCTTCACCCGAGAAAAGATTCGATTCG ATCAGCACTAGCGCTCTCAATGGCATGGGCCAAAATTTCTTCGACAAAAAGTACTTTGACAATTTTCCA GAAGGTGCTGTATCGAAGAGGTTCGACCCCATTTCTTCATCGGGGACCATAAGCGGATTACAACAGAA TTTCCTTAAGAAAAAAGACTCTGACTCCAGGTCGGCGGGCGGGATGGGAGGAGTCCGCCTGAGTTTTC CACGACAGACAGGAAGTGTGGACGGCATGACGTCATCTAAACGCCAGTTTGACTCCATCTCTGGGGGC AGCGGCATCGGCGGTCTGGGTCAGAACTTCCTGAACAGGCGACAGTTTGATTCGATAAACGAGGGGG ATATAGGCGGGATGGAGCAAAACTTTCTCAGTGGGAAACGATTCGAT ACCATTTCCAGTGGTAAAATG GCAGGATTCAATCAGAATTTTCTTGGCAAGCGACAGTTTGATTCCATTTCTACTGGGGAGATGAGCGG GATGGACCAAAACTTTCTGGGAAAACGCGGCTTCGATTCCATCAACAGCGACTCTCGCATTTCCGGAA TGGGGCAAAAGTATTTGGTTCGTCGTAGCTCCAGCCTCGAACCTCTACCCTCAGCAGAAGTGAGCGAG CAGCCCCATGACGTCTACAAACGCAGCCCAG AGGATGTGACGTCTCTGGAAGCCAGTCACGACTCGGC TGTTGTCACCGGCGATACTTCAGAACCGATCGGACCTGAGCATCGTTTTATAAAACGCAGCTTCGATTC TATTTCCAGATTTTCAGGAGGTATGGCGGGCATGAGTCAGAACTATCTGTCTCGCAGAGCGTCTAACA CTCGTGCTCTGGACTCGAACTCCCAACCGTCGTCGTCATCAGCAAGCTACACTAGCCGTGGCACTAAC GACGTGACTTCACT CGCCGGAGTAGACAACGATCACGAACTCTGGCGCATCCTGGACAAGAAAAACTT GGACTCAATCTCCAGATTGGGCGCTTATTTCCGGCTTTCATCAGAACTTCTTAGGCTTGCCGTCTGCCA AACGCGCGGGCCAGGGCGCGCGAGACTCCAGCCAACATCACCCCGCCACCGATCCTCTCTTCAATCGA TGGGATTACCAAAGATCGGGGTCCGCAGATTTCTCAGAGATTAA Protein MNQNYLSKRPLDSISGSKFHGMNQNYLSKRPLDSISSGFNGMRQNFLSKKSFDSISGSGLHGMGQNFLDG KRTFDSIGGGDLQGMGQNFLTSKRGFDPISTGSLHGMRQNFFGHKRSFDSISRNRLGNLRQNFLSKRADE SADEWSVNDASPEKRFDSISTSALNGMGQNFFDKKYFDNFPEGAVSKRFDPISSSGTISGLQQNFLKKKD SDSRSAGGMGGVRLSFPRQTGSVDGMTSSKRQFDSISGGSGIGGLGQNFLNRRQF DSINEGDIGGMEQNF LSGKRFDTISSGKMAGFNQNFLGKRQFDSISTGEMSGMDQNFLGKRGFDSINSDSRISGMGQKYLVRRSS SLEPLPSAEVSEQPHDVYKRSPEDVTSLEASHDSAVVTGDTSEPIGPEHRFIKRSFDSISRFSGGMAGMS QNYLSRRASNTRALDSNSQPSSSSASYTSRGTNDVTSLAGVDNDHELWRILDKKNLDSISRLGAYFRLSS ELLRLAVCQTRGPGRARLQPTSPRHRS SLQSMGLPKIGVRRFLRD Pleurin Gene Bank Accession Number: 56200040 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGCAGTCTCACGTGAGCAGCAGCCTACTGGCCCT GTGCGTCCTGACGGGCAGCGCATGCGCAATGTT CTACACCAAAGGCAGCGACAGCGACTACCCCCGGATCGGGCGTCGGAGCTTCTACACAACCGGAAAC GGAAACCATTACCCGCGAATAGGACGACGGGATTCCCCTGGCGCAGCTGCTGGCACGGACATTCTTTA CCCGAACACGGCTTCAGGGTTTTCCGATTTCTCGGCAGGCGCTCCGGCGGTGAAGAGAGGGATTTTCA CGCAGTCGGCGTACGGTTCT TACCCGAGGGTAGGACGAAGGAGTCAGGCGGGAGATGATGGTTCCCA GGAAGGTCTTCAGACTGCGAGATCTAAGATTTTCCAGAAGATTACGGGGCTGGACGGGCCGGTGGAA GAAGAGCTCGGGGATGGATCGGCCGAGAGATTGGAAGCTGACAACCAGATTGGGCTTGAGCTCATGT TCCTCGCCTTTGACCTAGATGGTGATCAAGCTTTGTCAAAATCAGAGTTCCAGTCCGGGATGGAAAAG TTCCGAC GACACAACCCACTTTGCTAA Protein: MQSHVSSSLLALCVLTGSACAMFYTKGSDSDYPRIGRRSFYTTGNGNHYPRIGRRDSPGAAAGTDILYPN TASGFSDFSAGAPAVKRGIFTQSAYGSYPRVGRRSQAGDDGSQEGLQTARSKIFQKITGLDGPVEEELGD GSAERLEADNQIGLELMFLAFDLDGDQALSKSEFQSGMEKFRRHNPLC Preprogonadotropin releasing hormo ne like protein

PAGE 110

110 Gene Bank Accession Number: 158906123 Reference: (Zhang et al., 2008) Prothoracicostatic peptide 2 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CAAAT GTCCGTTTGGGGTAAAAGAGATGAAGATGGCACTTAGATAAGCGGTGGAAACAAATGTCTGTT TGGGGAAAGAGGGATGGAGATGGCAACTTAGATAAACGGTGGAAGCAAATGTCCGTTTGGGGGAAGA GAGACGAGGATGGCAACTTAGATAAACGGTGGAAACAAATGTCTGTTTGGGGAAAGAGAGACGATGA CATAGGGGACTTAGATAAAAGATGGAAACAAATGTCGGTTTGGGGAAAACGCAATGGGG CTGATGTT GGTGGTGATAATTTGGACAAGAGGTGGAAACAAATGTCTGTGTGGGGCAAGAGGTTACAAGACCTGG ATTCAAACAAAAAGTGGGCTCAAATGTCAAGCTGGGGGAAGCGGGGGTTTGTGGGAGATGTAAGCGA CGTCGATAGCTTAGTTAACAAGAGATGGAAGCAGATAGCCGCATGGGGCAAACGACTGAGCCAGGAC GACGATCTCCAGAAAAAGTGGAAACAAATGCCTGTTTGGGGGAAGAG AGGCGATACCAACGACGTCG ACGATAAGAGATGGCGAGAAATGGCTGTTTGGGGGAAGAGAAACTCGGAAAATAACGACAACCGAA ACATCGTAAACGAAGGGAGCGACGAAAACACTGCGGACAAGCGATGGAAACAGATGTCAGTATGGG GGAAAAGGGATGCCGGGACGGCTCCAGCCGCCGTCATGGAAGCACCATCTCCAGTGTCATCATCATCA TCATCCTCATCATCATCACCAACATCTTCCTTATCG TCCCCCTCATTATCAGCCTCAGCCGCAGAAGCA GCAGCTGCTGCAGGGGCGCCGCGCGTGGACAAGAGGTGGCCCGATAGCTCCGTCTGGGGCAAGAGAA CGTACAGGCACATCAGGAACTGGCGAGACATGGACATGTGGGGCAAGAGGCCCAGCTGGTCGAGGAC AGGCTTCACCAGCTGGGGAAAACGCTCTGGGGCCGCTGTGGACCTGGCTCAGTTCAAAGACTATCTCC TAAACAGCTCCCAGCAACAGC AACTGCAACAGCAGCAGCAGCCGTTGATAGGTGAGTCGTCAACATA ATCAATAAAAATGAAAAATAAAATGACGAGGATGAATAAG Putative pheromone 2+Gene Bank Accession Number: n/a Reference: n/a Partial Clone: TGGCAATGGTTTAAAAGGCAGTTAAATCGCATCTTTTGGTTGAGACATATTTATTAGTTTTCTGATTCA TGTAATATCAGTA AAACTATATAGAGAGAGAGGATGGGAAAAGAAAGAGGGAGAGAGAGAGGGGGG GGGGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG AGAGAGAGAAGTAGAGACAGAGAGACGGAAAGGGGGAGATATATGGAGAGAAAGGGGGATAGTGA AATTGTCACTATTGGAATTAGGTTCTTTATACTATTGTCAAACGCATTGTACGTTCTTTTGTGGAAATGC GTTA ATTATATTAAAAGTAAGGTATCATTTCTAAATAGCGATTGTCAATCCTGAGCATGCATATATTAT TTTACCTATCTCAACAGAGAAGTTCTCGACGACCGCCTTCCCCACCACCATCACCCCACGCCGCGTCAA ACCAGTGACCACAGACCTCCCCAGCTCCCCACCGGAAGCGACCACCAACATCTGCGACTACCTGTGCT CCGTGCAGCTGGGTGGTGATGCCTGCCAGTGCAGTAACCCCAGTCTGCCCGGGA AGAAGTAGTCGGCG GTTCGAACCCCGCCCGAAACGATGCTCTCACCACAGAATCGTCGGCGCACCTAGCTAGACATCTAGAA CGTACCGCTGTTTGAACGACTTGTCCCGACACAAGGGGCGCTGTCTGTTCCAAAGACGTAGAGCTGAG ACTTCCGGTTGTTGTTTTATGTCGTGAATGTATCCAATACAAGACGGAGCATCTTTCGCAAACGTTTGA CTTTTGTTTGAGGTCTCTGTTTTGAATCCAATGGCTT TTTAAAGACTATATACAAAGTCTTCACATAATT TATTCTTCCATACGTTTTGCTTTGTCGGTTGTTGATACTTTATACGGCCTGTCCGGGTGCGTTTTGGAAG TGGCTGTTTTTTGTTGTTGTTTTTTTTGGTGCTTATTTTCCCTCTTCATAAAAGAGAGTAAGGGCAACTA TTTTGTTGCACTTATTGTTGTGTTTATTGTTTCATTTTGCTGGGGATGTTTTCCTCTTTTGGGAAATTGGG AACAACTGTGCT TTCGCACCTTCCTGCTTTTAATTTCATTTCTTTTCATTTCCTTCATTGATGTTGGTTTT AAATAAAAGCACATCCAATATCGCCCGGTTTGAGCTCATCTTTTGTCTTACAGATTTACCTAAGTTTTG AACTCTCTTTTTTTATAGGTCAATACTTGGTCAGTCTGTGCCAGCGTATATAAATCTTTGACAACCTATA CCAATACGTATCACTATTAACCCGTCTACCCTAAAGCTACACAGTTTTATCTGACTTT TGTGACCGTTTT GAGTTACTAAGACCAAGAGGCAGAGACAAATGTTCTATCAGCTCTTTTGAAATCGTAGACAATTCGAG CCGTATCATGATATTCTAAGAACTGCACGTGAAAAAGCTTGACGTGTTGTGAACTCAAACTTTCAGCG

PAGE 111

111 TCAAATTCTCAAATTAGGCCCTGAGCAGATAGAACTTGTGATTCTGGACAATAATCGTCCACTTATTTT CATAAGCTTGGGCTTTCCCCCTCGTTT R3 14 pept ide 2 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: ATTAGGAAGGGGGGCCGATCGCAGCGGCCATTTACCCCACTATAGGGGACCAAAAGGGGGGTCCCCG CCCCCCCCCCTAGGTTGGACGGATATGTTTAACTTTGAATTGAATTTCTTCGGGGCGGAGGCGGTCGGG GTTTAAATGCAAGAAACCCATAAGGCTTTCCAGGTTATTCCCCCTTCGGTTTTGGGATTTCCCTCCCCC CTGAGGATTATAATTTGGCAGCCCCAGTGAACTTAACTGGTCGGCGGGGGCTTAAAACATCCGCAATT AAAATGAATGGAAGGGCCCTAAAATTGCCCTCACCTAGTTTAAACAACCGTATGCCCTGCTCACCGTC GAACGCTCCGTATACCGGACTTTAGCATGGAGATTATCAGGGGAGAAAGGGGTGGTGTTTTGGGGTTT TCC TCTCTACAGCTTTTTTGACGCACCGAGCCCCCTCACCGATGGGGGGGCTCCAAGTTAGTTTGGCCG TTTTCCTCCGCCGCCAACCTTTGGATTCATTCCCGCTTGGGTCCTCTCGGTTCAACCAGAACCAAAGAC TGGCTTTTTTTGCTGTTTCCCGAAGGGAGGTTGTGAGTTTGTTTCTTTTTTTTGCGGGAAATCCTGCCCC TTTCATCCCCTTTCGGGGGCTTGGTGGTACAAAACCTCCGCATCCCACCCC TAAATTAATTGTTTTTAGT AGTTTTATAGGGGTATACAATTCATTTAATTTTTCTGGGGACGTTTTTTTTCCCTTTGACAGTTTTGGCA GATTTTAAAAACAAACGGGAAGCAGAAGAACCCTCCGCCTTCATGACCCCCCTTTCCAGACAAATTGG GCAAAAGCACCTTTGGGGTGCCGTCAACCCCGACCGTAAACCCGGCCCTGGGTCCGGCCGTCACGGCC GCTTCCTCATTTGTAACCGAAACCGATACGG GGGGGGGCATTTTTCCGGGGC Schitosomin like Gene Bank Accession Number: 56200042 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGATGAGACAACTGTTCGTCCT GGTCATCGTGGCCGGAGCCGTGCTCGCCGACAACTACTGGTGCCC CAAGGCCGGCGAGGCTTTCGAGTGCTTCGAGTCGTCCCCCACAGAAAGGTTTTGCCTGACCAACGGAC GCGAGACCGCTGTCATCTGCTCCAAGTGCAGGAAGAAGTTCGACTTCTGCCGTAACGACTTCGTCAAA TCCAAGAGAGCCCACACTGACTGTGGAGCCGGTTGGGAAAGCACTCCCTGCACCCACGACAACTCACA CGTGCCCGCTGTCTTCCCCGGGAAACTTTAA Protein: MMRQLFVLVIVAGAVLADNYWCPKAGEAFECFESSPTERFCLTNGRETAVICSKCRKKFDFCRNDFVKSK RAHTDCGAGWESTPCTHDNSHVPAVFPGKL Second (short fragments obtained from MS data) Gene Bank Accession Number: n/a Reference: n/a Partial Clone: NTGGGATT CGCAGTCGGTACTTTTTTTTTTTTTAGCCTCTTCTTTTTAGCCATTCCTAAAGAGTGAGAGT TTTTTTATAATTTAATTTTTTATGAACTTCAAAAGGTGGAAGGACAAGATTGAATGATTTAGTGCAGAG GACACTCAAAAAAAAAAAAAAATTCATCTCTGCTAATGAGCGATTTCTGCGAGACTAAAATTTGTTTT ATTGTGGGGTTGATTTGTAGCTCTACGTTCTGGGATTGTACGCCTCAAGAGTATAAT CACCCCTGGCTT GAATTTAAGCAGATCTCACTACGACTACTGCCAGTGTGGGTGGTGAATTGTTTCTGTGTCCAGAAAAT GTTTCCTTCACCACAAACATTTCTCAGTTCTGATCCGTTTATACTTCTTTTATGATTAATTCATTTCCTTT GTGCCGGTATCTCTCGTGATTCTCTAAAGAAAGAAAATTCACAAACTATTATTACAGTGTTCCTTTCAC CTGATGTATCCCGACAAAGGTACCTGTGGTTAGGCT ACCGGACCCGTGGTCGTGAATTGTGGGTCCCG GGGTTCGAGCCTCAACTCGGTCCGATGTTGCGTCCTTGGGGAAAGGCGTTTGAAGTTTCAAAAATGTC

PAGE 112

112 CTATTTGGCTTAGGCCTTTCGGAGAAGGACGTTAAACTAGAAAGCTCCTGCTTCTATCGACACTGTTAG GTTATAATTCAAGAGGCAGGAG Secretin ( a peptide hormone primary effect is to regulate the pH of the duodenal contents via the control of gastric acid secretion and buffering with bicarbonate ) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GATCAGTCAGTTTTCTGTGGCATAGTCT CTCAATTTGGACAGCTCATTTGTTGTACCGTCTTAAGTAAA ATAAAAACGAAAAAGTAA SFY1 like peptide* Gene Bank Accession Number: n/a Reference: n/a Partial Clone: GCGCTGACAGGATTGCTCTGCGCTTCCTGGCGGGAACAGTACAGTTCGCGTGGGGCGTGGTCAGTGGT ACAGCACGTGCTCCCCCGTCAGTGAGCTGGGCCAGTGTGTGCGAGG GGAAGCGTCCTTGGCAAGTGAG ATCACGGTCAGAGGTCGAGGCTTCCAACGGCGTGACACTAAGATCCTGTGTCAGTGCCCCATAGGGAA CGACCTCGAACAGAACCCCGTGGAAGTACGCAGAGAGGAAGACTCGCCGCGCATGACAATCATTGTC TTTTGTGGTGAGGATTCTCAAGCGACAGGCGAGATGAAAAAGAGAGGACGTGGCATGTCCGCGTTTTA CAAAAGAAACCATGGTCTCCAAAAGGGCTTC TTCTCTTCCTACAAACGTTACCCCAGCATCAGCGCGT TTTATAAGAGAAGCCCGGTATTGGGGACCGTGTTTGCGCGTCCCGACCGAAAGCTGCACTCTTTCTAC AAACGAAGTGTGGCGTCTGACGTCACTTCTGAAGAAGAGGACCAAGACGTAACGTCAACTAGCCGCTT TGCGCGAGATCTGAGTTCTTTCTACAAGAGAGGATTAGGAGTTGCATCACTGAGCTCGTTCTATAAGA GAGATGACGAAACAG CGGAGGAGGAAGATGGGGATGAGCCAGACAAACGAGGGGGCATGAGCGCTT TTTATAAGCGAGGTTTAAACGGCGTCAGCTCGTTTTATAAGAGAGGATTATCTCGTCAGAGCTCGTTTT ACAAGAGATTTCCTCAGTCTGTCAGCGCGTTCTACAAGCGAGAATTTGGTAACCCGGATAGCAGCTCT GCTCTATTAGATAATAGTTTGGATGGTCAGGAAAAGAGAGACTTTGGATCTGGGTTGAGTTCTTTTTA C AAAAAAAGCGGCCCGTTCAGCTTTTACAAACGCGGCATGAGCTCACTAAGCTCGTTCTACAAAAAGCG TGGACTTGACTCTGGCTTGAGTGCTTTCTACAAAAGAGTTCCTGGCTCGGCTTTGTCTTCGTTTTACAA ACGATCAGGCCTTAGCTCCTTCTACAAGCGACCTCACACGTCAGGTGGCATGTCCAGCTTTTACAAAC GAGCGAGAGGGTTATCCAGCTTCTACAAGAGGGATGATGATTCTGAAGAC ACAGCCATAGATTCTAAT CACGCCCCCATGATGTCCTCAGAGGATTCATATCACCGTAGC SFY3 like peptide* Gene Bank Accession Number: n/a Reference: n/a Partial Clone: LRGSYHRSKRSIKTDDHEATVQRRSTDALSSFYKRGLQGVSSFYKRASGQGVSSFYKRSPFGEMSNFYKRS PFNGVSNFYKRGAESYSSFYKRMLPGSQQSRFYKRTPNSLSS FYKRSPNNDVSSFYKRSPYDDVSSFYKRSP YDDVSSFYKRSPYDDVSSFYKRSRQSSFYKRFPQSVSAFYKREFGDPDSSSALLDNSLDGQEKRDFGSGLSS FYKKKRPVQLLQPRDELTKFVLQKSVDL Somatotropin I precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGGGA TTTTATAGATTTTTCTCTGCTGAAACTTTTCTTTCTACCAGAGGGCAGCTCCTTTTCAGCCTTTG CTCACCAAAACTGCTGCTATGTGGCCTTCTTCTATGAGATAGGGCACGACTGATGTGTAGAACACGGC

PAGE 113

113 CATTGTGGGTAACTTACCATAAATGGGAAGACTTCCCGCCCCTGGCTAATCTCTTGAACATGGCCGCC GATGCTGCTCTTTTTTTTTTCTTACAGCTAAATATTCTATTCATGTTAGATTTTT TCAGTCCTAAATATCA GGTTCACATAAAGTCATAAATTAACATTGGCCTCTTAGGTAAGTGTTGAAGTTATATAACGTGGGCATT AGAATTCGTGGGGATAGCCATCATCATCATTATAATCATCATAATTTTTCTGGTGGTCATATTAGGGTG GGGCTGTGTGCCTGCACGTGGTGGGGAGAGCAGTTGTCACATTCTATGGCCCTATTTATTGGCATCGTT CTCTGTGAACATTTGGTTGACTGGATTTGATTT ATTTGAAAACCATGAAGCATCGCT Somatotropin precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: ATAATGTTTCATTGAGATTGCCGGTTATCCTTAGATTATCCGGATTTTGCCCACCTCTAGTGCTTTGCCG CGCACGCCCCCCCCAAAAAATGCAAGCTTGATTTCCATGTTG GGAGATTTTTTTTTTTACAGAGAAGCA GAGTCCTTATGCTGGGACGTTGTGTTTCTCTCAGCCCAGGTTGGTCGTTACAGGCAGGATGAAGCCCGC CTCCTAAGTGATCTAAACTGTGTCCATAGGACCACAAAAACAACTTCATTTCATTAAACAAAAAAAAA AAAAAGTACCGACTGCG Suppressor of lurcher protein 1 ( Accessory protein required for glutamate gated currents. May participate in the gating of non NMDA (N methyl D aspartate) ionotropic glutamate receptors such as glr 1) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Prediction: GGTAGTAGGCATGGAATCCCGTCGAGTCGTAGA TGTCGTTAGATTTGAATATCACTCGGAAAAAGGAC CCATCTGACCAGATCTCTCGTTTCTCCTTTGATGGAGTGCCACATAATCTGGTCATCTTGCGGTCTTCGG TCTCAGCAAAGTTTGAGAAGGACACGTAGTCACTGTTGGTGCTTTCCTCACACCTAGGATTATTCCATC CACATCAAAGTCAACAAACTGGATGTGCACCCGCTCCTTGCCCCGTCCGTAGAACAAATAATGGCACT CTGTGTTACGGGGA TACTGCCCGTTGTAGTTTGGTGATGTGATCTCACCGGTGGACTGAACCGTACTCT GGTAGTTGAACACGCACGTTCCCCTGTTGTCTTGTTGCCCCTCCAGTATTCCAAAGTTTGTAACGAAAC TA Theromacin+Gene Bank Accession Number: 94471620 Reference: Kohn, A.B., Moroz L.L (2006). Unpublished. CDS: ATGGACAAGAAGGCA GCAAATGGAGGAAAAGAGAAGGGCCCCTTGGAAGCCTGCTGGGACGAGTGG AGCAGATGTACAGGTTGGAGCTCTGCTGGCACTGGAGTTCTTTGGAAATCTTGTGATGACCAGTGCAA AAAGCTGGGGAAAAGCGGTGGAGAATGTGTGCTCACTCCCTCTACTTGCCCATTTACACGTACCGACA AGGCTTACCAATGCCAGTGCAAGAAGTAA Protein: MDKKAANGGKEKGPLEACWDEWSRCTGWSS AGTGVLWKSCDDQCKKLGKSGGECVLTPSTCPFTRTDK AYQCQCKK Third (short fragments obtained from MS data) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned

PAGE 114

114 Prediction: GGCTCGACAAGTAGACCTGCTTCAATCCCAGAGGTGAATACTGAGACGAATAAGCATGCAGCTCTTAA TACTTCTTGCTGCCGTTGTGGGTGTATCCCTGGCCGCCCCAAGCCCTGATGTTTGCTCCAACGTCAACG CTACGGCTATTAAGTGTATGACAGACAACGGAATTCCTCTTCAAGATGTGCTCTCTATGCAGAAGGCT GCTTTGTCTGGGAACATTGCAGCAATGAACCTGACCCGCTTCTGTGGCGACAAAAAAGATGGCTACCT GAAAGGTGTGGAGTGTGTAATGAACTCCGTGGCTGTGTGTATCCCATCGT TCAAACAAACATTAGGAG ACTTGGACTTCAAAAAAATGCTGGATGTGCAATGTACCGACAAAAACATCAAGTACAACTGCCTGGGC CTGATGCAACTGCCTGCTAATAAAGCTGTTGTCCAGACCTGTGTCCAGAAAGCTTTTGAGAATCTGAA GGATTTGTCTTCAGACAAGAGAGTTTGTGAGGCGACCAAGAGCGCCTACGTGTGCGAGGATGCCATTG TGAGGTCATGTGATGTGCACACCGCTGATGTGTA CAAACAGTTCATTGACCCTTTCATGAAGAGGGAC TGCGATAACTTTGTCCTCGGGTGAATGTGACAAAGTGTCCAGGACAACCGGGGTATCAAAGGATAAAA AAGAAACTCGCAAC Whitnin precursor (SPTR) Gene Bank Accession Number: 56200044 + Reference: Moroz, L. L., Edwards, J. R. et al. (2006). Neuronal transcriptome of Aplysia : neuronal compartments and circuitry. Cell 127(7), 145367. CDS: ATGGAACTCCAAGCCTGTAACATTTTTGCCCTTTTCGTTGTGGTTGTGACGCTCTCAGTTGCCAGCAGT CTCCCTGCCAGCCGGACGGACGATGTGCTGCAGGAGGCCTCGGGTCTGGCTTTGAACAAACGGCCAAA GTACATGGACACCCGACGGGACCTTGACGTCTTTAAAG ATCTGGTGCTCATCAGCATTCAAGAGCTTG TGGACGAAAACCGCCTGAACCCCGCCTTGTTGCCAGAGGAAGATGCCCCCAAGCCCGTTGAGAAGCG AATGCGCTACATGGGAATCTGCATGAAGAAACAGTACAACAACTTCATCCCCTTCCCCTGCCTCAGGA GTGGTCGCTAA Protein: MELQACNIFALFVVVVTLSVASSLPASRTDDVLQEASGLALNKRPKYMDTRRDLDVFKDLVLISIQELVD ENRLNPALLPEEDAPKPVEKRMRYMGICMKKQYNNFIPFPCLRSGR Signaling Molecules Wnt 2 protein precursor Gene Bank Accession Number: 46981372 + Reference: Brown, B., Kohn, A.B et al. (2007). Unpublished. CDS: ATGAGATCATCACCAGGCTATCCACTCTTCATTTTGGAAGTGGTCCTACTCCTAGGGCT TGTTGCTAAG GAAGTTTCTGCATCTTGGTGGTTTATCAGCCAGCTAAACATCCATGCCCTGGGCCCTGGTGTGGTGTGT GACAACATCCCCGGTCTCGTGGGCAGACAGCGCAGGCTATGTCGTCATCACCCTGACGTCATGGTGCC GCTGAGCGAAGGCGTACGCCTCGGAGTGGAAGAGTGCCAGTTCCATTTCCGGAACCAGAGGTGGAAC TGTAGCACGCTGGAGAGGGATACCTCCTTGTTTGGGAAGGTC ATGCTAAGAGGCAGTCGCGAAGCTGC CTTTGTGTACAGCATCTCTTCCGCTGGCGTGGTCCACGCCATCACCCGTTCTTGCAGTCGTGGCCAGCT CATGCACTGCGCCTGCGACCCCACCAAAAGAGGATCTGGCAGAGACAAGGGAGGTGCCTTTGACTGG GGAGGATGTTCAGATAATGTTCGCTACGGCAGTACTTTCTCCAGGATGTTCATCGACGCTAAGGAACG CAAGCAGAGAGACGGGAGGGCTATGA TGAACTTACACAACAACAGAGCCGGCCGAAGGGCGGTGAA GAAGTTCCGCAAACTGGAGTGCAAGTGTCACGGTGTGAGTGGATCCTGTACCATCCGCACGTGCTGGC TGGCCATGCAGGAGTTCCGTCTGGTGGGGCAGCACCTCAAAACACGCTACAACGGGGCCACGCAGGT CATGATGAAGCAAAACGGCGCAAGTCTCATTGTGGCAGATAAGAACCACAAGCGTCCTACGCGCTCTG ACCTCGTCTACTT AGAATCTTCACCCGACTACTGCGTTGAGAATTCGGAAGTTGGATCGCTCGGGACTA CAGGGCGCACGTGCAACAAATCTTCTATGGGCACCGACGGTTGTGACATCATGTGTTGTGGGCGGGGC TACCACACACGCGTGGTGAAGCAACATTACAAGTGTGAGTGCAAGTTCCACTGGTGCTGCTTTGTACA CTGCAAGGATTGTCAAAGGTGGGTGGAGCTACACACCTGCAAAGGCCCCGCCCCTCAGGGTCTC ATTG GCTCCTGA

PAGE 115

115 Protein: MRSSPGYPLFILEVVLLLGLVAKEVSASWWFISQLNIHALGPGVVCDNIPGLVGRQRRLCRHHPDVMVPL SEGVRLGVEECQFHFRNQRWNCSTLERDTSLFGKVMLRGSREAAFVYSISSAGVVHAITRSCSRGQLMHC ACDPTKRGSGRDKGGAFDWGGCSDNVRYGSTFSRMFIDAKERKQRDGRAMMNLHNNRAGRRAVKKFRK LECKCHGVSGSCTIRTCWLAMQEF RLVGQHLKTRYNGATQVMMKQNGASLIVADKNHKRPTRSDLVYLE SSPDYCVENSEVGSLGTTGRTCNKSSMGTDGCDIMCCGRGYHTRVVKQHYKCECKFHWCCFVHCKDCQR WVELHTCKGPAPQGLIGS Frizzled related protein 2 Gene Bank Accession Number: 46981374 + Reference: Brown, B., Kohn, A.B. et al. (2007). Unpublis hed. CDS: ATGAGCGGGCGAATACAGCAGCCCAAATGCGTGGACATCCCCAGTAATCTAACTCTTTGTCAAGGAAT CGGATACGAGACGATGAGGCTTCCCAACCTGCTCGATCACGATTCTCTGAAAGAAGTGACACAACAAG CAGGTTCCTGGGTGCCTTTGACAAGGATCGCCTGCCACCCAGACACAAAGGTCTTCCTCTGTTCCCTGT TCTCTCCAGTCTGCTTAGACAGCCTAGATAGGTTGATCTAT CCGTGTAGGTCGCTGTGCCAGAACGTGA GGGCCTCGTGCGAGAGTCGGATGACTATACATGGCTTTGAGTGGCCAGCCATGCTGGAGTGTGAGAAG TTCCCACTCGACAACGACATGTGTATAATGCCCATACACGACATCAAACCTGACAACAACTGCACCGC TTGCAAGCACCCACAGACACACGAGGCTCTTATTGACCACTTTTGCCGGTCAGATGTTGCTATGCGGGT CACTTTGAAGGATCGCCGCGTTC TGGGCACCGACTTGGAGCTTGTCCTTCGAAAACGTCGCAAACTGT ACAAGTTCGAGAACATGGAGAAGAAAGAGGCGAAGAACCTGCCAGTCGTCATCGCTGGAGGTACTGC GTGCACATGTGAGACCATCAACGACACTCAAGGGAAGTACCTGCTCATGGGCAACAAACGCAACGGG AAAATCGTCATCAACTTCGGCATGGCTTGGAAGCGCAAGGATAAGGAGTTCCGTAAAGGTCTCCGCGC CATCAGAAAGGGCGACTGCTAA Protein: MSGRIQQPKCVDIPSNLTLCQGIGYETMRLPNLLDHDSLKEVTQQAGSWVPLTRIACHPDTKVFLCSLFS PVCLDSLDRLIYPCRSLCQNVRASCESRMTIHGFEWPAMLECEKFPLDNDMCIMPIHDIKPDNNCTACKH PQTHEALIDHFCRSDVAMRVTLKDRRVLGTDLELVLRKRRKLYKFENMEKKEAKNLPVVIAGGTACTCET INDTQGKYLLMGNKRNGKIV INFGMAWKRKDKEFRKGLRAIRKGDC Hedgehog Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGCTGTGAGGCAGTGAAATTGGGGACTGGTGCTGTCAGAGATGCTGGAGGGTGTCTGAAATATGATGA GGTCTACATGTTTGGCCACAAAGACGAGGTTGCCGTCTCCGAGTTTGTGATGCTGTT GACAGAGACAA AGTCTGTCTGTGTGAGTAGTGAACACAGCGTGTACTGCTCACGGGCTGGCCAGGAAATGTGTGTTGCT GCTGGTAATGTAAGAGTTGGGGATGAGCTTCTTGTACTGGACGCTTCGTCTGCACGTCTGGTGGCCGA GCCTGTGGTGAGTATTGGGTTTGAGAAGAAGCAAGGATTGTTTGCTCCGTTTACCCTGAGCGGCTCTAT CGTGGTGGACGGGACTCTGATGTCATGCTACGTGAACTCC CCGGCCCCTGCCACCGCCCATGCTCTGCT GTGGCCAGTGCGACGACTGTACAAGTTGTCTCCCTGGATGCTGGAGTTTATCAGCGGCTCTAGCAAAC AGGACACTATTCCTTGGTGGGCTAGGACCGCTCTCAGGTTTCTGTAGAAGAGAACTGTTTGACCCCCAT GAACCTGGTCGTGCACATTTGAATCAGTATTGGAAGTCTTTCAGAATCTTGTATGTGACCTGTAATTGC ATAGTTTCTGACATGTTGCAG ATCTGTATCTGTGTGTTGAAGCCCAAAATGCATGACTAGGGTCAACCC TGAACTAGGCCCTACCTGACCTGTCTTCCTTGAATTATCCTATCCTTACCATATTAAGTG Chordinlike protein Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned

PAGE 116

116 Predicted: GAGAGAAATACAATCCTTCTCAANTCGCAGG AGACGGGAGAAACAGATTTTGTTGTGTGATAACGTCA GATTATCTCCTCGAGGGAAACGCGCGCTGAGAAATAGTAAATAGTGAGCCGAGGAATTTATCTATCGT TTTGTGCTTCTTCGCGCAGTCTTGTAATTGAAGAGTGGGCCTCCTTCCAACTTTTCTTCCCTTCTTCTGTT TGACATCACTGACCGCAAGTCGAGTGTTCTCTGGTGTTGTGTGTTTCTTATAATTGTGGAACTACTGCA GAGGATTTGTT GTATGGACACTTCGGATTTTTCAGACGTTTGGAAATAATGCGCGAGTGTTTTGACTGA TTTCTGTACAGTACCTGGAAAATAAACTACGCACGTTCTGTGATTCTTACGGTACACTTGAAATTACTG ACAGACAGTTTTCCATTTCCTGACGCGACCCCGTTATAGGATGGGGCAACCGCCTACTCAAGCGGGCT GCTCTAGCAACTCGTGTGTCTATGAATGCGTCCGTGAGTCTGTTCGTGAGTCTGTACATCG TGGGTCAA GTCACATAAAGGTGGTCAACAGCTGAGGAGGTCTAGAGAGGACGAAACCAGAGACAGCTGCCAGCCG CCCATAACATGCAGAAGATGACTCAGATGCTGGTGTATAAAGTGCTCATCTTCGTCATGGTATATCGCT GCAGTGGAAAGCCTTCGGGAGGCCCGTGTGTTTTAGGCGGAGAGCAGTACAACGTTGGAGAGACGTG GCATCCTAAAGAGTTTTCTTCCACCGCCAACTCCTGTGTCCACTG CACGTGCCTAGAGGGCGGTCAGAT AAATTGTACCGGCGTGGACTGTCCTTCTCCAGAGTGCGAGGTGCCCAGATTTATCCACGGACAGTGTT GTCCTACATGTGATGTCCGGGGGGACGAGGCCGGAAGTCCCGAGACAGACATGCCCGGATGTGATTTC CATGGCGACCACTACGAGGACGGTGACATATTTCCGTCCAACAAGACGGCTTACAAACCCAAGCATGA CAACCAGTGTGTGTTGTGCGGGTGTTAT AGAGGTGAGGTCATCTGTCACCTGAAGACTTGTCTGCCCA GCCCCAGGTGTCGGCGGGTAGTCCGGGTGGATGACGACTGCTGCCTACAGTGTGAAGAGGAGTCCAG CATAGACGAATATTTCATGTCTATCGGGTCAGACAACATAAACCATACACTGGACGACGAAGACTGTC TGTCAGCGACAGGACGCCGTAAGAACGGCAGTACGTGGAAGCCAGTGGTGGGCGAGTACGGAGAGAT GCATTGTATCGTCT GCTCCTGTTTAGAAGGTCAGGTCGACTGCAAGCGTCTGACCTGCCCTGACGTCAC AACCTTGACCTGCGGACACCCAAGGCCACGACAGGACGGGTGCTGTAAGGAATGCCCTGAGAGCGAC AGACAGAGGGACAACAG Wnt 16 precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TTTT TTTCCCCCGTCTACTTAACATCATTTATGCAGCTGGTGACAGTAACGAAAGCACGACATTCCGCT TTTCGAAAGCATGGGCGTGTACCTACACAAAGCCACGATGGGTAGAAATAGCGCACTGAATGTCCATG CTGAAGTTCTCAGATTCTTGAACAGGACTCAGGAGTCCACAATCCTCTGGGATTTGATCACGCCTGTCA GAGTCCCAAATACTGCCAGAAGTGATTACACCGGTCAAATGACATGTGGTATTAATTGTTTTTTTAAAA GTAAACATGAAGGGACTTTCGTATCTAAAGTGAAGTACATTTTGGAAAAGTCCAACTTCATAGGAAAA GTTCCCAAAAATCATGACAGAGATTCCCAAAACAAAGGTGGAACCAGAATTTCTAAAATGAAGAAAA TAATTGTCTCTGAGGTGTACTCATGTGCAGTTTTCAGACTGGACCAAGAAGGCTGCACTCCGAAAGAG AAGGCTTCACCGAGTATTTAAATGGGAATAAGCCACAC TTTGGTGATCAGCTCGTCGAAAGAGCCAAA TTCCTCTTTGTCGGTGACTTTTAATTAAAATTTTGCATCTACATTTAATAGTGGGGTATTATTGGTATAG ATCTAGTATAAGCAGGGTGGTCCCATGCCCCTTTTTGGGTGACCACTGAATGCCCTTAATAATTTTCTT TTCAAAATCAGTACGTTGTCAAAAGTCCAGCATTTCTAGCAGAAAAAGGTCAGATTTTTGTGACACAT TCAACAAGGGCTGGTCATG TCATGCTGAGAATATTTCTACACTAAAACATGCAACGCCTGACAAATGA AATACAACACCACTAGTCTTTAATTTTTCAAACATAAAGACTTTGTAGAAGCAGCTCCGTCGGTACAA ACTGATTCACGTCAAGTGTTTAAGTATGCTCTTGCTAGGATTCATCATTTCCTATCACATCGTATTGCA AACTCCTGCTTTCGATTATACAATAAAAGCACAGGATGTTCACAAGGGCCACTGAAAAGTACTTCAGC CA AGAGAATTGGCGTTGATGGTAGGCTAATATATTGTTCTCACAGAGCAATACTGGCATCGCTCGAAA AATGGTTTCCTTCAGTGTAAAATAGTGTTACCAAGCTTATCCTCGGATACATTGTTTGAGAGGTTGTGA GGGAACGTTTCAATTTCGGCTTTAGAATGAGTTACAACTCCGTGGTCAGCAGTTTTCAAAGCTGTTGTT CGGGGACTTCATGCACCACCAATATCTTCCATAAATTCAATTGGACCTGCAT ACATTTAAGATCACGCA CAGCTCTCCTGAGTTAAATGATCGACCATCGGTGATCCATGCCAGACTCAGTGAAAGGTTTCCTCAAG AAGAGACAGACCGCATGCGCTTTAAAACTTTTGTCGGAAACTCGGCGGCGCAAGGTGCTCCATTATGT AAATGACACTTCACCACATGTCCTGTCACGGTATACTTCTAATCACAAGTGCACACTCTTAAAGTCAGG AAGGAATTCAGTCGCAGTGACAGAATTCCTTCTT CTCGGACAACGAGTTTCAATGTGCACGCCACGCC ATTTCTTGTTTCTCTTGGATTATAATATATACACAAAATCCAACACTTTTCGATTGACAGTGACGCAGG TGCTGTCACTTGCAGGTATGAATGTCAACCAAAGTTTCACAAGTCTTGCACTCAACGTAACAACACCA

PAGE 117

117 ATGGAACTTGCACCGGCATCTTTCTACGTGTCTGACGACCTGCGTGTTATAGCCACGCCCACAACACA GCAAGTCGCAGCTCTCT GCCCCGTTCGCCGTCCGGTTACACCGGCGTCCCGTCGTACCCAGAATCCCTC TCTTCGGGTTGTTCCGGCAGTAGTTCGCTCGAGCC Wnt inhibitory factor 1 precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TCATCACAGGAGTCCTCTGCTGCAGATTGCCCGTGAAGA GACCAAACATCGTAGTCAAGCCCCCAGGG CGTCTTCAGGTGTACAGAGACTTCATCTTCCTGCTGACCTGCCAGGCTTCCGGTATGCTCCCACCGCGT CTGCTCTGGTACAAAGATGGCCGCCGTCTCGCCAGTGGGAATTCCCGCATCTCTGTGCTCTCCTCGGGA GACCTGCTAGTGACGCTGGCCGTCGCTCGGACACCGGTCTGTACACCTGTGAAGCCATTAACGAGGAG GGCATTGATACAGCCAGCTCC TACGTCAGCGTAGCAGAGTACACGTCTGGGTGTGCAGACGACAGCAC TGACGGTCTCCACATGCACAGTGACATCCAGGCGTGTGCGGGTGAGTGGAAGTGGTCACGTGAAGTCC GCCAAATCACTATGTGCTCGCGGCTGGAGGGTGTGTAATCCTCGTGACCAACGGAGCCTCAAAGAGAT CACGTCCTTCGAGATGTTCGACCTGTCTGGCTGCTACGCTTACAATGCCGCCAGTCGGAAGAACAAGT GTAAG AGGTGCAAAAATAGCAAAATGTCCGGCATCGGGCGGGACTGCGGTCACGTGAGCTACTCCCA CACTTCCTGTCTGGCTCATGGTCGCATCGACGTCTTTGCGACCAACCAGACATCGTCGTGCCAGTACAC GCCAGGGCTGACCTCAGGCGTTCTCTGCTGCAAGAAACCCAAGAAGAGAAAAGGGGAGAAGTGTGTA CCGCGGTGTGAGCACAAGGGCGTGTGCATCTCACACAACCGCTGTCGATGTGCATCAGGTTACAAAGG CGCCCGCTGCCAACTGCCGGTGTGCTCACCCGGATGTGGCTCCAAGGGTCAGTGCATCCGGCCCAACA AGTGTCGCTGTAGTGCCGGCTACACCGGCCGCACGTGCAGGCGCAAGACCAAGCCCTGCAAGAGCAC CTGTCTCAACGGGGGGCGCTGCCGCAGGGGCAAGTGTAAATGTCCAGGGAGCTTCTGGGGGAAGGCT TGCCAGTACCCACTCCAGCACGTTTTGCTGACGCGACTCAACAG AACAGAGTAAAGATCACAATCAAC TCCCGCTGATCAGCATCACCGGAGCTCTTGCGTTCTCTGCTATTCGCACAACACTGCCAATAGATCGAG CGGTCCGGCAATCTGCACTTCTTGTCTCGAAAAGATAGTGCAATGTTTAGTGGAACATTTTTTTACAGC TGACTTCGTCAATGCAAGTTCATGACTGTGTCCGTTCGGTGTAAGTCCTAATCCAGGAGGCAGAGGAT TGGGCTCTTGTGTTCGCTGATTTACA GTAATATTTTGTTATGCTGTACCATCCTTGTTTGTTTACGATGT TGTAAAAGGAAAGGGAATGGCATAATATTCAGTACGCATTATCGTGAGTGATATGTTTCCTTTGTTCAT GAACATGACATTAATGTAGGTGTGTCTGCAGTGGGTGCCCTTATTGTGGATTGAGAGACGA Secreted frizzled related protein 3 Gene Bank Accession Number: n/a Reference: n /a Comment: Predicted only, not cloned Predicted: TATGATGGGAGTTGTGTTGTGTCGTTATCGTCTTTCCCATTGTCGTAATTGTCATTAGAGTCGCCTAGCG TGTCTCGACGGATGGAGGCAAAAATGGCATCATCATCATCATCGTCGTCCTCCTCATCATCGTGTGAG AGATCTGCTGGAGTGAGTCTTCTATCTCATATGGCGGGCGACATGCCAACGACGTCCTCCCATCGCCC T CGTGGTTCTGTATACTCCCCTCGTGCGTCGTTATCTTTCCTTTCTGTCCTCATTTTCCTGTATGTCTTCGC CGTTTGTCTAACTACCGCGGCCTCACAACAGTACAACAATGGTGAGGATAGCTTGTACAATTATTACT ACCACCATGACAGCAACAACTACAACGGCATGGATGAATACGTGTCGGACTGGAGCCAGCATGAGTG GGCCAAAATGAGCGGGCGAATACAGCAGCCCAAATGCGTGGACATCCCC AGTAATCTAACTCTTTGTC AAGGAATCGGATACGAGACGATGAGGCTTCCCAACCTGCTCGATCACGATTCTCTGAAAGAAGTGACA CAACAAGCAGGTTCCTGGGTGCCTTTGACAAGGATCGCCTGCCACCCAGACACAAAGGTCTTCCTCTG TTCCCTGTTCTCTCCAGTCTGCTTAGACAGCCTAGATAGGTTGATCTATCCGTGTAGGTCGCTGTGCCA GAACGTGAGGGCCTCGTGCGAGAGTCGGATGA CTATACATGGCTTTGAGTGGCCAGCCATGCTGGAGT GTGAGAAGTTCCCACTCGACAACGACATGTGTATAATGCCCATACACGACATCAAACCTGACAACAAC TGCACCGCTTGCAAGCACCCACAGACACACGAGGCTCTTATTGACCACTTTTGCCGGTCAGATGTTGCT ATGCGGGTCACTTTGAAGGATCGCCGCGTTCTGGGCACCGACTTGGAGCTTGTCCTTCGAAAACGTCG CAAACTGTACAAGTT CGAGAACATGGAGAAGAAAGAGGCGAAGAACCTGCCAGTCGTCATCGCTGGA GGTACTGCGTGCACATGTGAGACCATCAACGACACTCAAGGGAAGTACCTGCTCATGGGCAACAAAC GCAACGGGAAAATCGTCATCAACTTCGGCATGGCTTGGAAGCGCAAGGATAAGGAGTTCCGTAAAGG

PAGE 118

118 TCTCCGCGCCATCAGAAAGGGCGACTGCAAGCAGTTGTTCAAAACCGTCATCGACAACTTACATGAAG AT CAGAAGCAACCGGGGTCCT Secreted frizzled related protein 5 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TATGATGGGAGTTGTGTTGTGTCGTTATCGTCTTTCCCATTGTCGTAATTGTCATTAGAGTCGCCTAGCG TGTCTCGACGGATGGAGGCAAAAATGGCATCATCA TCATCATCGTCGTCCTCCTCATCATCGTGTGAG AGATCTGCTGGAGTGAGTCTTCTATCTCATATGGCGGGCGACATGCCAACGACGTCCTCCCATCGCCCT CGTGGTTCTGTATACTCCCCTCGTGCGTCGTTATCTTTCCTTTCTGTCCTCATTTTCCTGTATGTCTTCGC CGTTTGTCTAACTACCGCGGCCTCACAACAGTACAACAATGGTGAGGATAGCTTGTACAATTATTACT ACCACCATGACAGCA ACAACTACAACGGCATGGATGAATACGTGTCGGACTGGAGCCAGCATGAGTG GGCCAAAATGAGCGGGCGAATACAGCAGCCCAAATGCGTGGACATCCCCAGTAATCTAACTCTTTGTC AAGGAATCGGATACGAGACGATGAGGCTTCCCAACCTGCTCGATCACGATTCTCTGAAAGAAGTGACA CAACAAGCAGGTTCCTGGGTGCCTTTGACAAGGATCGCCTGCCACCCAGACACAAAGGTCTTCCTCTG TTCCCTGTTCTCTCCAGTCTGCTTAGACAGCCTAGATAGGTTGATCTATCCGTGTAGGTCGCTGTGCCA GAACGTGAGGGCCTCGTGCGAGAGTCGGATGACTATACATGGCTTTGAGTGGCCAGCCATGCTGGAGT GTGAGAAGTTCCCACTCGACAACGACATGTGTATAATGCCCATACACGACATCAAACCTGACAACAAC TGCACCGCTTGCAAGCACCCACAGACACACGAGGCTCTTATTGACCACTTT TGCCGGTCAGATGTTGCT ATGCGGGTCACTTTGAAGGATCGCCGCGTTCTGGGCACCGACTTGGAGCTTGTCCTTCGAAAACGTCG CAAACTGTACAAGTTCGAGAACATGGAGAAGAAAGAGGCGAAGAACCTGCCAGTCGTCATCGCTGGA GGTACTGCGTGCACATGTGAGACCATCAACGACACTCAAGGGAAGTACCTGCTCATGGGCAACAAAC GCAACGGGAAAATCGTCATCAACTTCGGCATGGCTT GGAAGCGCAAGGATAAGGAGTTCCGTAAAGG TCTCCGCGCCATCAGAAAGGGCGACTGCAAGCAGTTGTTCAAAACCGTCATCGACAACTTACATGAAG ATCAGAAGCAACCGGGGTCCT Interleukin 16 precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: AGCAGCAGACACAACTTACAGCAACACCAGCAGCTGCTCCTTCAGATCTCCNCAGCTGGAGTACAGAA GGTGATCATGCTCAAAGGGGCTACAGGAGTGGGCTTCTGTCTGGAAGGAGGCCTTGGCTCTCCCAAGG GCAATCTTCCCATTGCCATCAAGAGGATATTTAAAGGAGGTCCTGCAGAAAAATGTGGACAGCTGAAG GTGAAGGATGAGATCTTGGAGGTCAATGGAGTAGACTTTACAAACATGAGACACTATGAAGCATGGA ACCACCTAAAATTCCTTGATGATGGAGAAGTTCACATCACTGTGCGACGTCATTAGTATATTGATCAA GCATCTGTAGAGATTGTATTTGTTAACAGCTGAATTGTCACACTCTGTACATCCTGTTTATACTAACCA TATGTTTTGTGTGAGTATTTATTTAAAGAGCTTTTTGTATGTAAACCATATAAACAGGGACTGTTATGA TTTGTGTATTTCTTGAATCTGTATGATAATGTCTGTTCTCTTGTATTCTG TGATGTTGAAGAAGCCTACT GTGGGCTGGTGATACCTGAGAAACATATGTAAAGATCACATGGTTTTTAGTGTATACTTGAAATATTAT CTCTGACTATAGGGCTATTTATGGCTGACAGACAGAGCC Thrombospondin ( promotes the development of new synapses ) Ge ne Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GACCCCGCGAGAGAAATACAATCCTCTCAATCGCAGAGACGGGAGAAACAGATTCTGTTGGTGATAA CGTCAGATTATCTCCTCGAGGGAAACGCGCGCTGAGAAATAGTAAATAGTGAGCCGAGGAATTTATCT ATCGTTTTGTCCTTCTTCGCGCAGTCTTG TAATTGAAGAGTGGCCACCCCCTCCCACCTCTCCTCCCCAC

PAGE 119

119 CTCTGTTTGACATCACTGACCGCAAGTCGAGTGTTCTCTGGTGTTGTGTGTTTCTTATAATTGTGGAACT ACTGCAGAGGATTTGTTGTATGGACACTTCGGATTTTTCAGACGTTTGGAAATAATGCGCGAGTGTTTT GACTGATTTCTGTACAGTACCTGGAAAATAAACTACGCACGTTCTGTGATTCTTACGGTACACTTGAAA TTACTGA CAGACAGAAGATGACTCAGATGCTGGTGTATAAAGTGCTCATCTTCGTCATGGTATATCGCT GCAGTGGAAAGCCTTCGGGAGGCCCGTGTGTTTTAGGCGGAGAGCAGTACAACGTTGGAGAGACGTG GCATCCTAAAGAGTTTTCTTCCACCGCCAACTCCTGTGTCCACTGCACGTGCCTAGAGGGCGGTCAGAT AAATTGTACCGGCGTGGACTGTCCTTCTCCAGAGTGCGAGGTGCCCAGATTTATCCACGGACAGTGTT GTCCTACATGTGATGTCCGGGGGGACGAGGCCGGAAGTCCCGAGACAGGACATGGCCCGGATGTGAT TTCCAT Similar to Growth arrest specific protein (promote neuronal survival) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGAGGAA GATCATAGGAAAAGACAGTGGTGTGGGGTGAGGTGCCTGTGGGAAAGTTCACG AAATGAGTTGTTGTAGTGGTGCCCTTTGGTGATAGCTGAGAGCGGAAGAGTGGAGAAGAC ATCCTACCTCGTAGTGCTGTGAAACTCATGTGTCAGGATAACATGCCTCTGAGGTCTAAG ATGCGACCCCACTTCCTGCCTGCCCAAGCCCCACAAGTCACTGCGAGCCTGTCTTGATTC GTCACATCTGGCAGTAACA TATGCTTTGTTGAAGGAAATGGCCGTACCGACGAGTAGGCC TTACATAACCTGGCATCTGTTATGTCCAGTCTAACTGCTAGAATGTCGACCAGTTGGCGA TTTCCCCCGATCGAACAGTACATGATTGTTTTAGTCATGTCCACGTCCATTTCTCTTTAC GCAGACTTAACCGCAGTCTCAGACGAGAGCTGCCACGTAGCTCAACTTAACTGCCACGCG AGAAGCGGCTGCCAGAGGGCGCTCAACAACT TCTTCATTCACTGCCGATCTGTCATCAAA GGTGACTTCCGGGACGTCTGCCCTACGGACTGCAAAAATGCCCTTGTGTCTCTCCTGTCC ACGGAGGATGAAGCAGGACTGGCGTTCATCAACTGCGACTGCCATCAAGCAGGTCTTTGC TCAGAGCGGAAAGAGAGAGTTGAGGTCTGCAAGAGGGAAGTGTTGGCGTCCATGCACGTG CTTGAAGACAACGCTCCCCCCGTGTCCTGCAATCTGGCCAGAT GGATCTGCGAAGCTGAC ACGTCGTGCCAAACGGCTCTGCGTTACTACTACGACTACTGCTCCAAACTTTTCAAAGGG GTCAAGTGTACGTCGCGGTGTAAAAACAGTTTGTTCATTCTGTCTCGGCAACCTCACGCC AGCAAACTGAGGTCTTGCCTCTGTGACGGAACAGAAGACTACGACTGTCCCACGCTGAAG GTCAACACGGAGAATATGTGTTTCACTCCTAGGGCGCGTCGTCGCCATCGCAAAA GTGAC ATTGCCAGAAAAGGTGGGGCGAAAGTGCCGCAAGAAGGGCGCCCGAATGTGTCATCAAAA GATAAGCGCCGTGCTGGGCATGTAAAAAGAAGAACTCGTCGAAGAAAAGAGACGAAACCG GCCGCACAGACCACAGGGCCGACGCCAGCGCGCCAGGAGGAGG Similar to Growth arrest specific protein 8 (promote neuronal survival) Gene Bank A ccession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GTTACTGTTAGTGTTATACAACGCAGTACGTGACTCTGTGACCGTGAACCTGAGTGAGGT GAACGTGACCGTCCTATGAGTTGTGCCTTTGAATGGTTTTCGCAGTGGAATAGTGAAAGT TATTACTGGAGTGTTCCAGTTCCCAATACAGGATGAACTAAATTGATGTTT AGTTAGGTC AGAGACTATCCTCTTCGACATCCGGGTTCTGTTGCTCTGACAACCACCGTAAGCGTCCAT AACTGTGCTGTGGGACGACAACAAAGTTGCCGTAGTTTATCAAAATGCCTCCAAAAAAGA AGAGTGGCAAGAAAGGGAAGGGGAAGAAGGGGAAGAAGTCAGGAAAAACCCCCACTGTGA TAGATGGCATTCCCACAGAAGAGATGTCAAAGGAGCAGTTGGAGGAGCTCATTCAGAGGC TA CGTGAAGAGCTTGAGAGAGAAAGAGAAGAGAGAAATTACTTTCAGCTGGAACGTGACA AGGTTAACACATTTTGGGAAATTACAAAACGGCAGTTGGAGGAGAAGAAAGCAGAGCTCA GGAACAAAGATAGAGAGATGGAGGATGCTGAAGAACGCCATCAAATAGAGATCAAGGTGT ACAAGCAGAAGGTCAAGCACTTGTTGTACGAGCATCAGAACAATATCTCGGAACTGAAGG CCGAAGGCTCCGTG GCTCTGAAGCAGGCTCAGGATGGTCACAATTCGTCTGAGATGGAGC

PAGE 120

120 TCCGCAAAGACAAGAGAGCACTGAAGGTGGAGCTCAAGGAGCAGGAGCTAGCTCATGAAG ATGTCATCAAGAACCTGAAAAAGAGAAATGATGAAAACATGACCTCCCTTCGCAACAGCT TTGAGCGCCAAGCCAAAGAAATCGAGTCAAAATATGAGAAGAAGATGAGGGCTCTTCGTG ATGAGCTAGACTTGAGAAGAAAGACAGAAATTCATGAGATTGAGGAGAGGAAGAACGGCC AGATCAACACCCTTATGAAGAATCACGAGAAGGCGTTCAGCGATATTAAGAATTATTACA ATGACATTACATTGAACAACCCTGCCCTTATCAACACATTGAAGGGACAAAGTTGAAAAA AAAAAAAAAAGGACCGGCGGGCCCGGGGTGGACCCCCGGCTTAAACCCCAGGGGCGGCCG CCGCCGGGCGACCATATGGAAAACTCCCAACGGCGTGG AGGCAAACTTGGGTATTCCAAA GGGGCCCCAAAAACCTGGGGGAACCGGTCAAGGCGTCTTTTTGGCGGGGAAAANTTTNTT TTAGAAAACCCCCCCCAACCCCCCGGGGCCAGGGGGGGGGGGGAAAAGGGAGCCCCCGCC CAAATTGCGCCAAAAAGGGGGAAGAATAAAATATTGGGGGGAAAAACTTTTAAAAAAAAA AAAAAAAAAAAAAAA Secretory signaling peptides/prot eins found by homology search to Lottia genomic screening Strong Annotation Lottia Protein Model : jgi_Lotgi1_119019_e_gw1.30.87.1 NR Annotation: PREDICTED: similar to seleno protein N, 1 isoform 2 precursor [Canis familiaris] NR E value: 2.00E 85 Ac Annot ation: Seleno protein N precursor_[0.0]_[6]_APL_all_052305.8412.C1 Ac E Value: 7.00E 69 Sequence: MVLFTLLCCLFIIGRHDPTQIPEEIVINIGDDGVLLFQQHDRDNDGYLSIQEFEPLVYRLLEINVSGPVYDVPIS TDDEMITLKSYFIPIVKESMSKDLNDSVSIGLLRTMNSLHGLEKWQNVNLQWMNFGASHFSGFLPKDVDSI MLGSSYFIINVE KGLFNAALSSNRYYPPKVTTNESIIVHRLLTLFHPRPFVVSRFPAQSSVACVRAYNDKYLD IVFRIHAEFQLNEPPYHPFWFTPAQFTGNLIISKDGKHIQYFNLYVPNNKRLNIDMEWLNGPNESENMEVDI GFMPLMQLNSTQSSVPVKNLEEYELLEPLPERPQHKTEGENIEWMATIDLEDAKSSLEKALYPFKKVPYYN FTEAFKKAEDNKKLVHSILLWGALDDQSC* Lottia Protein Model : jgi_Lotgi1_121665_e_gw1.37.7.1 NR Annotation: hedgehog [Patella vulgata] NR E value: 1.00E 171 Ac Annotation: Sonichedgehog protein precursor (SHH) (HHG 1) [Contains: Sonic hedgehog protei_[3.65288E 39]_[1]_L7ALL ET3V2MC01DELVF_253 Ac E Value: 3.00E 32 S equence: MKLFFSFQSVCSWTFIFLFITSLTHACGPGRGSGSRRRPRKRTPLVFKQHVPNVSENTLGASGISDGKIRRNS EKFKNLVKNENPDIVFKNEEGDGSDYLMSRRCQDKLNSLAVSVMNNWKDVRLRVTEAWDDSPNSHAKD SLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESRGHIHCSVKSDSSVAIKIGGCFPPTGSVQTL HGWKTMGQLTVGDKVLSINSAGQLEYSPVIAFIDR NDLEFEKYLTLHTEDDTDITLTSKHLIYASGTNSSNF ESYDVVYADDIMEGDHVLITSSEKGAISPTRVVTISEKTLQGVYAPLTVNGNIVVDGVVVSCYAVVSDANL AHAVFAPMRGLHYLSQYVPWFLHSTQQENAPQNGVHWYAKMLYNIGSTFLNEKTLYIA* Lottia Protein Model : jgi_Lotgi1_159314_fgenesh2_pg.C_sca_20000054 NR Annotation: Bucca lin precursor [Contains: Buccalin D; Buccalin E; Buccalin F; Buccalin G; Buccalin H; Buccalin A; Buccalin I; Buccalin J; Buccalin K; Buccalin L; Buccalin B (BUCb); Buccalin M; Buccalin gene predicted acidic peptide A (BGPAP A); Buccalin N; Buccalin O; Bucc alin P; Buccalin Q; Buccalin R; Buccalin C; Buccalin S; Buccalin gene predicted acidic peptide B (BGPAP B)] gb|AAB27696.2| buccalin precursor [Aplysia californica]

PAGE 121

121 NR E value: 3.00E 66 Ac Annotation: Buccalin precursor [Contains: Buccalin D; Buccalin E; Bu ccalin F; Buccalin G; B_[0.0]_[43]_APL_all_052305.95.C2 Ac E Value: 4.00E 75 Sequence: MAARKHELVLVLTSVLCFVSSIVGDPNVPSDSQDNSALTQDDFAKRGMDKFGFAGGVGKRGLDKFGFTG QLGKRDMDSFGFAGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGF TGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQL GKRGLDQYGFAGQLGKRGLDQYGFTGQLGKRGLDQY GFAGQLGKRGLDHYGFAGQLGKRGLDQYGFAGQLGKRGFDQFGFAGQLGKRGLDHYGFAGQLGKRGLD QLGFTGQLGKRQMDIFGYRGQLGKRQSIDKYSFLGAGIGKRSVKNTAGIKKDDA* Lottia Protein Model : jgi_Lotgi1_163440_fgenesh2_pg.C_sca_39000174 NR Annotation: No match foun d NR E value: Ac Annotation: BEL 2PROTEIN_[0.749555]_[1]_APLCNSN01 F 043283 501 (antiapoptotic protein) Ac E Value: 1.00E 40 Sequence: MDLYGVVWLLAILQEVTSCFLTEKLPSLEGKEWFTKIRSGGSNDVIAYFDRNSVTFNDVKNTTNGETKWR CFMKIRGRYFLKNVRQGGGYLYKCMGIIVRSESAIQLEWSHVSRLADPAL CAEENINLDPWLLISYKTVVN DFTSCPVSGGFDIKHAGYYSRDVRDSRGYDIGCNLMNIPMRLEFDCIAGEGAIFNFRSKNCVPDIHFNIYQN TICVAKWSNKRHHFVLLRTKTGLEFWCARFPVGIENQNETELYLYSDVACLEGEYAEEFVKFMKFDLQRV MYTTICADEYPQCKEGTCNVFSKLECQKTCGLCNPHRPPGICTFPKRMQGTYLLHSKHGNQNVTLEGRTLN IENVGQFDCIVFEDSPLRSTKT YTTMSIFQNGCRPRYTCVRLKRLGPSALRYSLAQNLVWPIKKERIGANICN EDNFRADGDPIRDTFRSYKKTGKPVIKIHPKPRFINCGMNTSYTIRAELPDGSICHGGFYQHCKNETKLRFDF HTCNSFIKPKEDFNCAGIFRTNYWERAVLIQNVDNLNDIRCLIFNSLRPYQALIVVADECDKMASGLVDSNI RIPIMRLHIRSDVYPCKHIQLPLKDDVITNVVTTTEPIKMNVTLKESKNVNSFSVDLELS SRNSSGSRPFENKL NSLLLLFLCYVVFVKNL* Lottia Protein Model : jgi_Lotgi1_164016_fgenesh2_pg.C_sca_43000014 NR Annotation: chordin [Danio rerio] sp|O57472|CHRD_BRARE Chordin precursor (Protein chordino) gb|AAB93485.1| chordin [Danio rerio] NR E value: 1.00E 119 A c Annotation: Chordin likeprotein 1 precursor (Neuralin) (Ventroptin)_[1.94386E 18]_[6]_APL_all_052305.11591.C1 Ac E Value: 2.00E 24 Sequence: MSPMDLAVCLVLLAATVSSTKFSKIPLKAARENLFGIQPNRPGCILGSKFYGIGERWTPELLPVGEMYCVKC ECVPVERKGIIDMKGQTLCKNIKNLCPRPNCLNPVLPKGKC CKVCPDEVNSFEDSFSLGSKSSAPTMISSDSK RRVRNEFVSLLVGKNVRRESVKTSAVAAVYFSIVGRDEIRYSVRYFKLDRPKFLQITDANGNVLFERPIEKK RNGDKKFCGIWTKVPSAYIQYVREERLFAVITASRHFRGLVSGRIMVNRHAKREVFSSVLASPKANGIGGL LSVVYNPRTHLVDYVINLDGVFPGSSEEYFITISKKSKVLHQSKGKASSKTRFIRGSWTLKNKRQSKQLARG RLTIRLTST NGASVSGDIKPKVTCGVSQAVMSSGNSLEEMKLASSGTAVFELREKGSLDYKIRVTGLTSKVH RIRLEGAVNKKNRRQIIGSVHRNFKADENSFNGWCNGTFKKMTADDLHLFLNNKIFVNVATSENRISELRG RVIALPYHSHFEQVSADPAALGPKTITSNGHAGNAWISMGVGCSLHYDITTTTIAKHHDLTLSLVSPTDIDA YVTIEKISTTNLYEFQQASGSIAVVPESLFRDLDEGSALIQVISGDVILS GNVSVPNTCWQYSQDYDMDILVD EQTSEEVLAANRYKCVYEGKVYENGDSWLPDVNATCNTCSCNKGKIECHRLICPVVECANPIVLDGECCPT CPIETTDQTECKIDGDLRSHPVGSTWHPFLPLMGYAKCVICTCLPGGIYKCERKCPKLDCPASRRIRLNPKD CCQVCAPEEVKPEVVKPLQDVDMKGACSFKDQIYANGAKWHPRVIPFGYMKCIDCSCNNGTPSCSRPKCP KLSCSRKIREDGACCDTCAD SDGEEIVEEKKSDSCIFGGKKYDDGETFQPKTSSLSSCATCTCRKGSMKCSL TCPEKCKDPKHSNDPCCKHC* Lottia Protein Model : jgi_Lotgi1_164520_fgenesh2_pg.C_sca_45000185

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122 NR Annotation: PREDICTED: similar to filaggrin 2 [Rattus norvegicus] ( molecular regulation of epidermal terminal d ifferentiation) NR E value: 5.00E 37 Ac Annotation: A agglutininattachment subunit precursor_[2.05518E 9]_[1]_APLCNSN02 F 021300 501 Ac E Value: 2.00E 11 Sequence: MIKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDIVQMIKLTLSILDIVQIVKVTFSILDIVQIVKLTLSILDTVQ IVKLTLSILDIVQI VKLTLSILDTVQIVKLTLSILDTVQIIKLTLSILDIVQIVKLTLSILDTVQIVKLTLSILDTVQI VKLTLSILDTVQIVKLTLSILDTVQIVKVTFSILDTVQIVKLTLSILDTVQIVKLTLSILDTVQIVKLTFSIFDTV QIVKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDIVQIVKLTFSIFDTVQIVKLTLSILDIVQIVKLTLSIHVVC Lottia Protein Model : jgi_Lotgi1_1 68619_fgenesh2_pg.C_sca_77000042 NR Annotation: crossveinless [Saccoglossus kowalevskii] (a new family of twisted gastrulation like modulators of bmp signaling) NR E value: 1.00E 148 Ac Annotation: VonWillebrand factor precursor (vWF) [Contains: Von Willeb rand antigen II]_[0.0]_[6]_APL_all_052305.10450.C1 Ac E Value: 1.00E 44 Sequence: MYYTELSILAKCIALCILTCFPILIQAQLTGTVTRCYNEGGEVKIQGITTDPCISCYCMDGYVQCDKKKCDNL DGCHAILFDGPKRCCDTCKGCRLNGVKHESGKSWFDKRDRCTSYSCKAGVITKSKVQCHVPCSNPIKRKR QCCPSCKGCHFDGADRKNGEKFALASDPCVE CKCRKGSVTCMKRACPVLNCPDEVIYQPKGECCPKCKG MRKIFDFPGACYFAKRVYVDGFSFEPKSETRCTCTKGTVICEKATCPPVACPVEERVQKGSCQVCEPKRNC LYDGKIHMHRSKWQPRMCTQCSCQNGVTYCQRERCNNSLSCPNGYKLQFQPGECCPKCVEHDAVCRVFG DPHYRTFDGKMYNFQGTCKYMLSQDCQGKDFTIKVKNGVRLSSGFAWTQMVVVLMGDTRISFRQNLLIKI NRRRVQLP YTIPGKFSIRREGHSVTFRADIGLKVVWDGDSFLEVTASRKYKNRLCGLCGNYNGLETDDLIG RKGKNYLRGEEFGNSWRIGSKKACKTQPKVKNLQSICDKDFKAKVRANKECSVLYSRAFSSCRRVVDVTP YVTSCVTDMCDCPHGKKCSCESIQAYAHECKRAGHKVKWEKVSNCKAPQKNCPKGAYYSLCAPACPKTC SSNKPDGSCSKKCSPGCVCQNGAVLYKNRCISPEKCPKT* Lottia Prote in Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Euprymna scolopes] NR E value: 1.00E 122 Ac Annotation: Wnt 16protein precursor_[8.48178E 30]_[10]_APL_all_052305.8921.C1 Ac E Value: 7.00E 23 Sequence: MTVSPTVVWAAFSMALSSVVALGANII CNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTSAGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LPPFRKIGDSLKKRYKKSKIVVPYLGRRARTAVTLILKRAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK* Lottia Protein Model : jgi_Lotgi1_186451_estExt_Genewise1.C_sca_160091 NR Annotation: PREDICTED: similar to out at first [Nasonia vitripennis] (Vital for proper neuronal development and hatching) NR E value: 7.00E 69 Ac Annotation: Out at first protein_[0.0]_[4]_APL_all_052305.9349.C2 Ac E Value: 5.00E 90 Sequence: MSMSLIPSLSYSIFVCVLIKICHTQLVVNVKNGGGDILVESIQSNVSSDTITLEYPSSDGSLITQFIDFKSEAQIF

PAGE 123

123 RAYVPGEEERGQPPGSVQVLCFITRFSKNDFISSDAMSKLRQKNPTAIRTPEEERPQEVHSFDLSVDLNKSHV ISPHVFNICREAKESTFFNEDDLKTISRSLSKDYNTMMSAMIKLAPTKYGKCSDTSDIFKPCTCRYDACVGW YPCGLKYCRGKDSSGKVVSYRCGIKTCKRCLAFDHFVKQKFLCLWN Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301 NR Annotation: predicted protein [Nematostella vecte nsis] gb|EDO37057.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 109 Ac Annotation: MultipleEGF like domain protein 3 precursor (Multiple epidermal growth factor _[3.47103E 24]_[3]_APL_all_052305.12391.C1 Ac E Value: 1.00E 114 Sequence: MF LEVFNISATVLCILFLIGHRKCDTIQNSKHHAELMMDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVGVNGGFFNTTTGACLGNIVSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSA VGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLELGVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYGINCSTPCKPGYYGSNCAHQCYCLNHGTC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi 1_205842_estExt_fgenesh2_pm.C_sca_70027 NR Annotation: BMP2/4 [Patella vulgata] NR E value: 1.00E 159 Ac Annotation: Bone morphogenetic protein 4 precursor (BMP 4)_[8.29921E 13]_[3]_APL_all_052305.15764.C1 Ac E Value: 7.00E 27 Sequence: MIADFYRSVVLLLLVLVVN YTSSLSTNTLHPRKHGNEKVLAAVESNLLGLFGLKSRPVPGRRTPIPDYMLD LYKRLDSEPDFISPFAESKGKGIVEANTVRSFYHSDINFVKDCDERTCARIWFNISTIPFAEIMAASELRVFKD VYNHFKLTDANSLSGDRIKASLFKHRLEIHEIMRVSPDDSECISRLIDTKIVDTRNSSWESFDVHPAVLKWRR RPQYNYGLEVRIVSKSPWISTNSHVRLRRSAHMDEKNWQIQRPILVTYTHDGRNPNS RIKRASARNRRRKS RKRRRRKKKGNKNECKRHALYVDFGDVGWNDWIVAPTGYNAYFCRGECPFPMGQHLNSTHHAVMQTL VHSVNPSAVPKACCVPSELSAISMLYLDEWDKVVLKNYQDMVVEGCGCR* Lottia Protein Model : jgi_Lotgi1_208417_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Ac Annotation: Transforming growth factor beta induced protein IG H3 precursor (Beta IG H3) (K_[2.29414E 23]_[11]_APL_all_052305.8904.C1 Ac E Value: 2.00E 61 Sequence: MSRNLWIFLLILLSIIVIASSRRSRGRWYRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEEL DDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQ ATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSNNAFEKLEEKVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYST FPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW*

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124 Weak Annotation Lottia Protein Model : jgi_Lotgi1_110650_e_gw1. 12.27.1 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 1.00E 32 Ac Annotation: Slit homolog 2 protein precursor (Slit 2)_[0.0]_[4]_APL_all_052305.13937.C1 (axon guidance) Ac E Value: 3.00E 12 Sequence: MKIILVLLCFCHLIYSAKTCPSNCQ CHLTDKAVNCYKRELQFVPANIPLDTEILDLSSNDIANIDPTMFHNLT KLTDLVLNDNKISKLDPRTFQNLHKLEVIKLSQNFLSTIPEGLFDNLPIMVPKLMELNLSQNKIVKIENYVFR PLSMLSTVSLNDNPLKNADRLLSKNRRLSYIDMSECQLTTVPRGLPDSIEHLKLIKNNITKIKPKDFRNKNYL KILALDENEIEFIAPNSFKSLQKLIQLWLNGNRLRKFPSPISPSVTNVYLAKNNITKLSAAD F Lottia Protein Model : jgi_Lotgi1_113861_e_gw1.18.363.1 NR Annotation: PREDICTED: similar to tartan CG11280 PA [Apis mellifera] NR E value: 5.00E 27 Ac Annotation: Insulin like growth factor binding protein complex acid labile chain precursor _[1.10885E 21]_[2]_APL_all_052305.14739.C1 Ac E Value: 5.00E 18 Sequence: MVKPPTQNTRWWIIVWILLFQKYTCEPEVTCPHGCKCDVDVVNCLHSNLTSVPVGLSNGDRLQELKLGRN KITEIKLELLEYKELRLLDLISNNISTIDGSAFRTLYKLETLFLSRNFITKTDKNTFRNLTSLRFLDLSYNNLRC LKIGNFEDLQSLVNLDLSRNQISLIEAESFRCLENLEILKLPRNQLDQ FPWKIFQNLTSLKMLVLDDNLLTSLP ISAFSNLINLEYLSLTGNRLHELLPSSFMMSDEGAISLEQLYLRGTDLDTIPVKLFAKLKKLTVLDLSKTQIVS VKNENLNGLSSLESFIMTSSNNLTYIEKGVFQDLHSLRKIDISGNTLLSEIQFTTSSLRNLSLFDLSANSL Lottia Protein Model : jgi_Lotgi1_137329_e_gw1.113.40.1 NR Annotation: insulin like growth factor binding protein, acid labile subunit [Sus scrofa] gb|ABE73450.1| acid labile subunit [Sus scrofa] gb|ABE73451.1| acid labile subunit [Sus scrofa] NR E value: 1.00E 21 Ac Annotation: Slitprotein precursor_[2.64913E 12]_[2]_APL_all_052305.8468. C1 Ac E Value: 5.00E 13 Sequence: MSFVQLLPFFLFLTISQSSSQRWPLSGTCYPCSCDINQKGFGRVKYVNCSNLALTRIPRNLPLDLHTLDFSNN RILPQEIQQLCTFNSMQYVALSYNSLDSIPPEIFKDLENLHTLVLHGISSIPTTNIFEDLFNLEYLDINDCHVNQ IPDNWFRKLYSLKTLKLRQTGIRSIEPGVLDGLLNLQELDLSHNVMRTAQTESFKPIVQSIRRINFRGNMFKT I GDHLFEKMYNLVELDLSDNKLESIHKRSFLDLRQLINLDIRDNKLSNLPGGLFKNLRNLQTLNLAMNTFNN FPETLFKIGYLLKLDLSYNRIRRLPDSFVHSFPYLEFLNVDKNPLHCDCKSLDVKFYQPNLVII Lottia Protein Model : jgi_Lotgi1_152641_fgenesh2_pg.C_sca_2000061 NR Annotation: interleukin 17 [Crassostrea gigas] NR E value: 5.00E 07 Ac Annotation: CNSN01 F 034307 501 Ac E Value: 1.00E 06 Sequence: MVLMKVFICALLVNMVYSFTLIKNIEHILERERRSDECAVPNNIDELFTNLNSHVNRVEFLIAVNATHQPNA QGLIPPAAGDQTDTECPKEYTGKDTSNSGIWQRSTCPWFYSERNYGAEYYPSKVYNAACKCVDCLYSKSN SETGCSRIYRNINILKKTGCSNGFYTYEQR SISVSVGCTCAKLQANIG*

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125 Lottia Protein Model : jgi_Lotgi1_157019_fgenesh2_pg.C_sca_12000330 NR Annotation: No match found NR E value: Ac Annotation: Chordin likeprotein 2 precursor (Chordin related protein 2) (Breast tumor nove_[0.235318]_[1]_L7ALL ETFK5Z408E YR8G_257 Ac E Value: 2.00E 08 Sequence: MFSLVLLVVTPWLVFCMPPPPPAMCTCSDGTQVPPGGTHSISDCHSCYCDENNSEAQEIIAACLIVPCVDSV KMPGQCCPTCPNGENCRAEFKDDNGVERSEIIKVSDGVVKFNSTECQCDYQNYNGEPKANCVQVLSIEPVI AEKSTP* Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_710000 05 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 21 Ac Annotation: Fulicin precursor [Contains: Fulicin; Fucilin gene related peptide 1 (FGRP 1); _[0.00557519]_[20]_APL _all_052305.37.C1 Ac E Value: 2.00E 16 Sequence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSF IGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPMFLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNYFSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGS LSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Protein Model : jgi_Lotgi1_168377_fgenesh2_pg.C_sca_74000087 NR Annotation: TBL 1 NR E value: 2.00E 07 Ac Annotation: Dorsal ventralpatterning tolloid protein precursor (Mini fin protein)_[0.0204183]_[9]_APL_all_052 305.10078.C3 Ac E Value: 7.00E 14 Sequence: MLPHITQCVILFITFLCDISVLLPSRDGGRGLTYAQLVPHRTSNDVYMDPCKAAGFMGDIALSEEEFKRERK YLQQMNNTMFELQVEDPRTTYIGRKPSKYSGQPENTLKKLPMSELDVKKKKYLRKILKEKKQNLKSLCKK GFQTVVYTRQVPVGFKFISPFLYLLDLNIL* Lottia Protein Model : jgi_Lotgi1_1685 08_fgenesh2_pg.C_sca_76000035 NR Annotation: ependymin related protein precursor; EpenHg precursor [Holothuria glaberrima] NR E value: 3.00E 10 Ac Annotation: Mammalianependymin related protein 1 precursor (MERP 1) (UCC1 protein)_[0.0126998]_[10]_APL_all_0 52305.1963.C1 Ac E Value: 5.00E 19 Sequence: MMKYCLVVAVVVVGVVIGEPSKPGCCTPSQWEGQQGSTIGIVDGGIPSVRTSMVRISYDEKNQRIAYVTSE STPQGEKNYKIIYDYSRKFQFIVDLDTKHCTVSSLTDPFMKLCLPVDAQPKGKFYYGIDGNNLPAMAFSLN VNGTMITVVTTADNCIPIAEVLVDGQAQGVSQMHASGFVNLTSGIKDPSVFIPPGTCTQ

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126 Lottia P rotein Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chain; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica] NR E value: 4.00E 13 Ac Annotation: CNSN01 F 005950 501 (unknown) Ac E Value: 2.00E 11 Sequence: MEVTCKCPLVLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYCRNSQQRISIKK*

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127 Object 23. Controversial predicted neurosecretory produ cts All software programs are inherently flawed in that they can only use those parameters they are programmed with to make predictions about whether a protein may or may not be a secreted signaling molecules. Due to these flaws, our final list of predic ted secreted signaling molecules was manually screened for conserved motifs, signal peptides, and annotated to make the most conservative list of predicted neurosecretory products. Here, we show those transcripts predicted to be secretory molecules due to targeting to the classical signaling cascade but are unlikely to be secreted outside of the cell. Some of these predicted secretory molecules may be part of the secretory apparatus but do not encode for signaling peptides. Furthermore, many of these pre dicted molecules have motifs of non secretory products but may be secretory in our model species. Further experimentation is needed to determine the functional role of these products, so we have decided to leave these products in this supplemental section to provide a complete unbiased review of all predicted products for future researchers. Found by Homology Search 7B2 secretory granule neuroendocrine protein+Gene Bank Accession Number: 94471618 Reference: N/A CDS: ATGAACATCCTTCTCCTCGCCGCCACCCTTGTGGGTG TGACCTTGGCCAGCTATGACCCCTACGTAGAC ATGGCCCAGCTGTACCGGATGCAGCTTCTGGCCAACGCCTTTGACGACTACCTGCCTGAGAGCCAGCT TCTGGACAGCCGCAGCGAGGAGCCGTACTGGCCCGAGCTAGAGGACGTGGCTGAGCCGCAGGACGAC AAGGATGCCATCTATAACGACAGGTTCTACAGCGGGGCTCATCTCAGAGATCAGGAGCATTTGGAGCA TAGCGCTCTGCACGGTTATCA GTCGGTGTCTGGCGGTGCATCAGAAGTCCCCCCTAACCCCAAACAGG TCAAGTCGGACAAACAGCTGCCTGAGTACTGTAACCCACCCAACCCCTGCCCTGTGGGCAAGACAGCC AAGGACAACTGTGTAGAGAACTTCGACAACAGCGCGGAGAACAACGAGCGCCTATTGTCCCAACAAG ACTGTCCCTGCGACACCGAGCACATGTTCTCCTGCCCCGCCGGAAGCCAGACGGTGTCCTCCAAGGCC CAGAGC TCGGGCAACCAGCAGATGGCGCTCAACAAGGTCATGGACGAGATCGCCAAGATGGAGCATG ATGGGGAGAGCTTGGAAAACAACCCCACAATGTCGGAGACGCGGAAAAGAGTGACGCTGGTGGCAAA GAAGTCTCCACATATTATTCACAAACGATCTGAGCAATCAGACCACAGCAATCCTTTCCTGCAAGGAG CTCCTGTAGCTATTGCTGCTAAGAAAGACCCCAATACCGCACAGAGGGTCATTCCCCAGT GGGCCAGA TACGACCAGCCCTTGCGGTGA Protein: MNILLLAATLVGVTLASYDPYVDMAQLYRMQLLANAFDDYLPESQLLDSRSEEPYWPELEDVAEPQDDK DAIYNDRFYSGAHLRDQEHLEHSALHGYQSVSGGASEVPPNPKQVKSDKQLPEYCNPPNPCPVGKTAKDN

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128 CVENFDNSAENNERLLSQQDCPCDTEHMFSCPAGSQTVSSKAQSSGNQQMALNKVMDEIAKMEHDGESL EN NPTMSETRKRVTLVAKKSPHIIHKRSEQSDHSNPFLQGAPVAIAAKKDPNTAQRVIPQWARYDQPLR Acyl CoAbinding 0R protein Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CTTCTGATAACGGGCAAAGAACANGAAAGCTCAGAACTTCTGACAAACTGTCATCGTTCAGTTCAAAC TCT AGTAGTAGTACAAATTCCGGTCAAAAGCTTGAGAGCCTTTCTAAACCAATTCCCAAAAGAACATC ATTTGCCTTGAACAACAATGTGATCAAACCGGGCCCCCGATCAAACCACCGAAGAAAACGCCCCCTGC TGTCCCGGTGCGCAGTCCCAACACAGGGCTCACCCCAGGNCCTCCCACTGACTTGCCCTCAACACGGC TTATAATACTCGCAAAATTGGCAAGAAGCTGGGTATCCAGCTCAAGAAAGGTCCT TTAGGCCTGGGAT TTAGTGTGACATCTCGGGACAATCAGACAGATGGGAATACGCCCATTTACATCCGGAATATTCTGCCC AAGGGGGCTGCTGTACAGGATGGACAACTCAAGCCTGGGGACAGACTCTTGGAGGTGAATGGTGTAG AGATGACTGGCAACACTCAGGCTGAGGCCGTTTCCTTCCTTCGCATG Acyl CoAbinding 1F protein Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GACACACACACGCACACACACACACACACACCGGTAGGCCTCTAGACTAGAACTGATCTATCATTCGT CTCACCTGAAGGCCTTGTTGCACGGGACACGCCTTTTCACAGACCACTTGCTTTGTTGAGTTCATTGTG TTAACGTAAACGGTTAACTACACAAATCCATCGACATGGCAGACTTGGAAACTAAATTCAAGACAGCT GCTGATGAGGTGACAAAGCTGACCTCAGAGCCCTCCAATGAGGAAAAACTTGCTATTTATGGTCTCTT CAAACAGGGGACTGTCGGGGATTGCAACACAGAGAGGCCAGGAATGTTTGACATGAAAGGAAAGGCC AAGTGGGATGCCTGGAATGCATTGAAAGGCAAATCAAAAGAGGACGCACAGAATGAATACTGTTCCA AAGTTGAAGAACTGAAGGGGAAATACAACTAACTGACATTCC TTTCCTAGTTTTCTTCACCTTTTCAAT TTCATATCTCCATAGTGTTTGTGTTGTCAAGTTATCTTGTTGGATGATCACGTCATGTGTGCAGTGGCCC GCTGAGAAAATCTGGCACAGACAAACAACCTGGGGAACAACGAAGTTTTTTGGACTTCAGAGAAACT GTTAACATGACCAACTAATTAGGAATTTTTTGTCTTAATGAAGCTAGACAAGTATTGAGCCTGTGAAG ACACATCCATATATATTTTATCTC GCTTGTCAGAAAAAGACTACTTGTTGATACATTCGCTTATTTGTA GAGTCAGAGTTAGTCTTGTTACGTGAAGTTATGTGAGCATAATGGGTATTTAGTATTCTCATCCACTGC TGTCTGCTCAAGGTAAGAGATCGATCATGAAATGACTGCCTGTCAAATTATTGCTTTTGTGCTCATACT GACTTTTTGCCATGACTTTTATGACCAAAAAAAAGTACAGCAGGAGGCTTAACGTAACATTTCATTTTG GAGC ATTATTCTTTTGTTAGCATACTTTT Anaphylatoxin like 1 (Complement component 3) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CAAATGGCCTGGACACGCAAACGCCCCTTTTGGCAGATATACCCACGGCAAGGAAGGACCATGTGGT GATGGAGTCAGGTAGTGTCGTGTTTAGTGTCAGTTGCCCGGTACTGCCGAGATTATGTTCCTCAAAAA GCCATGACTCCGGGAAATATTTCCTCACCGTTTGAGGTGGAGCTTCTGCCACAAACAGTCCTTTATCTG TCTTTTTAACATGATCCCTGAACCCACTAGGAATTTGGGAGATAAATCCTCCAGGTCCTAATGGTCCAG TTGGTCCAACTGGACCTATGTTGGCTCCCCCTTTACGAATCGCCACTGTTCCTTCAAAGCGCCCTATGA TGGCGTTAGAATCTACGTTGTAAGGGGCGCTTGAGTCAACCAGGCGATGAATTAGACCAGAATTCTTG AATATTTCTGTTGCGTCCGATCCGTCTCCACTGTCTGAACCTTGGTCATGACGCACAAATTGGGAAGAC ATCGATTCTCTGGACAGAGTGACGTCCTTGTTCACCAGCAGCACAGCTTTGTCCACAGCCACCATGCCC ACTTTCATATTGCGGCCACCGATGACATTTAGCTTGTACCCATCCTTGGGCTT CACGCTCAGACTCCCG CCAAACATACCACCGGGCTTTTGGACTATGACCTCCTCCAGACACACGTCTGGGGTGTCGACCAGCAG

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129 AGAGTCCATAGCAATCTCATCGTCCACCATAAAATACGCTACCACTCTCATACTAGGGGTGAGTTCGC CGTGGAGTCGTTTCGGCAGACTCACAAGAGACCGTCCGTCCAGACTACGCATGGTGCTGGTAGTGTAG AGTACCTGTCCTCTGGAGATTACCAGTACTGTAATC GTGGTAAAGTCTTCTCCCACACCATACCAGTTG TCCTCAGGAGGTGTGTATCTCAGTAGGATCACCCAATCTTGAAATTTCTTGACATCTCCGTCCATGGCT ATGTGCAGGTATTGGGAGTGGGCGAGTTAAATTTTCGCACTGAGAAGGTCTTAAGCCTTTGTGTTTCAT GTCGGTGACCTGAG Apolipophorin precursor protein Gene Bank Accession Number: n/a Refer ence: n/a Comment: Predicted only, not cloned Predicted: AACTACGAACTGTCTGTTTTCTCGGAGAGATACGGAGCAGGAGCCATGCTGAGAGCATTGTTGTGTTT TCTTCATCTTCTTTCATTCCCAGATCTGCCATGCTGAACCTAACAGTGGACATGCTGGGTCAGTCGGTC AACGTGTTGGAAGTGGGAGGTCGTGTACAAGGCCTGGAGCTGTTGCTACAGAAGTACATGGGCAGCTT TTTCAGCGGCGAAGAGAACAAAGGGAAGAGTGGCCCCTCCTCAAGCTGCGTCCGTCAACAGAAGATG GACAAAATGAAGGATAAGTTTGCGGTGTCAAAAGATGATTTGGAGGCCTCTTTCTACGTGAGGATGTT TGGTAATGAGCTGGGCTTTGAGCATCTGAGTGGAGAAGAACTGATGAACATCAAGGACAAGTTCAAC CTCAAGACGATCATGAAAACTTTGGCAAAGGGAGACGATATCTCGTTC ACCAAGAGTGCCATGTTCAT GGACTCCAGCGTGGCTGTGCCAACTATTGCAGGTATGGCTCTGCGTCTCAGTGTGAATGGCACTGCGA CTGTTGAACTTGTTGCCAATGGAAAGATTGACATGAGAAAGATCTCCCGCAAAAACATGGCTGCCAAT TTCAATTTCAGGCCAAGTGCTGCTGTTCAGCTGTCCGGTTTGATGAGTGTGGAAGCGGGAGCGGCCCG GTCAGGACTGCGAATGACCACTACCCTGCACA CTACCAGTGCCACCACAGGTAGCTTGGATTTGACCA ATGGTCAGGTGGTCTCGGTAGACTTTGACACTCCTGTGGAGAAGATGGGGCTTCTTGATGTCAAGTCG GAATTCTTCATCCTTCACACTGAAGCAGAAAAGAGTGTTGAGATGAAACAGAACAGCCTCGTGAGTAA GCGTACATGCTCTCCCGACATGCTGCTGAAGGTCACCGGCTACGAGGTGTGCGGAGAGTTTTCTTTCAT GAGGCCGGCCACGCC AGACACCAGTCCCTATTTCCCCCTTACTGGGCCCGTGAAGTTGAGCCTGACCT TGGACAAGAAAGATGCGCCGAGTGGGTTTAAGCTTGTGGCAAAACAAGTTCAGAATACCGACACCTCT GAGTTCCACTTGTTAGTAGACACCCCGAGGTCGAATCAGGACAGAAAACTTACTGTTGACTACTTTCTC GACCAGCGTGCCAAGTCGCTGGAAGCAGAACTTGCCACCCCGTGGAAGAAAATGTTGGTTAAAGGA G CATACACAGTCGGAGATGACACACGCCTTCTTTCAGCCTCTGTGGTGGTTGATGGACGCGAATATGAA CTGCTCACGCAGTTAGACAAGGATGCAAAAGGGGTCAAAGGTCAAATGGACACCA Beta Amyloid precursor protein ( appear to be the main constituent of amyloid plaques in the brains of Alzheimer's disease patients )+Gene Bank Accession Number: 146230643 Reference: Brough, T., A.B. Kohn, L.L. Moroz (2007) unpublished. CDS: ATGGGGCTCGTTACAAATTGCGCTATTTTCTTAGGATTACTCCAGGTGTTGTACGCCGCCAGTGTGGAG GACAAGTATGAGCCGATGGTGGCCTTCATCTGTGAGAGGCCGGCTATGCACCGGGGGGTCAATGGATG GATCGCTGACAAGTCTACGGACTGCCTCAATAGGATGGAAG ATATCCTCTCTTACTGCCAGAGGATGT ACCCTGACCACAACATCACCAACGTAGTGGAGGCCTCCTACCTGGTCACCATCAAGGACTGGCCCATG ACCTCTGCTGACCGCATGCACCCGCACCGTGTCCGCCCCTTCAGATGTCTCGTGGGTGGCTTCCAGTCG GACGCTCTCCTAGTCCCCCAGCACTGTGTGTTTGACCACCGCCATGACCAGCGCGTGTGTCAGGGCTTC TCTCACTGGAACGTGGTGGCGGA CGAGTCCTGCCGCTCCAAGGACATGCACCTGGAGAGCTTTGGCAT GCTGCTCAACTGCAAGCTGGGCATGTTTAGCGGAGTGGAGTACGTCTGCTGTCCCAACCAGAACAAGC CCAACTACCACCCGCCACAGACCGACGACAAGCCTGACGACTGGCATGACAGCAGCGCCGAGGAGGA GGATGAGGAAGAGGAGAAGGAGGGCGAGGATTCGAACCCCACCGCCCCACAGAGCACATCCAGCGA GGAGAGCAGC GACTCTGGCGCCACCAGCAACAGCGACAGCGCGGTGACCATCAACGAAGACGACCTT GAGGAGGACGTGATGGACTTCTACGAGGCGTATCTGCGTGGCCAGGAGTTCCCCAGCAAGTACAACA ACGAACACAAGAAGTTCCTGGCCGCCAAGGATCGCATGAAGAAGAACCAGCAGCACAAGACTACCAA GCTTCTGCAACAATGGCAGGTGGCCAGGGACCATGTGGACGAGGTCAGGAAGGTGGATACCAAGA AG GCCGACAGTCTGAGCGGAGAGATCGCTTCGAGGTTCCAGAAGCTGTACGCCTCGTACGAGCAGGAAG ACGCGGCAGAGAAGCAGCAGCTGATGGCTCTGCACCAGCAACACGTACAGGCCGCCCTCAACGAGAG

PAGE 130

130 GAAGCGGGACGCCATGGACAACTACATGAAGGCTTTGGAGAAGGGAGATGTGGAGAAGATCATCAAA GACCTGCGTAGCTACTCAAGGCGGAGGAGAAAGGACCGCATGCACACTGTGAA CCACTTCGAGCACG TCAAGTACTCCAACGCTCGCGAGGCGCTGCGCATCCACCCGTTCATCATCAACCACCTGCGTCTCTCAG AGCAGAGGATCGACCAGGCCCTCGAGATGTTGTCCCGCTACCCAGACATCGAGGTCCAAGTCAGACCT GAAGTTGAGGAGTTCATGAAGCGTTTCGACGCCATCGCCAACAGCATCAAGAACGTGGTGCTGCCCCT GCCCAAGGTGGAAGAGGAGCCGCAGACCAAGTCGGCC TCGCAGGAAGCTCCACAAGACGCCAGCGAC AGCGTGAGCCCTGACGACGACATCAGAATAGACGACGCGGGCGACTTTGACCTTAGCAGCGAGCAGG TGGATGAGGAAGGCGACGACGTCTCGGAGGACGAGCACGACTACGAGAGAGGCAGCCAGTTCATCGC TCACCGCATGGACGACAAGATGCACGTGCGACAGGGCTTTGCCGAGTCGGCCGCAACAAGCAGCCAA ATGGGCAGCACCATCGGCATCGCGC TGGGAGGTGTCAGCGTGTTCGTCATCATCGTGGTTGCTGTGTTT ATGGTGAAGCGCAAGAACCGCAACCAGTACCCGTCCCCAGGATACGTGGAGGTTGACCCATCGGCCTC ACCGGAGGAGCGTCATGTCGCCAACATGCAGATGAACGGATACGAGAACCCCACGTACAAATACTTT GAG Protein: MGLVTNCAIFLGLLQVLYAASVEDKYEPMVAFICERPAMHRGVNGWIADKSTDCLNRMEDILSYCQRMY PDHNITNVVEASYLVTIKDWPMTSADRMHPHRVRPFRCLVGGFQSDALLVPQHCVFDHRHDQRVCQGFSH WNVVADESCRSKDMHLESFGMLLNCKLGMFSGVEYVCCPNQNKPNYHPPQTDDKPDDWHDSSAEEEDE EEEKEGEDSNPTAPQSTSSEESSDSGATSNSDSAVTINEDDLEEDVMDFYEAYLRGQEFPSKYNNEHKKFLA AKDRMKKNQQHKTTKLLQQWQVARDHVDEVRKVDTKKADSL SGEIASRFQKLYASYEQEDAAEKQQLM ALHQQHVQAALNERKRDAMDNYMKALEKGDVEKIIKDLRSYSRRRRKDRMHTVNHFEHVKYSNAREAL RIHPFIINHLRLSEQRIDQALEMLSRYPDIEVQVRPEVEEFMKRFDAIANSIKNVVLPLPKVEEEPQTKSASQE APQDASDSVSPDDDIRIDDAGDFDLSSEQVDEEGDDVSEDEHDYERGSQFIAHRMDDKMHVRQGFAESAA TSSQMGSTIGIALGGVS VFVIIVVAVFMVKRKNRNQYPSPGYVEVDPSASPEERHVANMQMNGYENPTYK YF Intersectin 2 (involved in endocytosis) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGAAAGACCTGGTGATCCTCGCTGTCCTCTCTCCTCCTTCCTGAAGTATGTGACGCACTTGCACC CAAT GACAGATGCCTTCTGGCAGCGGCCAAAGCGTAACGTCTCACAGTCAGACTACGTGTGGTACGACCACA CCCCCCT Laminin beta 1 chain precursor ( extracellular matrix glycoproteins, implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, s ignaling, neurite outgrowth and metastasis ) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GGCTCGAGCTGTGACCAGTCTGTTGACAACTACTGGAACCTGCAGAAGGACAACCCTCTTGGATGTCA AGCTTGTACGTGCAACCCAGATGGCACTATTCCAGACCTAGGATGTGATGATGAGACCGGCCTTTGCA GATGCAAGCGTTATGTCACTGGCAAAGATTGTGATGAGTGCTATCCTGGCTTCTACGGTCTGAGTGCTG ATGACCGCTACGGCTGCAAGTCCTGTGACTGTGATATTGGCGGAGCTGTCCGCTACACCTGTGACCAG GCTACTGGCCAGTGCGAGTGCCGGCATCACATCCGTGGAATGCGCTGTACTGAGGTTGATCCTGGTTT CTTCTTTGCTCACCTGGACCACTACAAGTTCGA GGCTGAGTACGGAACTGGCAGCGGTAATGCTCGTG TGTACATCAGGGAGCCGGTGGCTGGATCTCCCAGCTACTGGACAGGACCTGGCTACATGAAGGTGACG GAAGGAGACAGCATTGAGTTCACCGTCAACAACCTCCCATTCAGCACCTACTATGACATTGTCGTCAG ATATGACCCAAGGATGCCTGAGGTTTT

PAGE 131

131 Leucine Rich Repeat (LRR) containing proteins ( Leucine r ich repeat motifs have been identified in a large number of functionally unrelated proteins including ribonuclease inhibitor and toll like receptor s ) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GGGGAAGAACCACGATTACTGCCAACATAATTCAAAATGGCCGACTGTAAGATCTGCACTCTTGGAGA TTCTTCTTCTCTCA TTCGAACAAGTTCAAGGTCACGTCCTTTCATTTTTTCTTGCAGAGTCACAATTTCT GGGTCTTTCTCTTCCATGGCCTGTCTGTGTTTCAGTAATTGTTCTGACGTCTGACCGTCCACAGTGGACA GCTTCTCCCTGAGCTCGGCCTGCGTGGAACGTTCCTGCTCCAGTTTGGCCTGAGACTTGTTCAGCTCTA CTTTCACTTTGCCCACCTCCGACATGGTGTAGCTATTCTTCTCCGACACCTCCTGAGCCAT CTCGTCCAT CTGTCTCTTCATGTTCTCCAGCTCCAGCTGTAAGTTGCTGTTCTTGTGCTGTAGCTGCGTCAGCGAGGCT GTCTGCTGGTTGCAGTGGCTCTGGAGCTCGTCCTTGCTCGTTTGCAGCACTCGGATTTTCTCCTCTAATT GGACGTGACGTTGCTCCTCCTCCGACTTCAGTCGGTGCTTGAGAGATGATGTCTGCTACTCAGCATTCA ACTTATCAGCCATGTTAGCTGCCTTCAGACGGCTAATC TCTTCCCCCATGTCCAGTCTCTGTGACTCCAT TTTTGATATGCGGTCTCGCAGAATACGTTCCATTTCTTCACTCTCTTGTTTCATTCGGTTCATATCCTTCT GTTTGTACTGTTCCAGATCTCGCATGGCTTCCTTTTGTCTCAGCTTCTCCTGGGTAATCCTCTCATCAAA CTGTGAGCCTTTGAGCGTCAGCTCAGACTGTAGGTGAGTCACCTGTTCCTTCAGCTCGTAAATTTGTTC TTGCTGGTGACGGC ATTTACGTTCCAGGTCTTCAACCTTGTTTTGTAACTGGCCGTTCTTGTCCTGCGCC TCGGAGATTTCCCTCAGATACTTGATTTCTTCATTTGCTCTATCCTCTTTCTCTCGACGAAATTCATTTG AATGATTCACAGCCATTTCTTGCATTTCCGCCTTGAATCGATTGACAGTTGAACTGAACTCATTGACTT TCTGCTCTGATAACGCCAGTTCCGTCTCAGCCATAGATAATTTTGCTGCCATAGAGTTGAA TGCACCTT TCCTCTCATCAAGAGCCACTTGCAAGTTCCCAATCTGTTGGGTGGAGACTCTGTGAGATTTCCTCAAGA TTTCATCTTTCTGATCAATTGCAGACATTAAGTCATTGATCTGAAGTTTCTTATCAAATTCCAGATCACG GATGTGTTTTGCCATTGTCTGAGTCCTTTTGAGATTTTCATCTACAATGGTTTGTCGATCAGCATTGCCA GAAACTGCAGCATCAATTGATTTTGTGATGTCCGAANGG AACATTGTTTCCAGCCAGC Mannose binding lectin precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGTGTGGCTTTCTGTTTGATTTGTAGAGGTCCCGATTAATTTATAGAAGTCAAAATGGCAGTTCACGAT GCCTCGTAGAGCTACCTGGATCATCCTGTGTCAATTTTT GTGGGCTCTTGTTGCAGGAGAATGGAACAC CAAGGATTATCAAAGACGAGAACACAGCTTAGTGAAACCGTACCAAGGTTCTGGCATGGGTGTTCCTC AATGGGACTTCCTGGGTAGCACAATGGTGACGAACAACTACATCCGCTTGACTCCGGACAGGCAGAGC AAGATTGGAGCTATTTGGAACAATGTGCCATGCTTTGTACCAAACTGGGAGATTCATATCCAGTTCAA AGTCCATGGCAGTGGGAAGACA CTTTTTGGGGATGGCTTAGCTGTCTGGTATACCAAAGAGAGGATGA AAATTCGGAAATGTGTATGGCAATGCAGATCCCTTCACTGGCTTGGCCATTATTATGGACACCTACAG GAAACACAATGGCCCTCACAATCATAAGCAACCATACATTGCTGCTATGTTAAACAATGGCAGGCTCA CTTATGACCATGACACCGATGGGACACACACACAGCTGTCAGGATGTG Partitioning defective 3 homolog (PARD 3) (may be critical for cell migration) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Partial: CTTCTGATAACGGGCAAAGAACANGAAAGCTCAGAACTTCTGACAAACTGTCATCGTTCAGTTCAAAC TCTAGTAGTAGTACAAATTCCGGTCAAAAGCTTGAGAGCCT TTCTAAACCAATTCCCAAAAGAACATC ATTTGCCTTGAACAACAATGTGATCAAACCGGGCCCCCGATCAAACCACCGAAGAAAACGCCCCCTGC TGTCCCGGTGCGCAGTCCCAACACAGGGCTCACCCCAGGNCCTCCCACTGACTTGCCCTCAACACGGC

PAGE 132

132 TTATAATACTCGCAAAATTGGCAAGAAGCTGGGTATCCAGCTCAAGAAAGGTCCTTTAGGCCTGGGAT TTAGTGTGACATCTCGGGACAATCA GACAGATGGGAATACGCCCATTTACATCCGGAATATTCTGCCC AAGGGGGCTGCTGTACAGGATGGACAACTCAAGCCTGGGGACAGACTCTTGGAGGTGAATGGTGTAG AGATGACTGGCAACACTCAGGCTGAGGCCGTTTCCTTCCTTCGCATG Pituitary adenylate cyclase activating polypeptide precursor Gene Bank Accession Number: n/a Ref erence: n/a Comment: Predicted only, not cloned Predicted: TACGCCTCAAAGCAAAAATATAAGGTCCAATAAGAATAATACATGGTGTTTAGAATTAGTGTCTATCA TGTAGGGTAAGAACAATAAAAAATCCTGGGTGAGCAGGGAATAATGATATAGCTATAGGGCGGTCCT GGATCAGAGGTATTTGCAGTATCTCATGTCGTACGTTGTATGGGCATTGTGCAGATCTGAAA TAAATGT CTTGTGCTTTATCGCACCCAACACAGCTGGGAAAAATAAACAGACAACGGCCGGGGCTGATCTCTTTC CATTCACTTTATCCCACCCACATTAACGAAACGTTGCGAAAGAATAAATGCGTACC Protein deltex 2 (Deltex 2) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: G GATGCCAGCGTGCAGTGCCCGAGTTGTAAGACGATCTACGGAGAGAAGACAGGGATCTGCCCGCCG GGAAATATGCATTGGCGTATCTGTCCTGACCTTCACCTTCCAGGCTACGAGAACTACAGCACGATTAT GGTTACGTACAGCATATCTCCTGGTGTACAGGGCCCGGAGCATCCCCAGCCAGGCGCCAGGTATTCCA CGAGGGGCTTCCCGCGCAGAGGCTTCATACCCGACTGTGACCAGGGCAGGAAGC TCCTGAAG Protein NPC2 homolog precursor (Niemann Pick) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CTTACACAATCTGGGCCGGAATGATGGCGCAGAAGACATCATTGTTCGCTCCATCCACCAATTGCCAT TTGACAAAAACGTTGACAGAAGGGAAATAGCTTTTGA CTTCAAGAATTGGCGTGTAGTTTTGAGTCGT ACTGGCTTTGATGGGGCATTTAAGGCCTGAGTCTTTGCAGGCATCTGCGTTACTGAGAGGAAAGTTCA CATAGGGGAAATCTCCAATCTTGCCAGAAACTTTAGATGTTAACTTGGACTCGTCCAAATTGGCTTTAA AGGTGAAATCAATGGCCACTGAAGTGCCCCTTCTTCAGAATAGCCAAAGGTCCCTGGCAGGTTCCGTT Serine Protease In hibitor 85 Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TGTCTCCTGACAACTGTCCTCGCGTTTGGACTAGTAGAGGCGAACACACCCGATTACTGTAGACTTGCA GCAGACCCTGGACCTTGCGAGGCCGCTACGACCAGATACTACTACAACGATAAAGAAAAGTCATGCC AGCAATTTGTCTAT GGAGGGTGTGACGGTAATGAGAACAACTTCTCTACAGAGTCAGAGTGTCAAGCA GCATGCGTGTGTACTCAAGGATCAAGAAAAATTCGTCGCCTTGGGTGTACAAAACAAGTTTATTTCCA CAATGTTTACAGCGGCCAATGCAGGCTATACGGCCAGCGAAAAGGCGGAAAAAACCATAACGGCTGT AATGCAAAAGCTAATCGTTTCGCAAGCGCCGAGGAGTGCAATTCGGTATGTGCGACGTCACACTGCT A TCTGAGCCCGAAAAGAGGACCATGCAGAGCCCGTAACGAACGTTTCTTTTACAACGCACTCAGCGGCA AGTGCGAGAG

PAGE 133

133 Similar to cat eye syndrome chromosome region, candidate 5 isoform 2 precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: GTCGGCGAGCGCTTCATGCGCACTGCCGACAGCATAATATTGCTTGCAACATCTTTCATCAAGTGTAA GGAATAGTGCTGTCAATGTGAAAAAATTCCACACCCAAAGTGCTCTGTCAAACAAGTGTGAATTTGGA TTCTTGTTTGATATTGATGGAGTGATTGTTCGTGGAAAGAAACCTTTGCCTTTGGCAAAACAAGCTTTT CAACTATTAACAAAGGACAGACAATTCAAAGTTCCAACACTTTTCGTCACG AATGCTGGGAATTCTTT ACGGAGTAAAAAGGCTCAACAATTGTCGGAATGGTTAGATATTGAGGTGTCAGAAGAGCAAGTTGTG ATGTCCCATAGTCCCCTCAAGATGTTTCGGCAGTTTCATGACAAGCATTGTCTGGTCAATGGGCAAGGC CCCATCAAAGAGATTGCTCAGAACATCGGATTCACGCATGTAACCACCACAGAGGAGTTGAATCAACA ATTTCCAAATCTCGACAAAATGGATCATCTAAGGA GAAAACCTGCTCCTTGTGCATTTGAAGAATATTT TCCTCGAATTGATGCTGTGATT SN4* (hypothetical protein, unknown function) Gene Bank Accession Number: n/a Reference: n/a Partial Clone: TCGATGCAGTGAGGGATGCTGTGTCCACGAATACTGCGCATACAACAAAATATGCTACCCGAGAATCC ATTGCCATCTGGGCTGTCCAGA CGGCCATTGTGTGGGCAAGGGTAAAAACATCTCCAGACGCCAAACA AAAACTGCCCAATTCAGGGAAATCCAGAGCCACCAGCTCCTCCCCAAAGCCACAGCACGGACTCTCGG GCAAGGGGCCTATCATTTCCGAACGTTATTCTTATTCCGACGTGAATATGAAGGGGAACGACAACAGC AGGATAGACGAGGATGGGATATTTGTGGACAGAAAAGACCCGACCACGGTTGATGGGATCTGCATGC CTCTTCC GCCGTGCCGAGAGGGCGTTTTCTGTGGGACTGGGCATAGTTGTACCATGAACTATAACCTTG GATACAGTGTGTGTCAGTATAATCTAGATATCGGGAGCACTCTCTGTTACGATCGTCACGGGAAATTG GCCTTGACCCCCATGACGTGAGGTCAGGATGATAAAAGTATACAAAACGCAAAGAAAAAAAAAACTT TTGTAAATGTGTACGTCGATGAATAGATTATTTTTATATCATAATATCATTTTATTTTG ATGTTCAGAGA AATTCAAAAACATAAATGAGCATAAAGTACAATAATTTTGTCTGATATTGATTAAAATGCAGTACACA TTTTTAAAAAAAAAAAAAAAACTGAGTTTGGATCCTGCCATTTCATTACCTCTTTCTCCGCACCCGACA TAGA Stanniocalcin 2 precursor (STC 2) ( has an anti hypocalcemic action on calcium and phosphate homeostasi s) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: TTCTGGCTCTCCCGCGGGAACCCCCACGGCCTTAGCCGAGGGCAGAGCCGTGGAGGAGTCCTGCCTTG CTCTGGCCGACGAGGGAAGCTGTGAGTTTTACACCTGTTTTGAGAACCGCCTGCCCTGTGGCCGAGAC TGGTATATGCTGCGACATGGACA GTACTATTGCAACAAGATGCAGAGACTCAGTTCCACCTTCACAGA CGCGGGCCAGGCATTCCTCAGTGACGCCCAGCGTTGTTTGACCCAAGCTCTCAAGCCCCTGTACCAGA GGGACAATGTAGACTGTCACTCGCTGGAACACCAGGCTGTAGCCGCCATCACTCCTTGCTTCATTGAC AACGCGTTCTGCAGTGTGTTCCGTGACAATACTGACCAGTTTGCCAGGATCTACGATGTGGGCGATCT GTTCACG AGAGGCGCCGGAAAGGTGTGGCGTGAGATTGCGGCCCTAGCCTTGAGATGCACCCACGAG TACATCCGGGACTTCTCCTCTGAGACTCACAGCAACGTCGTGTCCTCAGTCAATGGCATGGTGGACGC CCTCAGGGATGCTGTCGAGGTGAACTAAGTCTCAGCTCAGACTGGTTCATTTCACACTCAACGAACAC CGCTACAACCACCATTTTGTTCTATGTTCTGTTGTGATTTATGATCGATAGTCTTAGAAC CCCAATATTC ATGGCTTCCATTATTTGACCCCCCCCCACCCCCTCCTCCGAAAAAAAGATAAAAATACATTAATTAATT AATAGTCTTGCTATTGAACCTTTAAAATTTTCAAATCTTTGTTCCAAACAAAATTTTCTCGAATACCAG AATCCCAATTCATGACTTCTAGAAAGGGAATTCAAAGAGTGGTGAAAAAATGTAATTCAACTTCGACG TAGACCAATTTACGCCATGGCTCTTTTCGAATTGAGATAACACACACAAAATACACTCTACAAAAGTC AATCCATAATTTGTTTGGAATTCAACTTCTAAGAAAAAAGAATTGAGGGCGTTTTATACTTCCCCGCAA

PAGE 134

134 AAAATGATATAATTTTCAAATCAACCATTTCAAGATCTGAACAGATTGACCCTTTTAGATACGGGTAC ACTTTTTATTCTATAAACCAGCAGATGAGTTTATTTCCCTTGTAATGCCAATACAGGTATACGTTCGTA CCTTTCGTCAGTGTTCGTGGTG CTGGAGTTATGTCCCTTTTGTCAAATTAAATGTCTTCAAATACATCTA CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGT Twisted Gastrulation Protein ( May be involved in dorsoventral axis formation) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: G CATGTGTTGTAAAATACTGCATCACAATTACTTTTGAAGTGCTGTTTTCATATCAGAAGCCAGTATGG CCAGACCTGAGACACTTTTGCTCCTCTTGCTGGGAACCGGAGCCTGCATCGTTTATGCATGCAACAACG CCATCTGTGGACCACGGGTCAGCAAGTGTATGCTGATCAAGTGTTGTGGCTGTGACATGTCAGACTTG AAGAACTGCACTTGCTGCCGCGACTGCCAGGTCTGCTTGAAGTCCCTCTAT ACTGAATGCTGTTCTTGT GTTGGTCTCTGTCCGCCTCCAGATCCTGAGGATGACCTTGCAAAAACGAGTTCTGTTGAGGATCTTCCA GATCCTATCCCTGACTTGTTTGATGTCTTGACGGAGGAGGAGGACCATTTCCAGCGATGGAGTACATA CACTTTTGCTGTCGACCTGGATAAATTGTTGTTCAAACCGGGCGGCGCCAAGTCCCCGAATGTCAACAT GAAGATCATTCAGACTGAAAAAGAGAAGGAAC CTCAGCGCAAGTTTGAACCATCAGAAGAAGAGGAT GCAATTCGTCTTGGTATTCGCAACTGCACTGTGGCTTTCTTCTCTGAGTGCATGTCCATGGGCAAGTGC AAGGCCTCATGCAGGTCCATGGGAGCTGCCAAGTACCGCTGGTTCCACGAACACGGCTGCTGTCAGTG TGTCGGCAGCTCCTGCTTTGACTATGGACTCAGCAACCCACAATGTCTGAGGTGTCCAGAAACGGCCG GTTTCAAGGAGCGCTA CATGTTTGAACACAAGGAGAATTCCATGTACCACAGCGATGGCTACGATAAA GTGGGAATGTCTCAGGCCTTTGTGGACACGGTATAGATGACAGATACGAAAGTGGGAGAGGTGACCTT GGAGCTCACAGAGAGGTGCATGAACTTGGTGCGTATGCACTCGCCAGGTGTGAGCAGAGATCCAGAA TGTGCTGTAGATCTTGTTTCGACGCCTGTCTTGTAAATCTGCTTACTGCAAATGACTATTATTAGATGT G ACAATCATCATAAAGTCTGAACACTGATTCTTGACATTTCTCCGTAAAAAATGGGATGATCAATCTTAA ACACATTATTAGCATGAAGAGACGAGACACTTTGTACGAAATTACACGGGAATAAAGCATGGTTACTG AGAAAGAAAGAATACCACTAGTTACTAACTGACAAAGTGAAGTTAAAGATCGTCATTTTATGCATAGA GGTTTAGGGCAGTTACTTTACTGATTTCTGAAAGCCAGATGCAGTGAAAC Venom protein 2 precursor Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CTCGTTATATTTAAAGGAGACTTGAACTACAGAAAACTTGTAGGAGATTTGAAGTGGGATTATACAAC CGATTTCCAGACGAGTTTAAGGGGGTTTCTACCAG Von Willebrand factor precurs or (Important in maintenance of homeostasis) Gene Bank Accession Number: n/a Reference: n/a Comment: Predicted only, not cloned Predicted: CGACGATCGAAAAGCGTAAAGACCGGTGAAGATCCACTGGCCGTCATAAGTCACGATGGTCAAGCTTT GTGCCCAGCCTGGGGCCAGAGTCACTACAGAACCTTTGACAACAAAATCTTCACCTTCCAAGGGACAT GCAAATACGTCTTGGCTCACGTGCCCAACTATTTCGAAATCATGACAGTCAACGACAAGATATGTGTG GATGGATTGAACTGCACTAGGGATATTGAGATATACTTCAACGACAACGTTGTCCGATTGAGGTCTGC ACAATCTGGGCCCATTGTGGAATGGAATGAGAGAAGGTTCAATATCCCAGGCTCCAGGACAGGCACT GTTATAGAAAGAGTTGGAAGCTACGTGACAGTCA AAAGTCCCTTGCAGTTCACGCTGAAGTGGGACGG TAGTCAGTCCGTGTTTATTACGGTGTCTGACAATCTTAAAGGAAAAGTCAGTGGCTTGTGTGGTCGTTA CAATGGAAACCCACGTGATGACTTTGAGAATGCCAGCGGAAAAGTTGTCACCTCAGTTTCCAGCTTTG GAAACAGCTGGAAGAAAACGAAGCTGTCAAATGGCCCTGTATGCTTGGACTTGCAACAAACCAGCGG ATGCAGCAACGATCTTGT CGAACAGAGTGCTCAAGCCAAATGTGGAGATCTTCTCACAGATCCCGCTT

PAGE 135

135 TCAAACTTTGCCATTCGGTAGTCGACCCACAACCTTATGTAGAGGCCTGCAAAACTGACTGCTGTAGC GCAGACTCTGCTGGGTGTATGTGCGAGACAATGCAAGCATATTCCAGAGCATGTGTGGACCTTGGTGT CAGACTCTCGTGGAGAAATCCAGGGCGTTGCGAAATCCAGTGTGATGGTGGTAAAGTATTCAAAGAAT GC GGCAGTTCATGCATCGAAGACTGTTCCAGTGCCAGTACTACCTGCTCTGACCCCACCTGCATCGATG GGTGCTTCTGTCCAGATGGCTATGCATTACACAATGGTGTATGCATACGGAAACAGGAATGTCCATGC ACATTGACTGCAGAGCAAGGAACTGGAAAAGAGTATCAACAAGGACACATCATCAAAAAAAAAAGTA CCGACTGCG Found by homology search to Lottia genomic screening Lottia Protein Model: jgi_Lotgi1_103075_e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mellifera] NR E value: 9.00E 50 Ac Annotation: Reticulocalbin1 precursor_[1.88764E 10]_[19]_APL_all_052305.1244.C1 Ac E Value : 7.00E 36 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRLE NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHPNEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLEDREVYLTE EENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL* Lottia Protein Model: jgi_Lotgi1_108564_e_gw1.8.40.1 NR Annotation: PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001179636.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 2.00E 86 Ac Annotation: Kinesin like motor protein C20orf23 (Sorting nexin 23)_[3.38462E 18]_[9]_APL_all_052305.1593.C3 Ac E Value: 6.00E 99 Sequence: MYLSCYLPVILLFIEDLKQAVVAMMLRKDEVEEKNKSLKAMLDREMEISSTL RAEIEEMKISFKLSKDKEIA KNETLQKENELLKHQLRKYINAVQLLRTEGAKDDTQGITLEDPQPIIPPAKPSIDYSHEASEYEKKLIQVAEM HGELMEFNELLHRQINCKEAVIRNLKEELTDLRGPLPYDAQSSDDSLSGDLESSLISRCLINIWIPSAFLGGSK TDSHHVYQVYVRIRDEEWNVYRRYSKFLDVHTRLKKVYPLIEKFEFPPKKTIGSKDPKVVTARRKMLQSY LRKVINHLLEKNADLSSNV SKEKLIAVLPFFK* Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Annotation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Ac Annotation: Tubulointerstitialnephritis antigen (TIN Ag)_[0.0]_[3]_APL_all_052305.11433.C1 Ac E Value: 8 .00E 78 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQCCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPWPLTTTTSRVPINILCNVYWFKCTKLHFSSHWECSNDDCLLEADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSNVVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFDEEPAVYHSVRIIGWGVERTIYGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWGQISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV*

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136 Lottia Protein Model : jgi_Lotgi1_134336_e_gw1.89.95.1 NR Annotation: apolipoprotein D [Branchiostoma belcheri tsingtauense] NR E value: 9.00E 41 Ac Annotation: CNSN01 F 078834 501 Ac E Value: 2.00E 25 Sequence: MERILTAVLLCIGVAVYA QVPGLGPCFDAKPQSTLNVSQYLGHWYEITKLPSFFEIEQVCLTANYSVKEDG HIKVFNQGKVNGKNVSSTGDAYVPDPSEPSKLIVKFPQAAPYGPYWVLDTDYTSYSLVFSCEQLGSFAHAK FLWILSRTPSIDPAVKDRLFKRLEKDGIGASGLRATKRSDCS* Lottia Protein Model : jgi_Lotgi1_154590_fgenesh2_pg.C_sca_6000093 NR Annotation: PREDIC TED: similar to meteorin, glial cell differentiation regulator like [Tribolium castaneum] NR E value: 3.00E 24 Ac Annotation: Meteorinprecursor (RJD6)_[1.49345E 11]_[5]_APL_all_052305.10513.C1 Ac E Value: 5.00E 43 Comments: Secreted, involved in both glial cell differentiation and axonal network formation during neurogenesis. Sequence: MDFLQATWLLVVMVIFHIYIITADQICNQCDCTIRDSTGKGNINVKPKCQSGKITWPDPVSAVRLELKPEVV GPFKACIALKSENIVTLVSREITTETVNYRTRSYDSKVDLLPWFSVEGQGKEHCVTSSESSVMLYLEIESTTE DSGLSRTFIYYDIELIPDNKFSPDPMEEC QPCSDDEILRSYCISDYVVVGRMKEVNFQSELDKTKVNIIVDQIV KTTDEDIFKRHDNLLEGALFTSVHCGVQKGPGQFLFTGRNRLGQPILTCAPFYDQWIQIQRLAQEKGTIECA YG* Lottia Protein Model : jgi_Lotgi1_160631_fgenesh2_pg.C_sca_25000236 NR Annotation: Substrate specific endoprotease Tex31 precursor emb| CAD36507.1| substrate specific endoprotease [Conus textile] NR E value: 4.00E 29 Ac Annotation: Delta like protein 3 precursor (Drosophila Delta homolog 3)_[5.19686E 34]_[5]_APL_all_052305.2047.C1 Ac E Value: 2.00E 05 Sequence: MNRLCFLLVVWQIALSQSKVVSSVEGFN GNLLRIVRSANNAAPQGNLNYNSNCNEAYKNISSSHSMCLVD VCTAVALSQEEIDAAVAAHNDYRKNVSPKAAAMQKMVWDDELAEIATKWAQQGRAGHDTKDARKTISL KGTYIGQNAAAGQANLIAAIKAWHDEVVDFKYGVGSINGKDVGHYTQVVHHQSTRVGCGKAHCPDSTY KSYYVCNYAIGQMGLTHPYKSGSSSCSDCPSSCSDNLCDCNGKICFNGGTLDINTCTCSCPSLYSGDQCQTL QCTKTE KSWCRRSYTVADCTKYSNFPIDCNMMCGVCPYPCDGKQCENGGTLDPNTCQCTCKDPYTGPTC ADKSCPGQEEWWCQKFFTAADCTKYSNFPTDCNIMCGVCPACKITA* Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sca_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus] NR E value: 1.00E 133 Ac Annotation: FlbAprotein_[0.0128025]_[2]_APL_all_052305.15119.C1 Ac E Value: 2.00E 84 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHLKK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAGTPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSS DNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKGDPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALELSKELN VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAATIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KKHDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQT STKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNTWPEKEECDSH

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137 LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHPLPWYPPICVTLLTINEYDEKTYDNLKSVGHP GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGARWVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYA DVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSVAPIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKSGECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGEDCNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLH STQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN HCSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPKSWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLMLLGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTLGNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model : jgi_Lotgi1_168805_fgenesh2_pg.C_sca_79000073 NR Annotation: PREDICTED: similar to plexin A CG11081 PA, isoform A [Apis mellifera] (a neuronal semaphorin receptor that controls axon guidance) NR E value: 1.00E 121 Ac Annotati on: PlexinA3 precursor (Plexin 4) (Transmembrane protein sex)_[1.07485E 23]_[4]_APL_all_052305.11722.C1 Ac E Value: 1.00E 27 Sequence: MEGGIRLQLFLLVSLICHTQCIKILHSFRDPGGNILQKLVIDDQTGNVFLGALNRLHKLDPDLNIVQSASTGP RLDNVECPPPLLPCDKPKTQLNSQIKGLVIDSASNSLILCSSLFHGSC QTLALNNITNVKKFFHKPLVPNDHH SCYMFLAPSINNTQALYIATAYSDLGDAAYRDLVPSISSRSLDNLDFIHRDTEGSTKLEILKEYRESFIVRQLY GFQNGGFVYFITLQRENPGSQKIVSRINRLCQQDRYFRSFVEIPIKCQKSDSDSYDVVQNAYFTNGKLYVIFS NTVSSAGAKKSTFCEYDIQDIDLEFNETVRNCYKAVGNFGPEHYKKIEPCPPLIFGSVDFCGESGEWKDYSA IEGSKPISATPL HFFTSDKPTAIFVHPEGNHRYAYIGTQDGHVIKLRFENGQVNQLFKITHSVGDIILQITNHPQ SNLLYILGSHKLSVLSLNHCDDLTSCDECMNDGDETCGWCVMDNRCTTKELCQSSVITPPWLSRTDQSCAS ISSLQPSSLSYKKFINGGDSRQIKFQVNKLKFESNRNLNCLFINGEKHDNTMAILDQKENTITCPLPRQLPTIP QGKDHEDLELQFHVEGKMIVKRDVSVFDCQGNNNCTSCSTSQFNCHWC PVSHSCVEQSESCPQGDSIKVN DRCPRLETAASDTDILVHSGELKTISVQVRALEANQRSNLKCHFDLSGQVQTVSAKISSSTLTCETIKLAYA DDIPYIVAGFKVTWGQQNHPLDNPQRMNVRIYKCASMVTNCGKCLSLDVEYECGWCEDQCSLQKKCQKS WLDQSETCPNPQILRYSPSKGPVKGKTEIQVNGINLGKEISDILGQVTVAGLPCDVMQEHYEPSSSFKCEVK GVTSAKSGTVKVVVNNKYTAE SDTVFEFVDPVLNHVDPKQGLMSGGTRVLIKGNNLDVGSDTQVIVGGN SVPVIK* Lottia Protein Model : jgi_Lotgi1_192261_estExt_Genewise1.C_sca_450209 NR Annotation: conserved hypothetical protein [Aedes aegypti] gb|EAT46740.1| conserved hypothetical protein [Aedes aegypti] NR E value: 1.00E 128 Ac Annotation: Ero1 likeprotein precursor (Endoplasmic oxidoreductin 1 like protein)_[2.65914E 25]_[1]_APLCNSN01 F 139925 501 (oxidoreductase activity) Ac E Value: 1.00E 38 Sequence: MTLYITSLVFFLFINGTLQVKNRSSKVADRCFCKLNGEIDDCSCKVETLSVLNNNKIYPRIKSLLSRNYFRYF KVNLKKKCPFWHDDSRCALKDCHVDTCKEEEIPSAIKKGSSAYRYSEEAQKEESCAEEKELSALNTTISDES IQAFKDWKEHDESQDLFCDIDDESSADSEYVDLLLNPERYTGYKGESPHRIWRSIYQENCFKPQTEYIYGPS KSSSPLCLEKRAFHRLISGLHTSINIHLCADYLYQENLGYGMKAHWKPNVAEFQKRFDPQTSNGEGPQRLK NLFFTYLVELRAIAKAAPYLQEED FFTADSDEDKDVRQGIDDLLNIISTFPDHFDESKLFAGNPNEAKLLKDE FRNHFRNVSRIMDCVGCDKCKLWGKLQTQGMGTALKILFSGDDIGPDSTVNAQQKKHFQLTRSEIVTLFN AFGRLSKSIHSLEMFKSLLSKS* Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 9 4 [Crassostre

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138 NR E value: 0 Ac Annotation: Heatshock protein HSP 90 beta (HSP 84) (HSP 90)_[0.0]_[23]_APL_all_052305.1.CB102 Ac E Value: 1.00E 105 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKN KEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDD EYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEAT EKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_kg.C_sca_820009 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Ac Annotation: Translocon associated protein, delta subunit precursor (TRAP delta) (Signal seq_[3.38716E 37]_[8]_APL_all_052305.232.C1 (bind calcium to the ER membrane) Ac E Value: 3.00E 46 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SYIQSEFVAACVAILVWWLAFSAKSKLSP* Lottia Protein Model : jgi_Lotgi1_211937_estExt_Gene wise1Plus.C_sca_110124 NR Annotation: PREDICTED: similar to SPFH domain family, member 1 [Monodelphis domestica] NR E value: 1.00E 124 Ac Annotation: ProteinC8orf2 (UNQ2441/PRO9924/PRO5003)_[0.0]_[11]_APL_all_052305.7501 .C1 Ac E Value: 1.00E 117 Sequence: MANPALALLSVSAVALAIFLNFSIHKIEEGHVGVYYRGGALLSSTSEPGYHFMLPFLTTFRSVQTTLQTDEV KNVPCGTSGGVIIYFERIEVVNKLDRAIVYETVRNYTADYDKTLIYNKIHHELNQFCSVHNLQEVYIDLFDQI DENLKKALQLDLNVMAPGLMVQAVRVTKPKIPESIRKNYEAMEGEKTKLLIAIQKQKLVEKEAETERKKA VIEAEKVALVAKIQWEQKVMEKESEKRMSEIEDSANTARAKAKADAEFYKTQKEIESNKLKLTKEFLEFTK YESISKNTKIYFGNSIPGIFSDKVEPQEIAKVVGERK* Lottia Protein Model : jgi_Lotgi1_116422_e_gw1.24.410.1 NR Annotation: PREDICTED: similar to CG14764 PA [Apis mellifera] NR E value: 7.00E 58 Ac Annotation: Retinitis pigmentosa 1 like 1 protein_[0.0216154]_[1]_APLPEG003 C 222184 501 Ac E Value: 3.00E 13 Sequence: MKTKNFYILVLLISVTSVALWLVSYPSPTFRNIMSTTQNNIRKIPKHMQMSKSPDLEVDKKYLDLLGFDSES KPKNDIVVIGSALMPADTENTILFLKSAQEYVPNLKLVLFDMGLSKADKQTLTNCRNETKHCEIRKFNFDQ YPSHISDFANKSYKPICIQLLLKEFGTVIWADTS ELFASSDIDNTMKQAKTEGLVAWTIEDATSSLTHPKMFD YFKTDQKKYFFHHAVQTSHIILVNTDKIHENVMLPWVKCALVEECINPMGAQNSGCNYQRKPKFRYTGCH WYDMSALNVILGLAFDFDEAYSGKDKIFWTKFDQEKAKAENMTDGAVHKVSVPHRVYF*

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139 Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found N R E value: Ac Annotation: HISTIDINE RICH GLYCOPROTEIN PRECURSOR_[7.36731E 10]_[4]_APL_all_052305.6806.C1 Ac E Value: 1.00E 16 Sequence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_149784_fgenesh2_kg.C_sca_15000101 NR Annotation: PREDICTED: similar to chromosome 19 open reading frame 10 [Canis familiaris] NR E value: 9.00E 20 Ac Annotation: Structural maintenance of chromosome 1 (DA box protein SMC1 )_[3.12337]_[10]_APL_all_052305.5506.C1 Ac E Value: 2.00E 18 Sequence: MLISNDFKCLFMLILGFIQWVSSYEMTRDYEEASHFDVMPGGAVLSHEKQLDGFTCKFTYACQGGTKEGW QLTINTNTDKTKYLCSVQRPDGKSYLFFQSFKLEFTGLEVKEGYAMGANFKPLSPSEYQVDFQKHSISDVA GKFGSQLEKVELYGEKGKAEL* Lottia Protein Model : jgi_Lotgi1_152847_fgenesh2_pg.C_sca_2000267 NR Annotation: SARP 19 precursor [Littorina littorea] NR E value: 5.00E 08 Ac Annotation: CNSN01 F 148317 501 Ac E Value: 2.00E 07 Sequence: MIAFICVLGFISGCLAAPCTHTNSAEHTEEQVIARVIALADHNNDGILSVTDLAAYFVINFDHNHNSEV DKN EFILQWHETFHDEKAFAEHVFTHFDTNADNVLNLSDLFALNIRLDTDGNGTVTNKELESYLQLIYNAC* Lottia Protein Model : jgi_Lotgi1_158797_fgenesh2_pg.C_sca_18000147 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E value: 5.00E 92 Ac Annotation: KILLERTOXIN KHR PRE CURSOR (KILLER OF HEAT RESISTANT)_[0.162197]_[4]_APL_all_052305.10913.C1 Ac E Value: 1.00E 15 Sequence: MKIHILAVLFSFFSATNAATLRFVSQTPLSITINKDGSYTVNVNGEPWLESSAIYFYAEGRRVSMGDGSLKIA STFMDNGYDSVGQYGKTCWRFEIESKYIDTCFKQYMEPTPFIIFEQHYLNDTENTASIDYDYTIAGFPAFTAP PTGKLGYLHYAGSMTGDSDKSFGIWQEKAKMLTGVAGGPLVLFDKDADDCLILSPLNQFMAASSLFDGQ NYSTGVLGGVNFVPKDFVLQTVLFYGKGINHAMSDWGLLLTQYYGKDDSYRKSDLTLNYIGYWTDGGA YYYYNTEPNKTYQQTILDVRQNAKDIGMPYKFVQYDSFFYYKGPHGGVKTWVPMPDLFPDGFQYLYNQT GWPAGAHNRWWSPKTTYARQNGGNFDFIVEEAKAVPTDQAFWDYLFDSSKKWGLTL YEQDWLDNEFAG VKALLTQIDLGRTWLMQMGRAAQENGITIQYCMSNPRHIMQSVEIPVVTQARASGDYRAGKDNWKIGISSI MVDALGVAPFKDNFWSSPDQPGNPAYPDLKETHADLQVAVSTLSTAPVAPGDMLNKTNVPLVMQCCNA DGMLLQPSKPAKAIDAQILQLAFGGDVGPQGDIWTTFSNIAGFYFGIILGTDMTADYNLTPSKASFKAFLPS EVFPRFASIPKSQEFSASNPLVITTNCTKTNIC LYYTSPVFSPGGTNVLIYGEENKIVPMSPKRVTDITLDRDL KLTLTGAAKESVRFGFILPDQYITITCQLGQSGSAVLSFNERSCNGY*

PAGE 140

140 Lottia Protein Model : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO44886.1| predicted protein [Nemat ostella vectensis] NR E value: 2e 07 Ac Annotation: Low density lipoprotein receptor related protein 1B precursor (Low density lipo_[3.7488E 24]_[1]_APLCNSN01 F 143892 501 Ac E Value: 2.00E 06 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNTINDCDDGS DENNCACLEKLGMEDGTI LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEGLNAFSNNSLYRSLFFPSLVTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGKFGCLSKLGSGGCKSFYLALGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVRKTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVLRTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSAT NDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDNFNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWSDEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model : jgi_Lotgi1_153437_fg enesh2_pg.C_sca_3000340 NR Annotation: hypothetical protein PB105991.00.0 [Plasmodium berghei strain ANKA] emb|CAI04701.1| conserved hypothetical protein [Plasmodium berghei] NR E value: 8.00E 09 Ac Annotation: DAZ associated protein 1 (Deleted in azoosper mia associated protein 1) (Proline_[0.0]_[6]_APL_all_052305.517.C1 Ac E Value: 3.00E 09 Sequence: MKTMVAIIGVIIVSCVLHTSGLPLFKNILNERSVEYGGESGNGFKNERAVGYGGESGNGFLNERSVEYGGES GNGFQNERAVGYGGESGNGFLNERSVEYGGESGNGFQNERAVEYGGESGNGFLNERSVEYGGESGNGFQ NERAVGYGGESGNGFLN ERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAIEYGGESGNGFLNERAVE YGGESGNGFLNERAIEYGGESGNGFQNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNERSVQNSGKSG NGFLNERSVQQGGKSGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNE RSVQYGGESGNGFLNERSVQYGGESGNGFLNERSVEYGGESGNGFLNERSVEYGGESGNGFLN ERSVEYG GEAGNGLK* Lottia Protein Model : jgi_Lotgi1_160696_fgenesh2_pg.C_sca_25000301 NR Annotation: No match found NR E value: Ac Annotation: Translocated actin recruiting phosphoprotein (Tarp protein)_[0.451769]_[276]_L7ALL contig08326_921 Ac E Value: 8. 00E 06 Sequence: MKLLLLLTVLIPLCVSVPTKPGCCVPPVWEGIQGMFSGDGSGPAPELIQQSALISYDEKRQMININITQSIHGL TSDVRILTDYKKGVQYLINSKSRSCTTSKVPQTFQRFCIPDYFYYKGSFNVAFTLRASTEQFIVPLIWSKCKS NSSNFFKMRKACFSRDIQLVVGTCDCLFN* Lottia Protein Model : jgi_Lotgi1_163475_fgenesh2_pg.C_sca_40 000029 NR Annotation: Myc homolog [Crassostrea virginica] NR E value: 8.00E 08 Ac Annotation: 104120_3728_3444 (Unknown)

PAGE 141

141 Ac E Value: 1.00E 11 Sequence: MDLSYLYYGMFWFSCLLVSTVSAEFSYEEVTYRIPPTPTGFYDQNCTDKISDCEEFGIASCFKPYESWAREN CAKTCEFCQGKPEKPPTCTDVEKGCDLYTTSI CTDPSFISWAKAKCRRFCRMCPEQVLAELDLMTTTLTPV DCFDKVVCERYGKKACTGDFASWATDNCQKYCGFCSTGVTKSKRLCVDKLPNCPQYDSRICTDKKYYSW TDENCRSTCKFCQDKLVPFLIPTPQAVRTPKPPTT* Lottia Protein Model : jgi_Lotgi1_166720_fgenesh2_pg.C_sca_60000088 NR Annotation: No match found NR E value: Ac Annotation: ProteinC 20orf129_[0.888824]_[1]_APLPEG002 C 006682 301 Ac E Value: 8.00E 23 Sequence: MMLLIKTGVLLIILFSLNTLCDSCACEFHPVLSGKWFFTGENKNINIKIHSKIIVFKVSNYLKLRYKCTERYG NIYLLRILCLFLEVDFLSDLFMKGGFTSIHVEGRGFDSFIFSPKNVTRKYANIDNTCDSEISRERKFIYRKGGC RVPRDMV GTWGFTFRQSSAITMGKRNLTIHFLNNDNLTLNCEARDRNMFLFRRSSYINSSTDAIICVQIDPV LDDPFYDYEFNRIITGNAVENLVRTVPRGKKLYINSDCDMIHSPARPEYIYTVDNIL* Lottia Protein Model : jgi_Lotgi1_166891_fgenesh2_pg.C_sca_62000015 NR Annotation: PREDICTED: similar to scavenger receptor class F, member 1 isoform 1 precursor [Canis familiaris] NR E value: 9.00E 05 Ac Annotation: Multiple EGF like domain protein 3_[0.0]_[1]_APLCNSN01 F 105232 501 Ac E Value: 3.00E 13 Sequence: MATGLYFILSIVTCIINNCVIGLEICEDHYVSCPSQDCGIFPLEPLFRLHCMKTCGVCTDVNRASRKPTAQS SV DVNGMASHAVDGNTDLNICSKTNSSQPDTQWWCSDLQQVYEIDAIKIYSPVIGTKCNTGYYSQSSTSCSPC DTCSENSCEPTNGMCWDGCKQGFKGETCRQCEDGLFGPTCRFACNTTKCQSNCDRLTGICSECDTGYYGK NCELVCDKDCREGKCLKTDGSCTTDGKGSVVSTERAPVQHYTENINKYLVGVIGTFIGIVVTILLVLTYRCF KKRRSDTIYELPTRPPSKQELDFPLDPQYISSGVSTTSSRYTEVHPDSGSSHGSHYLARRNLSQYTHKLRPPA ELPENEKRRLQAEMRQSKISGPRGKYMEYPNDAYDRDGSDYEAVKTEI* Lottia Protein Model : jgi_Lotgi1_171718_fgenesh2_pg.C_sca_119000006 NR Annotation: PREDICTED: similar to orphan G protein coupled receptor HG38 [Gallus gallus] NR E value: 1 .00E 15 Ac Annotation: Coiled coildomain containing protein 22_[0.568464]_[1]_L7ALL ETMYUMG01BM4OM_257 Ac E Value: 1.00E 05 Sequence: MAPIYLTFIASLLLASSTFCEQLCLEGCKCEDHPSEKLGPHLMIDCSNLGSSSVPRDLNKFQNYGRPMHLKL SKNIIARVSKADFPENLPIIALDFNQNRGVMITDDAFMNIKGLRYLGLND ISMPFTDTMKYLKGMTKLRYLY LNKNYQFGEGLVPVSLFSGLELKLKDLSLHDCYLKGIENGALANVVITGSLDIGANQLDHIPEEVKTLKNLT KLDVSQNSIKSIPSNTFEGMSKLRTLSLSNNYINGQSFKTDSFNGLKLKELRLEKSELNTIPSAALSSLSSLKIL KLNNSPIKTIPKHSFNGSYCLEVLDLTNVLVEIENTHLDGLESCLQKLYLNNMQIREIPKFFKDFKVLNYVSL SGNSISEIKMDDLV GTTITELDLTENPLHRIEKGSFEEFPHAISLIISDTRLSNLSFVRDYEYGTIKTIKLGNVLP MCDCELAFAEYRVLEIHGNCNFNNTKNVAISTDEFMSVTSHCWPDGKMPQKKDVWAVVGDNSAGTFSTS ILSYFCVFLFFKVLNIV* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_sca_130000017 NR Annotation: predicted protein [Nem atostella vectensis] gb|EDO35011.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 26

PAGE 142

142 Ac Annotation: Cellwall protein DAN4 precursor_[8.10371E 4]_[9]_APL_all_052305.453.C4 Ac E Value: 5.00E 19 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDA REKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPHIGNRENYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG RYTYRTTNIAQDISCCYVGRTC LHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIKSFVSYGTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPIRGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTI LTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMYMFPEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTPNYPFDYPLNTECNWLFVAPEGSEVML NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEM AHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYDNFDDCEELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD GFTNWRSTYP VSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADRKSS CTRNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESFTTQCPA GQICTQNCQHFSRACRRSGEVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGPTCYFFSLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTSAGNTESSSTGASRSSPSTDAFVSEDTTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model : jgi_Lotgi1_172 798_fgenesh2_pg.C_sca_142000011 NR Annotation: gb|AAA30047.1| calcium binding protein [Strongylocentrotus purpuratus] NR E value: 7.00E 07 Ac Annotation: P protein [Includes: DNA directed DNA polymerase ; RNA directed DNA polymerase _[0.795798]_[34]_APL_a ll_052305.384.C8 Ac E Value: 3.00E 24 Sequence: MRADSIQVRGLKALVLILLVLYEGHTKETCRTGVKTDKNLRGVNLDKTSGLPFYWFDLHRASDGYHSEYT YTFRLHSSSEASPFSDAVLQVETANNCGTGDLVVNTTFLYQPRNCPKMIMTEYKKPMTNVPFKWRAPKCG CIKFRATVIRNGVTYFYDDENISDGNLVKTVCPHGVRVAEQLVEVDEKTTDSTTKTLSTVTSKMDRAA LLD LLCKITSTLNNLELLKRPEFIQRRKLQNSSLVKLLNIELALEQRKADISSCCHKKGEDKTICFDNVRRYRIDQ LCGDGQPRIPFVEQKRNYMRKRESECCWRIGQPRYQCFSKSSEQLTEYSVDYSLDESDPANDIADYPKEVE RYEMASNTKFFSPPPTLKPDNQNIETLTKLEETTTPTSVTKSVTLPPIRQPSARPHRQTTPTSVLTHHRQRPVP AVKTKLRQSRTVGQGDMGYNLRKQAKYTRKTTNLR HSASSVEITVNTLKQKLWKSSLKLECCQQGRIFAA KTGRTDVWDRCRSAAKDFRSSVVRGKKICSNTFQKCCVEQFITATAKASTIPKPNTPKGSPSTKQLVTTTSL RTNIKKENYRRAPLDVDSSREADEKYASMESKAEDDLMEQDDKASAEDAGEHVDDDGDDVSDDSDNDD QGEDGDQDEDDDQDDDDKDGDVDDDKVDEINNDEKNRKRKFIDYPREDPSADTFHNLQTKRKSTPNTDI AHRDSNHRNLKI ESAKRKRNRRRHNRKLRRRKHKSD* Lottia Protein Model : jgi_Lotgi1_201804_estExt_Genewise1.C_sca_1950010 NR Annotation: DICTED: similar to putative porin precursor [Strongylocentrotus purpuratus] ref|XP_001192936.1| PREDICTED: similar to putative porin precursor [Strongylocentrotus purpuratus] NR E value: 8.00E 19 Ac Annotation: WAP four disulfide core domain protein 2 precursor (Epididymal secretory protei_[1.82442E 12]_[6]_APL_all_052305.6277.C1 Ac E Value: 3.00E 12

PAGE 143

143 Sequence: MRLLIFTLSLLVAVVRSVDASQCPEPPSGFGPCVIT DANCLTDSECSNGQICCPHLCGKICKDKVSLTVVKPG TCPSNAAISCFVGSVADTCSSDSDCPGNQKCCNLCGKNCSPPQGSAPQLPPIGLPAPPPVCPPVCAIACEYGN VLDQNGCPTCSCKKTPCKGEAPLTLDRNGNTINCGRGGVRCPLNSECKIHPADAYALCCPIEPDDPKPGVCP KLPNPLPRCLIFQNDCKQDSECPGDQKCCSVTCGIGCIDPVKLCPVVDCLQYCEFGYRLDSNGCQTCQCKK SPC EDEAPMTLDSTGNTINCGRGGVRCPSNSECKIHPADRYAVCCPKVCPPVCEIYCEFGNVLDENGCKTC KCKKSPCEDGAPMTLDSNGNTINCGRGGVRCPLNSECKIHPADRYAVCCPKVCPPVCAIYCQYGNVLDEN GCATCTCKKTPCKGEAPLTLDRNGNTINCGRGGVRCPLNSDCKIHPADAYAVCCPKEPSPVCKFGSCPVPP SSFLLRCASIDRCKYDSECEGTKKCCKHPCGRQCRDIVKSKPGVCPK KADPTLVLCKRRGDDCYEDATCPG SQKCCQGACGNACMDPRLTSTDHAGTCPALDPKIQSLILCASLDTECELDSDCKVDQKCCKDYCGIKCVKS VPKPIIKKGTCPRLRYRCRRKRRDFCRSDGDCPGDRKCCRLPCGYKCRRPKRIPIIRKECPAVNTHPIYKLFC SPPLNPPCQVDRDCPQGKICCPGVCANRCLKAPTKPVCDPNPCGDAQIKCQYGQTKDENGCLLCQCKSYDF KMS* Lottia Prot ein Model : jgi_Lotgi1_205030_estExt_fgenesh2_kg.C_sca_1120002 NR Annotation: PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001188672.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 3.00E 20 Ac Annotat ion: Sec sixty one protein homolog_[0.842179]_[2]_APL_all_052305.13357.C1 Ac E Value: 4.00E 07 Sequence: MKLMFLGFIGILMLSTSNNASASPWFCKGLECPKFNLLNKTKDYEVRQYGSLTWVTTSHIGQWKSDILGEL LFKNLFSYIEGNNTMKKQLDLAVPLLTKLESAGDRGSIYTMHLLIPRENIDHLPSPLNTNLTFITIPPSTYYIRS FDGVA YDRQYMKVLQELVNAINDTGAYPDDFFYTATYDHPQVKENRHNEVWLLKKEKIP* Unknown Function Lottia Protein Model: jgi_Lotgi1_105864_e_gw1.4.718.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO49128.1| predicted protein [Nematostella vectensis] NR E value: 3.00E 15 Ac Annotation: CNSN01 F 034625 501 Ac E Value: 3.00E 09 Sequence: ILTCLAFSLVWHSHLSGILTCLAFSLAWHSHLPGILTCLAFSLAWHSRLPGILTCLAFSLAWHSHLPGTLACL AFSLVWHSRLPGTLACLAFSLDWHSHLPGILTCLAFSLDWHSHLTGILSCLAFSLDWHSHLTGILTCLAFSL VWHSHLTGILT* Lottia Protein Model: jgi_Lotgi1_107224_e_gw1.6.194.1 NR Annotation: No match found NR E value: Ac Annotation: PEG002 C 001338 501 Ac E Value: 2.00E 06 Sequence: CFLSVCLCSSICFTTECCFLSACLRVCFTTECCFLSVCLLFKRSRVCFTTECCFLSPCLRVCFTTECCFLSVCLL FKRSRVCFTTECCFLSVCL* Lottia Protein Model : jgi_Lotgi1_122612_e_gw1.41.141.1

PAGE 144

144 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO49127.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Ac Annotation: 307649_3461_0681 (unknown) Ac E Value: 3.00E 08 Sequence: MSYRRCFG ISCSSMSFAVDWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILLVDVV DWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILMVDVVEWCILLVDVEDWCILLV DGVEWCILLVDGVEWCILMVDGVAWCMLLVDGVEWCILMVDVVEWSILLVDVEDWCILLVDGVEWCIL LVDVVDWLILFGISRFKLELLHLEI* Lottia Protein Model : jgi_Lotgi1_ 132441_e_gw1.79.137.1 NR Annotation: PREDICTED: similar to CG11081 PA [Nasonia vitripennis] NR E value: 4.00E 72 Ac Annotation: CNSN01 F 064139 501 (unknown) Ac E Value: 5.00E 42 Sequence: MAWKIYVLFVCVLCLRFILAARENYIKATFEGAKDRGIFNKLVVNRETNELYLAAVNHLYKLSPNLE VIQE VVTGPHLDDPSCTSLTNLTCDPDLKSCACKLRPYYTKALVVDYVNKKLITCSTLYHGHCDKLDLDNITNKQ MFPFMPMVANNATASTVMFIAPGPAVVKDEPQRQVLYVAASYTRTGLKAYREQVPAFCSRSLEDFNLLLK NAFGSSSIEIESQHRDTFPVRYIYGFGSDNFSYVLSIQKANVQTDRYVTKISRICQSDDDYYSYAEVGLSCTM QNTEFNLLQAAYVGKSGTKLARSLGIPTTEDVLYTVFS IGDSNSANPSATSAMCVYSLRDIRKKFTENIQEC FSGIGNTGPDHMIQSSKCLKVVSSHFFVIFFLESFESADLSVL* Lottia Protein Model : jgi_Lotgi1_143169_e_gw1.194.72.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO36917.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Ac Annotation: CNSN01 F 096652 501 (unknown) Ac E Value: 1.00E 12 Sequence: MVKVCSIPLTVVKVCSIPLTMVKVSSIPLTISKVCSIPLTMVKVSSIPLTMVKVCSIPLTMVKVCSIPLTIVKVS SIPLTIVKVCSIPLTMVKVSSIPLTIIKVCSIPLTVVKVCSIPLTVVKVCSIPLTMVKVSSIPLTMVKVCSIPLTVV KVSSIPLTMVKVCSIPLTVVKVCS Lottia Protein Model : jgi_Lotgi1_145599_e_gw1.360.11.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO49883.1| predicted protein [Nematostella vectensis] NR E value: 6.00E 16 Ac Annotation: CNSN01 F 006444 501 (unknown) Ac E Value: 4.00E 10 Sequence: LVVYCHCGLVVYCHWELVVYCHWGLVVYCHWDLEVYCIVYCYWGLEVYCHWGLEVQCYWCLEVYCH CDQVVYCSWELVVYCHWGLVVYCYWGLEVYCHWGLEVQCYWCLEVYCHCDQVVYCSWELVVYCHW GLVVYCHWGLVVY* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: L OC431836 protein [Xenopus laevis] NR E value: 1.00E 140 Ac Annotation: CNSN01 F 041871 501 (unknown)

PAGE 145

145 Ac E Value: 7.00E 24 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFE TVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* Lottia Protein Model : jgi_Lotgi1_157590_fgenesh2_pg.C_sca_14000213 NR Annotation: No match found NR E value: Ac Annotation: CNSN01 C 005830 5 01 (unknown) Ac E Value: 5.00E 11 Sequence: MLMFTSLLFVCFASIYAGESVYPDCQNPDNNERIPNGSKFKLENSGDCIEFTCKDGGYSASSVECDNNGECV AVGRTVEENCRSKKCVYDKFVGFQVTETKCEDDEGQCHSPGEEFTRTYQGTTYSKTTCTVDGNLVKYSYK N* Lottia Protein Model : jgi_Lotgi1_161950_fgenesh2_pg.C_sca_31000192 NR Annotation: RE49668p, putative [Brugia malayi] NR E value: 3.00E 08 Ac Annotation: CNSN01 F 103859 501 (unknown) Ac E Value: 9.00E 12 Sequence: MKLGGDYGILCVLFTILTPVISIDCYACTSLGGNNEGCEDSFERTLKTVHFIARDCYYGYFRGTHCYKLKGE RADGSSIMVRHCSDSHWGSHCGDIKYTNEHNVEEKI KGCLETCDHDGCNTATQKQFNFSLVIMAVLMTSA LLL* Lottia Protein Model : jgi_Lotgi1_165259_fgenesh2_pg.C_sca_50000100 NR Annotation: hypothetical protein CHLREDRAFT_144736 [Chlamydomonas reinhardtii] gb|EDP05543.1| predicted protein [Chlamydomonas reinhardtii] NR E v alue: 6.00E 23 Ac Annotation: 220720_2775_1358 (Unknown) Ac E Value: 6.00E 10 Sequence: MKVLLVLDICCLIGLVSVGEAVCPDNCNYNGVCDVKHSRCNCYEGFTGSDCKTDCRCNGHGTCQSGSVC KCDEGWKYSGGQCVWDCHCLNGAKCIGPGECGCVHNCKMGNCRNGQCQCWNGYKGSDCSEYDPTM* Lottia Protein Model : jgi_Lotgi 1_166645_fgenesh2_pg.C_sca_60000013 NR Annotation: No match found NR E value: Ac Annotation: CNS003 F 114184 501 (unknown) Ac E Value: 9.00E 13 Sequence: MGASILLGLTILLSYVNQVHLQMFHPRDISRGGQQQEEQLRRGYLIQILKSFLNRRQEDATEKRKRSCNLNL GFHCQTDEYSSIADMYDFLQSALSPGKRK RNVKIVSIEGS*

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146 Lottia Protein Model : jgi_Lotgi1_167022_fgenesh2_pg.C_sca_63000026 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO43826.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 08 Ac Annotation: CNSN01 F 102728 501 (un known) Ac E Value: 7.00E 34 Sequence: MVAVRIRKYKNLLFFALLFAGVLSISTIFYSNHYQIMNTGTRSDVNTGFSYSNANSSKYVIYICDGKNSCGG YGDRQKGIVASYIISVMMNRQFGVIMNDSCDIKQMFKPNQINWIVDNNDIKSKTSKRIKALDGYSDKLRKS MIEMDLDQEYKEEVIYFSLNLEFVFYLRQNKRYEKQLKWLEHLSMSEIYNVVWQSIFKLRPYIREKLDPFL DLKKQGLKLISAQIRLGKNPTIPHDSRVVNSLNNMNVLWQFYKKYNDSSKYRIFISTDSDTVREKARSIFPD VYSDVPGKVFHVERSNKTDICNGWRKVILDQVILSLSDVLVISNSGFGRIAAFFRQNENDLYCLNLDKIRRC TTQSEIFVDKTW* Lottia Protein Model : jgi_Lotgi1_168040_fgenesh2_pg.C_sca_71000102 NR Annotation: No match found NR E value: Ac Annotation: PEG003 C 229928 501 (unknown) Ac E Value: 1.00E 17 Sequence: MVGTRYLQLATSLAVFLLVLFLTCTQAAPVDDFETDNEALRKALFIARLLSSSDRLKNTKPEGSNLDFSELA SIPFSNQQKRYRPPMQGRSGGMSLCLWKVCPAAPWLVSKRSEKTWDKNNMLGK* Lottia Protein Model : jgi_Lotgi1_170051_fge nesh2_pg.C_sca_92000069 NR Annotation: hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] gb|EAR97557.1| hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] NR E value: 3.00E 08 Ac Annotation: PEG001 C 000321 501 (unknown ) Ac E Value: 2.00E 18 Sequence: MKMINIFIVVYCLISPCLGFWLSSKTVDPKVDISTECINKALTGDCGFFTCFEERLPCGEYGYAESYGGKYC WQFQQSHHLFTKKGVEFVEKLTRCHMNRSITSYRQNQIECFSHYDQSFVIMGDCYVESGFCDVVIDNFMSL ARILEPKDFTNYRVVREVFRAAKQCPNNISGGIISKFLSYKRGSSL* Lottia Protein Model : jgi_Lotgi 1_171167_fgenesh2_pg.C_sca_110000033 NR Annotation: No match found NR E value: Ac Annotation: CNSN01 C 005636 501 (unknown) Ac E Value: 2.00E 23 Sequence: MAISHDWSFTLLWLIWIFTLLFSDSYGKRTQNYYMEGLDCGDSKHIGGATVYSNFRGDVSTVYGNDIECQ MTFKAENKDWRLMLRIIELDIPDRTSTGLC NDALYVYDESSIYARAMEEANGNTGLCGNILPPTLYSTGQY LTVHFSVSLQNQKDIV* Lottia Protein Model : jgi_Lotgi1_171450_fgenesh2_pg.C_sca_115000014 NR Annotation: No match found NR E value: Ac Annotation: CNSN01 F 059701 501 (unknown) Ac E Value: 1.00E 09

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147 Sequence: MKRFTLLAL LLVAILYISQSESKCQGVTKCMVNLFTCKREQISVNIAADCCKEYFECFNECKPGSRPPCKNT APGKRGSWNKRYSSRYGSDILGDIISGY* Lottia Protein Model : jgi_Lotgi1_171757_fgenesh2_pg.C_sca_119000045 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37253.1| predicted protein [Ne matostella vectensis] NR E value: 7.00E 24 Ac Annotation: CNSN01 C 000842 501 (Unknown) Ac E Value: 2.00E 07 Sequence: MWKIFSFHWIYFLFSLFCLLHSVTCVKKVSKTPRKSKESEVSKESTKRSASLDDDPSLWPGHLQPLGSSQQV HKVDAYKKYLKPEEFLNSYAAPYLPVLFQAVEQTSIAYEKWGNITYFTVLPQTLTNWVHISPEVGQVSFRK FLLNYTDKGSVFNNKVPFFMRSAKWEPYRDDVSVPKCLHCEPILNSFSTHKLWISHTDQSSPIHRRNKDTIH CTFVGSQEFTLISHIEYGDKVPVDYTTIDINKVDFTKFPTLREVEYHKVRVKAGDCLYIPTKWYWQVRSTG KTYSADIEWYHRNKTVKSSNCKTKTSISSLNSVVFKDIDDPTTIETDVLLHYFTTYLLNVPKFTLPQFERHLK KDKKFMEDLIEWTDELQHITKEVFETLDVNKDSR FSMADLESLSQPMLEDIRGLFLDRLQDYDDILKDQQE EAKESKKQSEKENKAKDAPATTGPVDDYLKNYIGQLEDVLKESIEEMSVTGHLPDIKKKFAEKQAQGKTG NADKPKQSTKPKTEREKAKEKLEQDRQRRKEKQEAENIKEKTKDDEKYLIVDDSVEEEIMADDDDDSTTG STNKRTPTADKADTKDNKSHTKDNKADTRHVDKKTGRKGQDGKEAKQKNTGSKIKTEL* Lottia Protein Mod el : jgi_Lotgi1_173456_fgenesh2_pg.C_sca_159000027 NR Annotation: No match found NR E value: Ac Annotation: CNSN01 C 007362 501 Ac E Value: 8.00E 06 Sequence: MWSGVWCGVVYGVMWSGVWCGVWSGVERSVVWRGVWSDVDGVMWKECGVVYGVVWCMKWCGV WSDVERSVVWCMKWCGVWSDVERSVVWCMEWCGVWSGV Lottia Protein Model : jgi_Lotgi1_175543_fgenesh2_pg.C_sca_302000002 NR Annotation: No match found NR E value: Ac Annotation: PEG003 C 220719 501 (unknown) Ac E Value: 3.00E 08 Sequence: MNTMFLLVVFLLIPATSAFDSTQTNRTIVNSTITTKINGSSIGLGTCSEFTPQSCSGVCGQFF ELWCSCDEYCQ VYRRCCFDYEAVCKEESEKSKQDHENLMNVTVECDSNMSGVHVPQQRISPASG* Lottia Protein Model : jgi_Lotgi1_176704_fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTED: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Ac Annotation: CNSN01 F 096594 501 (unknown) Ac E Value: 9.00E 12 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDDVNVCHGELPANAMCSGENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lottia Protein Model : jgi_Lotgi1_204607_estExt_fgenesh2_kg.C_sca_690020

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148 NR Annotation: ctg4a [Aedes aegypti] gb|EAT44181.1| ctg4a [Aedes aegypti] NR E value: 2.00E 45 Ac Annotation: CNSN01 F 087652 501 (unknown) Ac E Value: 2.00E 30 Sequence: MAFMFFQTLLIIVSIGYLHADDSDGVQEATLCEVCRILAEELQLRLDETGKSKEVIETGHGLDTKKKKKYNF SELRLIEALQEPNICDRILEYSVHKEK TGIERFAKGRSQTMEALHGLVNKGVKVELGMPHEMWDKPSVEIT QMQRKCYALVEEYDEDIEDWYYNHQDQPFLDYFCRNLVLNPQNNECLDEDMRSQNEGSSSEMKGDKGK KEEL* Lottia Protein Model : jgi_Lotgi1_204654_estExt_fgenesh2_kg.C_sca_720011 NR Annotation: PREDICTED: hypothetical protein [Monodelphis domes tica] NR E value: 8.00E 93 Ac Annotation: 190207_2814_0323 (unknown) Ac E Value: 2.00E 12 Sequence: MSLLIATGLFCVIYLLVKFLNISQPCHLPELYFKDKTSNFIQTVFTMCPILFESYIPPLLWGKSGHLQTFIYAK MGRIMSPLPNGKRIELIRPDNATMSFDVFQPHYEHPTGRDYTLAVCPGIANSSESLYIRTLVDHAQYNGFRV AVLNHLGALR KVKLTSPRIFNYGETGEYNCMIDELKRLYPNTSIIAVGCSMGANIVIKYLGECKQHENKVIG ALSFCQGYDVNDAKPFLLGWESCRRLYCYFMAINLRQLLRSHQDILFTEQAQSEYGTVELKKLYSATSLLA IDELYSVNAFKFKCCEEYYHWASCKQYIQNITKPIFILNAEDDPIVPKQLYTTPLKYQENHDNCLFIVTKHGG HLGFFEGGFIKPNTITWLDRAVVQFSNVLCELDCKERKS* Lottia P rotein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 10 Ac Annotation: 061241_2672_2657 (unknown) Ac E Value: 3.00E 11 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSCCMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMWRAVFSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI Lottia Protein Model : jgi_Lotgi1_98330_gw1.10.599.1 NR Annotation: hypothetical prote in NEMVEDRAFT_v1g147894 [Nematostella vectensis] gb|EDO28414.1| predicted protein [Nematostella vectensis] NR E value: 5.00E 06 Ac Annotation: No_Hit_[N/A]_[1]_APLCNSN01 F 143489 501 (unknown) Ac E Value: 3.00E 07 Sequence: TVCSLGTHCLSITVCSLGTHCLSITVCSLGTH CLSITVFCSLGTHCLSITVCSLGTHCLSITVCSLGTH CLSITVCSLGTHC

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149 Object 24. P redicted Lottia secreted signaling proteins Strong Annotation Lottia Protein Model: jgi_Lotgi1_103075_e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mel lifera] (Reticulocalbin 1 precursor) NR E value: 9.00E 50 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRLE NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHP NEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLEDREVYLTEEENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL* Lottia Protein Model : jgi_Lotgi1_119019_e_gw1.30.87.1 NR Annotation: PREDICTED: similar to seleno protein N, 1 isoform 2 precurs or [Canis familiaris] NR E value: 2.00E 85 Sequence: MVLFTLLCCLFIIGRHDPTQIPEEIVINIGDDGVLLFQQHDRDNDGYLSIQEFEPLVYRLLEINVSGPVYDVPIS TDDEMITLKSYFIPIVKESMSKDLNDSVSIGLLRTMNSLHGLEKWQNVNLQWMNFGASHFSGFLPKDVDSI MLGSSYFIINVEKGLFNAALSSNRYYPPKVTTNESIIVHRLLTLFHPRPFVVSRFPA QSSVACVRAYNDKYLD IVFRIHAEFQLNEPPYHPFWFTPAQFTGNLIISKDGKHIQYFNLYVPNNKRLNIDMEWLNGPNESENMEVDI GFMPLMQLNSTQSSVPVKNLEEYELLEPLPERPQHKTEGENIEWMATIDLEDAKSSLEKALYPFKKVPYYN FTEAFKKAEDNKKLVHSILLWGALDDQSC* Lottia Protein Model : jgi_Lotgi1_121665_e_gw1.37.7.1 NR Annotatio n: hedgehog [Patella vulgata] NR E value: 1.00E 171 Sequence: MKLFFSFQSVCSWTFIFLFITSLTHACGPGRGSGSRRRPRKRTPLVFKQHVPNVSENTLGASGISDGKIRRNS EKFKNLVKNENPDIVFKNEEGDGSDYLMSRRCQDKLNSLAVSVMNNWKDVRLRVTEAWDDSPNSHAKD SLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESRGHIHCSVKSDSS VAIKIGGCFPPTGSVQTL HGWKTMGQLTVGDKVLSINSAGQLEYSPVIAFIDRNDLEFEKYLTLHTEDDTDITLTSKHLIYASGTNSSNF ESYDVVYADDIMEGDHVLITSSEKGAISPTRVVTISEKTLQGVYAPLTVNGNIVVDGVVVSCYAVVSDANL AHAVFAPMRGLHYLSQYVPWFLHSTQQENAPQNGVHWYAKMLYNIGSTFLNEKTLYIA* Lottia Protein Model : jgi_Lotgi1_1 59314_fgenesh2_pg.C_sca_20000054 NR Annotation: Buccalin precursor [Contains: Buccalin D; Buccalin E; Buccalin F; Buccalin G; Buccalin H; Buccalin A; Buccalin I; Buccalin J; Buccalin K; Buccalin L; Buccalin B (BUCb); Buccalin M; Buccalin gene predicted aci dic peptide A (BGPAP A); Buccalin N; Buccalin O; Buccalin P; Buccalin Q; Buccalin R; Buccalin C; Buccalin S; Buccalin gene predicted acidic peptide B (BGPAP B)] gb|AAB27696.2| buccalin precursor [Aplysia californica] NR E value: 3.00E 66 Sequence: MAARKHEL VLVLTSVLCFVSSIVGDPNVPSDSQDNSALTQDDFAKRGMDKFGFAGGVGKRGLDKFGFTG QLGKRDMDSFGFAGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGF TGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFAGQLGKRGLDQYGFTGQLGKRGLDQY GFAGQLGKRGLDHYGFAGQLGKRGLDQYGFAGQLGKRGFDQFGFAGQLGKRGLDHYGFA GQLGKRGLD QLGFTGQLGKRQMDIFGYRGQLGKRQSIDKYSFLGAGIGKRSVKNTAGIKKDDA* Lottia Protein Model : jgi_Lotgi1_163440_fgenesh2_pg.C_sca_39000174

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150 NR Annotation: No match found NR E value: Sequence: MDLYGVVWLLAILQEVTSCFLTEKLPSLEGKEWFTKIRSGGSNDVIAYFDRNSVTFNDVKNTTNGETKWR CFMKIRGRYFLKNVRQGGGYLYKCMGIIVRSESAIQLEWSHVSRLADPALCAEENINLDPWLLISYKTVVN DFTSCPVSGGFDIKHAGYYSRDVRDSRGYDIGCNLMNIPMRLEFDCIAGEGAIFNFRSKNCVPDIHFNIYQN TICVAKWSNKRHHFVLLRTKTGLEFWCARFPVGIENQNETELYLYSDVACLEGEYAEEFVKFMKFDLQRV MYTTICADEYPQCKEGTCNVFSKLECQKTCGLCNPHRPPGICT FPKRMQGTYLLHSKHGNQNVTLEGRTLN IENVGQFDCIVFEDSPLRSTKTYTTMSIFQNGCRPRYTCVRLKRLGPSALRYSLAQNLVWPIKKERIGANICN EDNFRADGDPIRDTFRSYKKTGKPVIKIHPKPRFINCGMNTSYTIRAELPDGSICHGGFYQHCKNETKLRFDF HTCNSFIKPKEDFNCAGIFRTNYWERAVLIQNVDNLNDIRCLIFNSLRPYQALIVVADECDKMASGLVDSNI RIPIMRLHIR SDVYPCKHIQLPLKDDVITNVVTTTEPIKMNVTLKESKNVNSFSVDLELSSRNSSGSRPFENKL NSLLLLFLCYVVFVKNL* Lottia Protein Model : jgi_Lotgi1_164016_fgenesh2_pg.C_sca_43000014 NR Annotation: chordin [Danio rerio] sp|O57472|CHRD_BRARE Chordin precursor (Protein chordino) gb|AAB9348 5.1| chordin [Danio rerio] NR E value: 1.00E 119 Sequence: MSPMDLAVCLVLLAATVSSTKFSKIPLKAARENLFGIQPNRPGCILGSKFYGIGERWTPELLPVGEMYCVKC ECVPVERKGIIDMKGQTLCKNIKNLCPRPNCLNPVLPKGKCCKVCPDEVNSFEDSFSLGSKSSAPTMISSDSK RRVRNEFVSLLVGKNVRRESVKTSAVAAVYFSIVGRDEIRYSVRYFKLDRPKFLQITDANGNVLFERPIEKK RNGDKKFCGIWTKVPSAYIQYVREERLFAVITASRHFRGLVSGRIMVNRHAKREVFSSVLASPKANGIGGL LSVVYNPRTHLVDYVINLDGVFPGSSEEYFITISKKSKVLHQSKGKASSKTRFIRGSWTLKNKRQSKQLARG RLTIRLTSTNGASVSGDIKPKVTCGVSQAVMSSGNSLEEMKLASSGTAVFELREKGSLDYKIRVTGLTSKVH RIRLEGAVNKKNRRQIIGSVH RNFKADENSFNGWCNGTFKKMTADDLHLFLNNKIFVNVATSENRISELRG RVIALPYHSHFEQVSADPAALGPKTITSNGHAGNAWISMGVGCSLHYDITTTTIAKHHDLTLSLVSPTDIDA YVTIEKISTTNLYEFQQASGSIAVVPESLFRDLDEGSALIQVISGDVILSGNVSVPNTCWQYSQDYDMDILVD EQTSEEVLAANRYKCVYEGKVYENGDSWLPDVNATCNTCSCNKGKIECHRLICPVVECANP IVLDGECCPT CPIETTDQTECKIDGDLRSHPVGSTWHPFLPLMGYAKCVICTCLPGGIYKCERKCPKLDCPASRRIRLNPKD CCQVCAPEEVKPEVVKPLQDVDMKGACSFKDQIYANGAKWHPRVIPFGYMKCIDCSCNNGTPSCSRPKCP KLSCSRKIREDGACCDTCADSDGEEIVEEKKSDSCIFGGKKYDDGETFQPKTSSLSSCATCTCRKGSMKCSL TCPEKCKDPKHSNDPCCKHC* Lottia Pr otein Model : jgi_Lotgi1_164520_fgenesh2_pg.C_sca_45000185 NR Annotation: PREDICTED: similar to filaggrin 2 [Rattus norvegicus] ( molecular regulation of epidermal terminal differentiation) NR E value: 5.00E 37 Sequence: MIKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDI VQMIKLTLSILDIVQIVKVTFSILDIVQIVKLTLSILDTVQ IVKLTLSILDIVQIVKLTLSILDTVQIVKLTLSILDTVQIIKLTLSILDIVQIVKLTLSILDTVQIVKLTLSILDTVQI VKLTLSILDTVQIVKLTLSILDTVQIVKVTFSILDTVQIVKLTLSILDTVQIVKLTLSILDTVQIVKLTFSIFDTV QIVKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDIVQIVKLTFSIFDTVQIVKLTLS ILDIVQIVKLTLSIHVVC Lottia Protein Model : jgi_Lotgi1_168619_fgenesh2_pg.C_sca_77000042 NR Annotation: crossveinless [Saccoglossus kowalevskii] (a new family of twisted gastrulation like modulators of bmp signaling) NR E value: 1.00E 148 Sequence: MYYTELSI LAKCIALCILTCFPILIQAQLTGTVTRCYNEGGEVKIQGITTDPCISCYCMDGYVQCDKKKCDNL DGCHAILFDGPKRCCDTCKGCRLNGVKHESGKSWFDKRDRCTSYSCKAGVITKSKVQCHVPCSNPIKRKR

PAGE 151

151 QCCPSCKGCHFDGADRKNGEKFALASDPCVECKCRKGSVTCMKRACPVLNCPDEVIYQPKGECCPKCKG MRKIFDFPGACYFAKRVYVDGFSFEPKSETRCTCTKGTVICEKATCPPVACP VEERVQKGSCQVCEPKRNC LYDGKIHMHRSKWQPRMCTQCSCQNGVTYCQRERCNNSLSCPNGYKLQFQPGECCPKCVEHDAVCRVFG DPHYRTFDGKMYNFQGTCKYMLSQDCQGKDFTIKVKNGVRLSSGFAWTQMVVVLMGDTRISFRQNLLIKI NRRRVQLPYTIPGKFSIRREGHSVTFRADIGLKVVWDGDSFLEVTASRKYKNRLCGLCGNYNGLETDDLIG RKGKNYLRGEEFGNSWRIGSKKACKTQ PKVKNLQSICDKDFKAKVRANKECSVLYSRAFSSCRRVVDVTP YVTSCVTDMCDCPHGKKCSCESIQAYAHECKRAGHKVKWEKVSNCKAPQKNCPKGAYYSLCAPACPKTC SSNKPDGSCSKKCSPGCVCQNGAVLYKNRCISPEKCPKT* Lottia Protein Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Euprymna scolo pes] NR E value: 1.00E 122 Sequence: MTVSPTVVWAAFSMALSSVVALGANIICNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTSAGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LPPFRKIGDSL KKRYKKSKIVVPYLGRRARTAVTLILKRAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK* Lottia Protein Model : jgi_Lotgi1_186451_estExt_Genewise1.C_sca_160091 NR Annotation: PREDICTED: similar to out at first [Nasonia vi tripennis] (Vital for proper neuronal development and hatching) NR E value: 7.00E 69 Sequence: MSMSLIPSLSYSIFVCVLIKICHTQLVVNVKNGGGDILVESIQSNVSSDTITLEYPSSDGSLITQFIDFKSEAQIF RAYVPGEEERGQPPGSVQVLCFITRFSKNDFISSDAMSKLRQKNPTAIRTPEEERPQEVHSFDLSVDLNKSHV ISPHVFNICREA KESTFFNEDDLKTISRSLSKDYNTMMSAMIKLAPTKYGKCSDTSDIFKPCTCRYDACVGW YPCGLKYCRGKDSSGKVVSYRCGIKTCKRCLAFDHFVKQKFLCLWN Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37057.1| predi cted protein [Nematostella vectensis] (growth factor) NR E value: 1.00E 109 Sequence: MFLEVFNISATVLCILFLIGHRKCDTIQNSKHHAELMMDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVGVNGGFFNTTTGACLGNI VSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSAVGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLELGVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYGINCSTPCKPGYYGSNCAHQCYCLNHG TC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi1_205842_estExt_fgenesh2_pm.C_sca_70027 NR Annotation: BMP2/4 [Patella vulgata] NR E value: 1.00E 159 Sequence: MIADFYRSVVLLLLVLVVNYTSSLSTNTLHPRKHGNEKVLAAVESNLLGLFGLKSR PVPGRRTPIPDYMLD LYKRLDSEPDFISPFAESKGKGIVEANTVRSFYHSDINFVKDCDERTCARIWFNISTIPFAEIMAASELRVFKD VYNHFKLTDANSLSGDRIKASLFKHRLEIHEIMRVSPDDSECISRLIDTKIVDTRNSSWESFDVHPAVLKWRR RPQYNYGLEVRIVSKSPWISTNSHVRLRRSAHMDEKNWQIQRPILVTYTHDGRNPNSRIKRASARNRRRKS RKRRRRKKKGNKNECKRHALYVD FGDVGWNDWIVAPTGYNAYFCRGECPFPMGQHLNSTHHAVMQTL VHSVNPSAVPKACCVPSELSAISMLYLDEWDKVVLKNYQDMVVEGCGCR*

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152 Lottia Protein Model : jgi_Lotgi1_208417_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVIAS SRRSRGRWYRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIA DMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSN NAFEKLEEKVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* We ak Annotation Lottia Protein Model : jgi_Lotgi1_110650_e_gw1.12.27.1 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 1.00E 32 Sequence: MKIILVLLCFCHLIYSAKTCPSNCQCHLTDKAVNCYKRELQFVPANIPLDTEILDLSSNDIANIDPTMFHNLT KLTDLVLNDNKISKLDPRTF QNLHKLEVIKLSQNFLSTIPEGLFDNLPIMVPKLMELNLSQNKIVKIENYVFR PLSMLSTVSLNDNPLKNADRLLSKNRRLSYIDMSECQLTTVPRGLPDSIEHLKLIKNNITKIKPKDFRNKNYL KILALDENEIEFIAPNSFKSLQKLIQLWLNGNRLRKFPSPISPSVTNVYLAKNNITKLSAADF Lottia Protein Model : jgi_Lotgi1_113861_e_gw1.18.363.1 NR Annotat ion: PREDICTED: similar to tartan CG11280 PA [Apis mellifera] NR E value: 5.00E 27 Sequence: MVKPPTQNTRWWIIVWILLFQKYTCEPEVTCPHGCKCDVDVVNCLHSNLTSVPVGLSNGDRLQELKLGRN KITEIKLELLEYKELRLLDLISNNISTIDGSAFRTLYKLETLFLSRNFITKTDKNTFRNLTSLRFLDLSYNNLRC LKIGNFEDLQSLVNLDLS RNQISLIEAESFRCLENLEILKLPRNQLDQFPWKIFQNLTSLKMLVLDDNLLTSLP ISAFSNLINLEYLSLTGNRLHELLPSSFMMSDEGAISLEQLYLRGTDLDTIPVKLFAKLKKLTVLDLSKTQIVS VKNENLNGLSSLESFIMTSSNNLTYIEKGVFQDLHSLRKIDISGNTLLSEIQFTTSSLRNLSLFDLSANSL Lottia Protein Model : jgi_Lotgi1_137329_e_gw1.113.40. 1 NR Annotation: insulin like growth factor binding protein, acid labile subunit [Sus scrofa] gb|ABE73450.1| acid labile subunit [Sus scrofa] gb|ABE73451.1| acid labile subunit [Sus scrofa] NR E value: 1.00E 21 Sequence: MSFVQLLPFFLFLTISQSSSQRWPLSGTCYPCSCD INQKGFGRVKYVNCSNLALTRIPRNLPLDLHTLDFSNN RILPQEIQQLCTFNSMQYVALSYNSLDSIPPEIFKDLENLHTLVLHGISSIPTTNIFEDLFNLEYLDINDCHVNQ IPDNWFRKLYSLKTLKLRQTGIRSIEPGVLDGLLNLQELDLSHNVMRTAQTESFKPIVQSIRRINFRGNMFKT IGDHLFEKMYNLVELDLSDNKLESIHKRSFLDLRQLINLDIRDNKLSNLPGGLFKNLRNLQTLNLAMNTF NN FPETLFKIGYLLKLDLSYNRIRRLPDSFVHSFPYLEFLNVDKNPLHCDCKSLDVKFYQPNLVII Lottia Protein Model : jgi_Lotgi1_152641_fgenesh2_pg.C_sca_2000061 NR Annotation: interleukin 17 [Crassostrea gigas]

PAGE 153

153 NR E value: 5.00E 07 Sequence: MVLMKVFICALLVNMVYSFTLIKNIEHILERERRSDECAVP NNIDELFTNLNSHVNRVEFLIAVNATHQPNA QGLIPPAAGDQTDTECPKEYTGKDTSNSGIWQRSTCPWFYSERNYGAEYYPSKVYNAACKCVDCLYSKSN SETGCSRIYRNINILKKTGCSNGFYTYEQRSISVSVGCTCAKLQANIG* Lottia Protein Model : jgi_Lotgi1_157019_fgenesh2_pg.C_sca_12000330 NR Annotation: No match found NR E va lue: Comment: Similar to Chordin Sequence: MFSLVLLVVTPWLVFCMPPPPPAMCTCSDGTQVPPGGTHSISDCHSCYCDENNSEAQEIIAACLIVPCVDSV KMPGQCCPTCPNGENCRAEFKDDNGVERSEIIKVSDGVVKFNSTECQCDYQNYNGEPKANCVQVLSIEPVI AEKSTP* Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71 000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 21 Sequence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPMFLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQ NYFSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Protein Model : jgi_Lotgi1_168377_fgenesh2_pg.C_sca_74000087 NR Annotation: TBL 1 NR E value: 2.00E 07 Sequenc e: MLPHITQCVILFITFLCDISVLLPSRDGGRGLTYAQLVPHRTSNDVYMDPCKAAGFMGDIALSEEEFKRERK YLQQMNNTMFELQVEDPRTTYIGRKPSKYSGQPENTLKKLPMSELDVKKKKYLRKILKEKKQNLKSLCKK GFQTVVYTRQVPVGFKFISPFLYLLDLNIL* Lottia Protein Model : jgi_Lotgi1_168508_fgenesh2_pg.C_sca_76000035 NR Annotati on: ependymin related protein precursor; EpenHg precursor [Holothuria glaberrima] NR E value: 3.00E 10 Sequence: MMKYCLVVAVVVVGVVIGEPSKPGCCTPSQWEGQQGSTIGIVDGGIPSVRTSMVRISYDEKNQRIAYVTSE STPQGEKNYKIIYDYSRKFQFIVDLDTKHCTVSSLTDPFMKLCLPVDAQPKGKFYYGIDGNNLPAMAFSLN V NGTMITVVTTADNCIPIAEVLVDGQAQGVSQMHASGFVNLTSGIKDPSVFIPPGTCTQ Lottia Protein Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chain; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica] NR E value: 4.00E 13

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154 Sequence: MEVTCKCPLVLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYCRNSQQRISIKK*

PAGE 155

155 Object 25. C ontroversial predicted Lottia secreted s ignaling proteins All software programs are inherently flawed in that they can only use those parameters they are programmed with to make predictions about whether a protein may or may not be a secreted signaling molecules. Due to these flaws, our final list of predicted secreted signaling molecules was manually screened for conserved motifs, signal peptides, and annotated to make the most conservative list of predicted neurosecretory products. Here, we show those transcripts predicted to be secretory mo lecules due to targeting to the classical signaling cascade but are either unlikely to be secreted outside of the cell, or unlikely to serve as neurosecretory signaling molecules. Some of these predicted secretory molecules may be part of the secretory ap paratus but do not encode for signaling peptides. Furthermore, many of these predicted molecules have motifs of non secretory products but may be secretory in our model species. Further experimentation is needed to determine the functional role of these products, so we have decided to leave these products in this supplemental section to provide a complete unbiased review of all predicted products for future researchers. Annotated Lottia Protein Model: jgi_Lotgi1_108564_e_gw1.8.40.1 NR Annotation: PREDICTE D: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001179636.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 2.00E 86 Sequence: MYLSCYLPVILLFIEDLKQAVVAMMLRKDEVEEKNKSLKAMLDREMEISSTLRAEIEEMKISFKLSKDKEIA KNETLQKENELLK HQLRKYINAVQLLRTEGAKDDTQGITLEDPQPIIPPAKPSIDYSHEASEYEKKLIQVAEM HGELMEFNELLHRQINCKEAVIRNLKEELTDLRGPLPYDAQSSDDSLSGDLESSLISRCLINIWIPSAFLGGSK TDSHHVYQVYVRIRDEEWNVYRRYSKFLDVHTRLKKVYPLIEKFEFPPKKTIGSKDPKVVTARRKMLQSY LRKVINHLLEKNADLSSNVSKEKLIAVLPFFK* Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Annotation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQCCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPWPLTTTTSRVPINILCNVYWFKCTKLHFSSHWECSNDDCLL EADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSNVVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA

PAGE 156

156 SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFDEEPAVYHSVRIIGWGVERTI YGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWGQISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV* Lottia Protein Model : jgi_Lotgi1_134336_e_gw1.89.95.1 NR Annotation: apolipoprotein D [Branchiostoma belcheri tsingtauens e] NR E value: 9.00E 41 Ac Annotation: CNSN01 F 078834 501 Ac E Value: 2.00E 25 Sequence: MERILTAVLLCIGVAVYAQVPGLGPCFDAKPQSTLNVSQYLGHWYEITKLPSFFEIEQVCLTANYSVKEDG HIKVFNQGKVNGKNVSSTGDAYVPDPSEPSKLIVKFPQAAPYGPYWVLDTDYTSYSLVFSCEQLGSFAHAK FLWILSRTPSIDPAVKDRLFKRLE KDGIGASGLRATKRSDCS* Lottia Protein Model : jgi_Lotgi1_154590_fgenesh2_pg.C_sca_6000093 NR Annotation: PREDICTED: similar to meteorin, glial cell differentiation regulator like [Tribolium castaneum] NR E value: 3.00E 24 Comments: Secreted, involved in both glial cell differentiation and axonal network formation during neurogenesis. Sequence: MDFLQATWLLVVMVIFHIYIITADQICNQCDCTIRDSTGKGNINVKPKCQSGKITWPDPVSAVRLELKPEVV GPFKACIALKSENIVTLVSREITTETVNYRTRSYDSKVDLLPWFSVEGQGKEHCVTSSESSVMLYLEIESTTE DSGLSRTFIYYDIELIPDNKFSPD PMEECQPCSDDEILRSYCISDYVVVGRMKEVNFQSELDKTKVNIIVDQIV KTTDEDIFKRHDNLLEGALFTSVHCGVQKGPGQFLFTGRNRLGQPILTCAPFYDQWIQIQRLAQEKGTIECA YG* Lottia Protein Model : jgi_Lotgi1_160631_fgenesh2_pg.C_sca_25000236 NR Annotation: Substrate specific endoprotease Tex31 precursor emb|CAD36507.1| substrate specific endoprotease [Conus textile] NR E value: 4.00E 29 Sequence: MNRLCFLLVVWQIALSQSKVVSSVEGFNGNLLRIVRSANNAAPQGNLNYNSNCNEAYKNISSSHSMCLVD VCTAVALSQEEIDAAVAAHNDYRKNVSPKAAAMQKMVWDDELAEIATKWAQQGRAGHDTKDARKTISL KGTYIGQNAAAGQANLIAAIKAWHDEVVDFKYGVGSINGKDVGHYTQVVHHQSTRVGCGKAHCPDSTY KSYYVCNYAIGQMGLTHPYKSGSSSCSDCPSSCSDNLCDCNGKICFNGGTLDINTCTCSCPSLYSGDQCQTL QCTKTEKSWCRRSYTVADCTKYSNFPIDCNMMCGVCPYPCDGKQCENGGTLDPNTCQCTCKDPYTGPTC ADKSCPGQEEWWCQKFFTAADCTKYSNFPTDCNIMCGVCPACKITA* Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sca_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus] NR E value: 1.00E 133 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHLKK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAG TPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSSDNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKGDPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALELSKELN VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAATIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KK HDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQTSTKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNTWPEKEECDSH LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHPLPWYPPICVTLLTINEYDEKTYDNLKSVGHP

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157 GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGAR WVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYADVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSVAPIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKSGECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGED CNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLHSTQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN HCSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPKSWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLMLLGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTL GNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model : jgi_Lotgi1_168805_fgenesh2_pg.C_sca_79000073 NR Annotation: PREDICTED: similar to plexin A CG11081 PA, isoform A [Apis mellifera] (a neuronal semaphorin receptor that controls axon guidanc e) NR E value: 1.00E 121 Sequence: MEGGIRLQLFLLVSLICHTQCIKILHSFRDPGGNILQKLVIDDQTGNVFLGALNRLHKLDPDLNIVQSASTGP RLDNVECPPPLLPCDKPKTQLNSQIKGLVIDSASNSLILCSSLFHGSCQTLALNNITNVKKFFHKPLVPNDHH SCYMFLAPSINNTQALYIATAYSDLGDAAYRDLVPSISSRSLDNLDFIHRDTEGSTKLEILKEYRESFIVRQLY G FQNGGFVYFITLQRENPGSQKIVSRINRLCQQDRYFRSFVEIPIKCQKSDSDSYDVVQNAYFTNGKLYVIFS NTVSSAGAKKSTFCEYDIQDIDLEFNETVRNCYKAVGNFGPEHYKKIEPCPPLIFGSVDFCGESGEWKDYSA IEGSKPISATPLHFFTSDKPTAIFVHPEGNHRYAYIGTQDGHVIKLRFENGQVNQLFKITHSVGDIILQITNHPQ SNLLYILGSHKLSVLSLNHCDDLTSCDECMNDGDETC GWCVMDNRCTTKELCQSSVITPPWLSRTDQSCAS ISSLQPSSLSYKKFINGGDSRQIKFQVNKLKFESNRNLNCLFINGEKHDNTMAILDQKENTITCPLPRQLPTIP QGKDHEDLELQFHVEGKMIVKRDVSVFDCQGNNNCTSCSTSQFNCHWCPVSHSCVEQSESCPQGDSIKVN DRCPRLETAASDTDILVHSGELKTISVQVRALEANQRSNLKCHFDLSGQVQTVSAKISSSTLTCETIKLAYA DDIPYI VAGFKVTWGQQNHPLDNPQRMNVRIYKCASMVTNCGKCLSLDVEYECGWCEDQCSLQKKCQKS WLDQSETCPNPQILRYSPSKGPVKGKTEIQVNGINLGKEISDILGQVTVAGLPCDVMQEHYEPSSSFKCEVK GVTSAKSGTVKVVVNNKYTAESDTVFEFVDPVLNHVDPKQGLMSGGTRVLIKGNNLDVGSDTQVIVGGN SVPVIK* Lottia Protein Model : jgi_Lotgi1_192261_est Ext_Genewise1.C_sca_450209 NR Annotation: conserved hypothetical protein [Aedes aegypti] gb|EAT46740.1| conserved hypothetical protein [Aedes aegypti] NR E value: 1.00E 128 Sequence: MTLYITSLVFFLFINGTLQVKNRSSKVADRCFCKLNGEIDDCSCKVETLSVLNNNKIYPRIKSLLSRNYFRYF KVNLKKKCPFWHDDSRCALKDCHVDTCKEEEIPSAIKKGSSAYRYSEEAQKEESCAEEKELSALNTTISDES IQAFKDWKEHDESQDLFCDIDDESSADSEYVDLLLNPERYTGYKGESPHRIWRSIYQENCFKPQTEYIYGPS KSSSPLCLEKRAFHRLISGLHTSINIHLCADYLYQENLGYGMKAHWKPNVAEFQKRFDPQTSNGEGPQRLK NLFFTYLVELRAIAKAAPYLQEEDFFTADSDEDKDVRQGIDDLLNIISTFPDHFDESKLFAGNPNEAKLLKDE FRNHFRNVSRIMDCVGCDKCKLWGKLQTQGMGTALKILFSGDDIGPDSTVNAQQKKHFQLTRSEIVTLFN AFGRLSKSIHSLEMFKSLLSKS* Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVA KDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDEYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS

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158 FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATEKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSL EVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_kg.C_sca_820009 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEV TVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SYIQSEFVAACVAILVWWLAFSAKSKLSP* Lottia Protein Model : jgi_Lotgi1_211937_estExt_Genewise1Plus.C_sca_110124 NR Annotation: PREDICTED: similar to SPFH domai n family, member 1 [Monodelphis domestica] NR E value: 1.00E 124 Sequence: MANPALALLSVSAVALAIFLNFSIHKIEEGHVGVYYRGGALLSSTSEPGYHFMLPFLTTFRSVQTTLQTDEV KNVPCGTSGGVIIYFERIEVVNKLDRAIVYETVRNYTADYDKTLIYNKIHHELNQFCSVHNLQEVYIDLFDQI DENLKKALQLDLNVMAPGLMVQAVRVTKPKIPESIR KNYEAMEGEKTKLLIAIQKQKLVEKEAETERKKA VIEAEKVALVAKIQWEQKVMEKESEKRMSEIEDSANTARAKAKADAEFYKTQKEIESNKLKLTKEFLEFTK YESISKNTKIYFGNSIPGIFSDKVEPQEIAKVVGERK* Lottia Protein Model : jgi_Lotgi1_116422_e_gw1.24.410.1 NR Annotation: PREDICTED: similar to CG14764 PA [Apis me llifera] NR E value: 7.00E 58 Sequence: MKTKNFYILVLLISVTSVALWLVSYPSPTFRNIMSTTQNNIRKIPKHMQMSKSPDLEVDKKYLDLLGFDSES KPKNDIVVIGSALMPADTENTILFLKSAQEYVPNLKLVLFDMGLSKADKQTLTNCRNETKHCEIRKFNFDQ YPSHISDFANKSYKPICIQLLLKEFGTVIWADTSELFASSDIDNTMKQAKTEGLVAWTIEDATSSLTHPKMFD YFKTDQKKYFFHHAVQTSHIILVNTDKIHENVMLPWVKCALVEECINPMGAQNSGCNYQRKPKFRYTGCH WYDMSALNVILGLAFDFDEAYSGKDKIFWTKFDQEKAKAENMTDGAVHKVSVPHRVYF* Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Sequence: CVMLYMLCKCSSLDCVK LYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_149784_fgenesh2_kg.C_sca_15000101 NR Annotation: PREDICTED: similar to chromosome 19 open reading frame 10 [Canis famil iaris] NR E value: 9.00E 20 Sequence: MLISNDFKCLFMLILGFIQWVSSYEMTRDYEEASHFDVMPGGAVLSHEKQLDGFTCKFTYACQGGTKEGW QLTINTNTDKTKYLCSVQRPDGKSYLFFQSFKLEFTGLEVKEGYAMGANFKPLSPSEYQVDFQKHSISDVA GKFGSQLEKVELYGEKGKAEL*

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159 Lottia Protein Model : jgi_Lotgi1_152847_fgenesh2_pg.C _sca_2000267 NR Annotation: SARP 19 precursor [Littorina littorea] NR E value: 5.00E 08 Ac Annotation: CNSN01 F 148317 501 Ac E Value: 2.00E 07 Sequence: MIAFICVLGFISGCLAAPCTHTNSAEHTEEQVIARVIALADHNNDGILSVTDLAAYFVINFDHNHNSEVDKN EFILQWHETFHDEKAFAEHVFTHFDTNADNVLNLSDLFALNIRLDTDGNGTVTNKELESYLQLIYNAC* Lottia Protein Model : jgi_Lotgi1_158797_fgenesh2_pg.C_sca_18000147 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E value: 5.00E 92 Sequence: MKIHILAVLFSFFSATNAATLRFVSQTPLSITINKDGSYTVNVNGEPWLESSAIYFY AEGRRVSMGDGSLKIA STFMDNGYDSVGQYGKTCWRFEIESKYIDTCFKQYMEPTPFIIFEQHYLNDTENTASIDYDYTIAGFPAFTAP PTGKLGYLHYAGSMTGDSDKSFGIWQEKAKMLTGVAGGPLVLFDKDADDCLILSPLNQFMAASSLFDGQ NYSTGVLGGVNFVPKDFVLQTVLFYGKGINHAMSDWGLLLTQYYGKDDSYRKSDLTLNYIGYWTDGGA YYYYNTEPNKTYQQTILDVRQNAKDIGMPYKFVQYDSFFYYKGPHGGVKTWVPMPDLFPDGFQYLYNQT GWPAGAHNRWWSPKTTYARQNGGNFDFIVEEAKAVPTDQAFWDYLFDSSKKWGLTLYEQDWLDNEFAG VKALLTQIDLGRTWLMQMGRAAQENGITIQYCMSNPRHIMQSVEIPVVTQARASGDYRAGKDNWKIGISSI MVDALGVAPFKDNFWSSPDQPGNPAYPDLKETHADLQVAVSTLSTAPVAPGDMLNKTNVPLVMQCCNA DGMLLQPSKP AKAIDAQILQLAFGGDVGPQGDIWTTFSNIAGFYFGIILGTDMTADYNLTPSKASFKAFLPS EVFPRFASIPKSQEFSASNPLVITTNCTKTNICLYYTSPVFSPGGTNVLIYGEENKIVPMSPKRVTDITLDRDL KLTLTGAAKESVRFGFILPDQYITITCQLGQSGSAVLSFNERSCNGY* Lottia Protein Model : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO44886.1| predicted protein [Nematostella vectensis] NR E value: 2e 07 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNTINDCDDGSDENNCACLEKLGMEDGTI LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEGLNAFSNNSLYRSLFFPSLVTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGKFGCLSKLGSGGCKSFYLALGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVR KTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVLRTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSATNDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDN FNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWSDEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model : jgi_Lotgi1_153437_fgenesh2_pg.C_sca_3000340 NR Annotation: hypothetical protein P B105991.00.0 [Plasmodium berghei strain ANKA] emb|CAI04701.1| conserved hypothetical protein [Plasmodium berghei] NR E value: 8.00E 09 Sequence: MKTMVAIIGVIIVSCVLHTSGLPLFKNILNERSVEYGGESGNGFKNERAVGYGGESGNGFLNERSVEYGGES GNGFQNERAVGYGGESGNGFLNERSVEYGGESGNGFQNE RAVEYGGESGNGFLNERSVEYGGESGNGFQ NERAVGYGGESGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAIEYGGESGNGFLNERAVE YGGESGNGFLNERAIEYGGESGNGFQNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNERSVQNSGKSG NGFLNERSVQQGGKSGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNE

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160 RSVQYGGESGNGFLNE RSVQYGGESGNGFLNERSVEYGGESGNGFLNERSVEYGGESGNGFLNERSVEYG GEAGNGLK* Lottia Protein Model : jgi_Lotgi1_160696_fgenesh2_pg.C_sca_25000301 NR Annotation: No match found NR E value: Sequence: MKLLLLLTVLIPLCVSVPTKPGCCVPPVWEGIQGMFSGDGSGPAPELIQQSALISYDEKRQMININITQSI HGL TSDVRILTDYKKGVQYLINSKSRSCTTSKVPQTFQRFCIPDYFYYKGSFNVAFTLRASTEQFIVPLIWSKCKS NSSNFFKMRKACFSRDIQLVVGTCDCLFN* Lottia Protein Model : jgi_Lotgi1_163475_fgenesh2_pg.C_sca_40000029 NR Annotation: Myc homolog [Crassostrea virginica] NR E value: 8.00E 08 Sequence: MDLSYLYYGMFWFSCLLVSTVSAEFSYEEVTYRIPPTPTGFYDQNCTDKISDCEEFGIASCFKPYESWAREN CAKTCEFCQGKPEKPPTCTDVEKGCDLYTTSICTDPSFISWAKAKCRRFCRMCPEQVLAELDLMTTTLTPV DCFDKVVCERYGKKACTGDFASWATDNCQKYCGFCSTGVTKSKRLCVDKLPNCPQYDSRICTDKKYYSW TDENCRSTCKFCQDKLVPFLIPTPQAVRTPKPPTT* Lotti a Protein Model : jgi_Lotgi1_166720_fgenesh2_pg.C_sca_60000088 NR Annotation: No match found NR E value: Sequence: MMLLIKTGVLLIILFSLNTLCDSCACEFHPVLSGKWFFTGENKNINIKIHSKIIVFKVSNYLKLRYKCTERYG NIYLLRILCLFLEVDFLSDLFMKGGFTSIHVEGRGFDSFIFSPKNVTRKYANIDNTCDSEISRERKFI YRKGGC RVPRDMVGTWGFTFRQSSAITMGKRNLTIHFLNNDNLTLNCEARDRNMFLFRRSSYINSSTDAIICVQIDPV LDDPFYDYEFNRIITGNAVENLVRTVPRGKKLYINSDCDMIHSPARPEYIYTVDNIL* Lottia Protein Model : jgi_Lotgi1_166891_fgenesh2_pg.C_sca_62000015 NR Annotation: PREDICTED: similar to scavenger receptor class F, member 1 isoform 1 precursor [Canis familiaris] NR E value: 9.00E 05 Sequence: MATGLYFILSIVTCIINNCVIGLEICEDHYVSCPSQDCGIFPLEPLFRLHCMKTCGVCTDVNRASRKPTAQSSV DVNGMASHAVDGNTDLNICSKTNSSQPDTQWWCSDLQQVYEIDAIKIYSPVIGTKCNTGYYSQSSTSCSPC DTCSENSCEPTNGMCWDG CKQGFKGETCRQCEDGLFGPTCRFACNTTKCQSNCDRLTGICSECDTGYYGK NCELVCDKDCREGKCLKTDGSCTTDGKGSVVSTERAPVQHYTENINKYLVGVIGTFIGIVVTILLVLTYRCF KKRRSDTIYELPTRPPSKQELDFPLDPQYISSGVSTTSSRYTEVHPDSGSSHGSHYLARRNLSQYTHKLRPPA ELPENEKRRLQAEMRQSKISGPRGKYMEYPNDAYDRDGSDYEAVKTEI* Lottia P rotein Model : jgi_Lotgi1_171718_fgenesh2_pg.C_sca_119000006 NR Annotation: PREDICTED: similar to orphan G protein coupled receptor HG38 [Gallus gallus] NR E value: 1.00E 15 Sequence: MAPIYLTFIASLLLASSTFCEQLCLEGCKCEDHPSEKLGPHLMIDCSNLGSSSVPRDLNKFQNYGRPMHLKL S KNIIARVSKADFPENLPIIALDFNQNRGVMITDDAFMNIKGLRYLGLNDISMPFTDTMKYLKGMTKLRYLY LNKNYQFGEGLVPVSLFSGLELKLKDLSLHDCYLKGIENGALANVVITGSLDIGANQLDHIPEEVKTLKNLT KLDVSQNSIKSIPSNTFEGMSKLRTLSLSNNYINGQSFKTDSFNGLKLKELRLEKSELNTIPSAALSSLSSLKIL KLNNSPIKTIPKHSFNGSYCLEVLDLTNVLVEIENTHL DGLESCLQKLYLNNMQIREIPKFFKDFKVLNYVSL SGNSISEIKMDDLVGTTITELDLTENPLHRIEKGSFEEFPHAISLIISDTRLSNLSFVRDYEYGTIKTIKLGNVLP

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161 MCDCELAFAEYRVLEIHGNCNFNNTKNVAISTDEFMSVTSHCWPDGKMPQKKDVWAVVGDNSAGTFSTS ILSYFCVFLFFKVLNIV* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_sc a_130000017 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO35011.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 26 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDAREKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPHIG NRENYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG RYTYRTTNIAQDISCCYVGRTCLHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIKSF VSYGTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPIRGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTILTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMYMF PEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTPNYPFDYPLNTECNWLFVAPEGSEVML NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEMAHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYDNF DDCEELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD GFTNWRSTYPVSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADRKS S CTRNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESFTTQCPA GQICTQNCQHFSRACRRSGEVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGPTC YFFSLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTSAGNTESSSTGASRSSPSTDAFVSEDTTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model : jgi_Lotgi1_172798_fgenesh2_pg.C_sca_142000011 NR Annotation: gb|AAA3004 7.1| calcium binding protein [Strongylocentrotus purpuratus] NR E value: 7.00E 07 Sequence: MRADSIQVRGLKALVLILLVLYEGHTKETCRTGVKTDKNLRGVNLDKTSGLPFYWFDLHRASDGYHSEYT YTFRLHSSSEASPFSDAVLQVETANNCGTGDLVVNTTFLYQPRNCPKMIMTEYKKPMTNVPFKWRAPKCG CIKFRATVIRNGVTYFYDDENISD GNLVKTVCPHGVRVAEQLVEVDEKTTDSTTKTLSTVTSKMDRAALLD LLCKITSTLNNLELLKRPEFIQRRKLQNSSLVKLLNIELALEQRKADISSCCHKKGEDKTICFDNVRRYRIDQ LCGDGQPRIPFVEQKRNYMRKRESECCWRIGQPRYQCFSKSSEQLTEYSVDYSLDESDPANDIADYPKEVE RYEMASNTKFFSPPPTLKPDNQNIETLTKLEETTTPTSVTKSVTLPPIRQPSARPHRQTTPTSVL THHRQRPVP AVKTKLRQSRTVGQGDMGYNLRKQAKYTRKTTNLRHSASSVEITVNTLKQKLWKSSLKLECCQQGRIFAA KTGRTDVWDRCRSAAKDFRSSVVRGKKICSNTFQKCCVEQFITATAKASTIPKPNTPKGSPSTKQLVTTTSL RTNIKKENYRRAPLDVDSSREADEKYASMESKAEDDLMEQDDKASAEDAGEHVDDDGDDVSDDSDNDD QGEDGDQDEDDDQDDDDKDGDVDDDKVDEINNDEKNR KRKFIDYPREDPSADTFHNLQTKRKSTPNTDI AHRDSNHRNLKIESAKRKRNRRRHNRKLRRRKHKSD* Lottia Protein Model : jgi_Lotgi1_201804_estExt_Genewise1.C_sca_1950010 NR Annotation: DICTED: similar to putative porin precursor [Strongylocentrotus purpuratus] ref|XP_001192936.1| PRE DICTED: similar to putative porin precursor [Strongylocentrotus purpuratus] NR E value: 8.00E 19 Sequence: MRLLIFTLSLLVAVVRSVDASQCPEPPSGFGPCVITDANCLTDSECSNGQICCPHLCGKICKDKVSLTVVKPG

PAGE 162

162 TCPSNAAISCFVGSVADTCSSDSDCPGNQKCCNLCGKNCSPPQGSAPQLPPIGLPAPPPVCPPVCAIACEYGN VLD QNGCPTCSCKKTPCKGEAPLTLDRNGNTINCGRGGVRCPLNSECKIHPADAYALCCPIEPDDPKPGVCP KLPNPLPRCLIFQNDCKQDSECPGDQKCCSVTCGIGCIDPVKLCPVVDCLQYCEFGYRLDSNGCQTCQCKK SPCEDEAPMTLDSTGNTINCGRGGVRCPSNSECKIHPADRYAVCCPKVCPPVCEIYCEFGNVLDENGCKTC KCKKSPCEDGAPMTLDSNGNTINCGRGGVRCPLNSECKIHPADRYAVCCPKVCPPVCAIYCQYGNVLDEN GCATCTCKKTPCKGEAPLTLDRNGNTINCGRGGVRCPLNSDCKIHPADAYAVCCPKEPSPVCKFGSCPVPP SSFLLRCASIDRCKYDSECEGTKKCCKHPCGRQCRDIVKSKPGVCPKKADPTLVLCKRRGDDCYEDATCPG SQKCCQGACGNACMDPRLTSTDHAGTCPALDPKIQSLILCASLDTECELDSDCKVDQKCCKDYCGIKCVKS VPKPIIKKGTCPRLRYRC RRKRRDFCRSDGDCPGDRKCCRLPCGYKCRRPKRIPIIRKECPAVNTHPIYKLFC SPPLNPPCQVDRDCPQGKICCPGVCANRCLKAPTKPVCDPNPCGDAQIKCQYGQTKDENGCLLCQCKSYDF KMS* Lottia Protein Model : jgi_Lotgi1_205030_estExt_fgenesh2_kg.C_sca_1120002 NR Annotation: PREDICTED: hypothetical protein [Str ongylocentrotus purpuratus] ref|XP_001188672.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 3.00E 20 Sequence: MKLMFLGFIGILMLSTSNNASASPWFCKGLECPKFNLLNKTKDYEVRQYGSLTWVTTSHIGQWKSDILGEL LFKNLFSYIEGNNTMKKQLDLAVPLLTKLESAGDRGSIYTMH LLIPRENIDHLPSPLNTNLTFITIPPSTYYIRS FDGVAYDRQYMKVLQELVNAINDTGAYPDDFFYTATYDHPQVKENRHNEVWLLKKEKIP* Lottia Protein Model: jgi_Lotgi1_150026_fgenesh2_kg.C_sca_22000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO30774.1| predicted protein [Ne matostella vectensis] NR E value: 3.00E 25 Sequence: MLVYLLLAALLTDFSSAGVFYKDCGSVDGTISNIEVSSCASEPCQFQKNTNISVTIDFTSKTVVSSATTVVHG IIAGIPVPFTSVQPDACKDMSCPLASGTKYTYKNQVAVLPIYPTIQLVVQWEVKDQDGKDLLCFKIPVRITN* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_pg.C_s ca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purpuratus] NR E value: 1.00E 26 Sequence: MMKILEILLCLSFFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCSLKCSGR CRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSCTKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCNDGCKQDTGYCNKCNKGVYGNVCDKNCNKGCLDGCNIDGSCKCKSTYYGTNCSKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQCPSSCP ICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEKCNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNGDCDRSTGNCGKCNEGYFGDNC NQNCSINCNGGCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICL QTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Unknown Function Lottia Protein Model: jgi_Lotgi1_105864_e_gw1.4.718.1 NR Annotation: predicted protein [Nematostella vectensis] gb| EDO49128.1| predicted protein [Nematostella vectensis] NR E value: 3.00E 15 Sequence:

PAGE 163

163 ILTCLAFSLVWHSHLSGILTCLAFSLAWHSHLPGILTCLAFSLAWHSRLPGILTCLAFSLAWHSHLPGTLACL AFSLVWHSRLPGTLACLAFSLDWHSHLPGILTCLAFSLDWHSHLTGILSCLAFSLDWHSHLTGILTCLAFSL VWHSHLTGILT* Lottia Prot ein Model: jgi_Lotgi1_107224_e_gw1.6.194.1 NR Annotation: No match found NR E value: Sequence: CFLSVCLCSSICFTTECCFLSACLRVCFTTECCFLSVCLLFKRSRVCFTTECCFLSPCLRVCFTTECCFLSVCLL FKRSRVCFTTECCFLSVCL* Lottia Protein Model : jgi_Lotgi1_122612_e_gw1.41.141.1 NR Annot ation: predicted protein [Nematostella vectensis] gb|EDO49127.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Sequence: MSYRRCFGISCSSMSFAVDWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILLVDVV DWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILMVDVVEWCILLVDVEDWCILLV DGVEWCILLVDGVEWCILMVDGVAWCMLLVDGVEWCILMVDVVEWSILLVDVEDWCILLVDGVEWCIL LVDVVDWLILFGISRFKLELLHLEI* Lottia Protein Model : jgi_Lotgi1_132441_e_gw1.79.137.1 NR Annotation: PREDICTED: similar to CG11081 PA [Nasonia vitripennis] NR E value: 4.00E 72 Sequence: MAWKIYVLFVCVLCLRFILAARENYIKATFEGAKDRGIFNKLVVNRETNELYLAAVNHLYKLSPNLEVIQE VVTGPHLDDPSCTSLTNLTCDPDLKSCACKLRPYYTKALVVDYVNKKLITCSTLYHGHCDKLDLDNITNKQ MFPFMPMVANNATASTVMFIAPGPAVVKDEPQRQVLYVAASYTRTGLKAYREQVPAFCSRSLEDFNLLLK NAFGSSSIEIESQHRDTFPVRYIYGFGSDNFSYVLSIQKANVQTDRYVTKISRICQSDDDYYSYAEVGLSCTM QNTEFNLLQAAYVGKSGTKLARSLGIPTTEDVLYTVFSIGDSNSANPSATSAMCVYSLRDIRKKFTENIQEC FSGIGNTGPDHMIQSSKCLKVVSSHFFVIFFLESFESADLSVL* Lottia Protein Model : jgi_Lotgi1_143169_e_gw1.194.72.1 NR Annotation: predicted protein [Nemato stella vectensis] gb|EDO36917.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Sequence: MVKVCSIPLTVVKVCSIPLTMVKVSSIPLTISKVCSIPLTMVKVSSIPLTMVKVCSIPLTMVKVCSIPLTIVKVS SIPLTIVKVCSIPLTMVKVSSIPLTIIKVCSIPLTVVKVCSIPLTVVKVCSIPLTMVKVSSIPLTMVKVCSIPL TVV KVSSIPLTMVKVCSIPLTVVKVCS Lottia Protein Model : jgi_Lotgi1_145599_e_gw1.360.11.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO49883.1| predicted protein [Nematostella vectensis] NR E value: 6.00E 16 Sequence: LVVYCHCGLVVYCHWELVVYCHWGL VVYCHWDLEVYCIVYCYWGLEVYCHWGLEVQCYWCLEVYCH CDQVVYCSWELVVYCHWGLVVYCYWGLEVYCHWGLEVQCYWCLEVYCHCDQVVYCSWELVVYCHW GLVVYCHWGLVVY*

PAGE 164

164 Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.00E 140 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECL KLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* Lot tia Protein Model : jgi_Lotgi1_157590_fgenesh2_pg.C_sca_14000213 NR Annotation: No match found NR E value: Sequence: MLMFTSLLFVCFASIYAGESVYPDCQNPDNNERIPNGSKFKLENSGDCIEFTCKDGGYSASSVECDNNGECV AVGRTVEENCRSKKCVYDKFVGFQVTETKCEDDEGQCHSPGEEFTRTYQGTTYSKTTCTVDGNLVKYSYK N* Lottia Protein Model : jgi_Lotgi1_161950_fgenesh2_pg.C_sca_31000192 NR Annotation: RE49668p, putative [Brugia malayi] NR E value: 3.00E 08 Sequence: MKLGGDYGILCVLFTILTPVISIDCYACTSLGGNNEGCEDSFERTLKTVHFIARDCYYGYFRGTHCYKLKGE RADGSSIMVRHCSDSHWGSHCGDIKYTNE HNVEEKIKGCLETCDHDGCNTATQKQFNFSLVIMAVLMTSA LLL* Lottia Protein Model : jgi_Lotgi1_165259_fgenesh2_pg.C_sca_50000100 NR Annotation: hypothetical protein CHLREDRAFT_144736 [Chlamydomonas reinhardtii] gb|EDP05543.1| predicted protein [Chlamydomonas reinhardtii] NR E value: 6.00E 23 Sequence: MKVLLVLDICCLIGLVSVGEAVCPDNCNYNGVCDVKHSRCNCYEGFTGSDCKTDCRCNGHGTCQSGSVC KCDEGWKYSGGQCVWDCHCLNGAKCIGPGECGCVHNCKMGNCRNGQCQCWNGYKGSDCSEYDPTM* Lottia Protein Model : jgi_Lotgi1_166645_fgenesh2_pg.C_sca_60000013 NR Annotation: No ma tch found NR E value: Sequence: MGASILLGLTILLSYVNQVHLQMFHPRDISRGGQQQEEQLRRGYLIQILKSFLNRRQEDATEKRKRSCNLNL GFHCQTDEYSSIADMYDFLQSALSPGKRKRNVKIVSIEGS* Lottia Protein Model : jgi_Lotgi1_167022_fgenesh2_pg.C_sca_63000026 NR Annotation: predicted protein [Nematos tella vectensis] gb|EDO43826.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 08 Sequence: MVAVRIRKYKNLLFFALLFAGVLSISTIFYSNHYQIMNTGTRSDVNTGFSYSNANSSKYVIYICDGKNSCGG YGDRQKGIVASYIISVMMNRQFGVIMNDSCDIKQMFKPNQINWIVDNNDIKSKTSKRIKALDGYSDKLRKS

PAGE 165

165 MIEMDLD QEYKEEVIYFSLNLEFVFYLRQNKRYEKQLKWLEHLSMSEIYNVVWQSIFKLRPYIREKLDPFL DLKKQGLKLISAQIRLGKNPTIPHDSRVVNSLNNMNVLWQFYKKYNDSSKYRIFISTDSDTVREKARSIFPD VYSDVPGKVFHVERSNKTDICNGWRKVILDQVILSLSDVLVISNSGFGRIAAFFRQNENDLYCLNLDKIRRC TTQSEIFVDKTW* Lottia Protein Model : jgi_Lotgi1_ 168040_fgenesh2_pg.C_sca_71000102 NR Annotation: No match found NR E value: Sequence: MVGTRYLQLATSLAVFLLVLFLTCTQAAPVDDFETDNEALRKALFIARLLSSSDRLKNTKPEGSNLDFSELA SIPFSNQQKRYRPPMQGRSGGMSLCLWKVCPAAPWLVSKRSEKTWDKNNMLGK* Lottia Protein Model : jgi_Lotgi1_170051_f genesh2_pg.C_sca_92000069 NR Annotation: hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] gb|EAR97557.1| hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] NR E value: 3.00E 08 Sequence: MKMINIFIVVYCLISPCLGFWLSSKTVDPKVDISTECINKALTGDCGFFTCFEERLPCGEYGYAESYGGKYC WQFQQSHHLFTKKGVEFVEKLTRCHMNRSITSYRQNQIECFSHYDQSFVIMGDCYVESGFCDVVIDNFMSL ARILEPKDFTNYRVVREVFRAAKQCPNNISGGIISKFLSYKRGSSL* Lottia Protein Model : jgi_Lotgi1_171167_fgenesh2_pg.C_sca_110000033 NR Annotation: No match fou nd NR E value: Sequence: MAISHDWSFTLLWLIWIFTLLFSDSYGKRTQNYYMEGLDCGDSKHIGGATVYSNFRGDVSTVYGNDIECQ MTFKAENKDWRLMLRIIELDIPDRTSTGLCNDALYVYDESSIYARAMEEANGNTGLCGNILPPTLYSTGQY LTVHFSVSLQNQKDIV* Lottia Protein Model : jgi_Lotgi1_171450_fgenesh2_pg.C_sca_115000014 NR Annotation: No match found NR E value: Sequence: MKRFTLLALLLVAILYISQSESKCQGVTKCMVNLFTCKREQISVNIAADCCKEYFECFNECKPGSRPPCKNT APGKRGSWNKRYSSRYGSDILGDIISGY* Lottia Protein Model : jgi_Lotgi1_171757_fgenesh2_pg.C_sca_119000045 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO37253.1| predicted protein [Nematostella vectensis] NR E value: 7.00E 24 Sequence: MWKIFSFHWIYFLFSLFCLLHSVTCVKKVSKTPRKSKESEVSKESTKRSASLDDDPSLWPGHLQPLGSSQQV HKVDAYKKYLKPEEFLNSYAAPYLPVLFQAVEQTSIAYEKWGNITYFTVLPQTLTNWVHISPEVGQVSFRK FLLNYTDKGSVFNNKVPFFMRSAKWEPYRDDVSVPKCLHCEPILNSFSTHKLWISHTDQSSPIHRRNKDTIH CTFVGSQEFTLISHIEYGDKVPVDYTTIDINKVDFTKFPTLREVEYHKVRVKAGDCLYIPTKWYWQVRSTG KTYSADIEWYHRNKTVKSSNCKTKTSISSLNSVVFKDIDDPTTIETDVLLHYFTTYLLNVPKFTLPQFERHLK KDKKFMEDLIEWTDELQHITKEVFETLDVNKDSRFSMADL ESLSQPMLEDIRGLFLDRLQDYDDILKDQQE EAKESKKQSEKENKAKDAPATTGPVDDYLKNYIGQLEDVLKESIEEMSVTGHLPDIKKKFAEKQAQGKTG NADKPKQSTKPKTEREKAKEKLEQDRQRRKEKQEAENIKEKTKDDEKYLIVDDSVEEEIMADDDDDSTTG STNKRTPTADKADTKDNKSHTKDNKADTRHVDKKTGRKGQDGKEAKQKNTGSKIKTEL*

PAGE 166

166 Lottia Protein Model : jg i_Lotgi1_173456_fgenesh2_pg.C_sca_159000027 NR Annotation: No match found NR E value: Sequence: MWSGVWCGVVYGVMWSGVWCGVWSGVERSVVWRGVWSDVDGVMWKECGVVYGVVWCMKWCGV WSDVERSVVWCMKWCGVWSDVERSVVWCMEWCGVWSGV Lottia Protein Model : jgi_Lotgi1_173484_fgenesh2_pg.C_sc a_161000006 NR Annotation: No match found NR E value: Sequence: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFPWAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPG GNETFTFDFISIMLGKKQFE* Lottia Protein Model : jgi_Lotgi1_175543_fgenesh2_pg.C_sca_302000002 NR Annotation: No match found NR E value: Sequence: MNTMFLLVVFLLIPATSAFDSTQTNRTIVNSTITTKINGSSIGLGTCSEFTPQSCSGVCGQFFELWCSCDEYCQ VYRRCCFDYEAVCKEESEKSKQDHENLMNVTVECDSNM SGVHVPQQRISPASG* Lottia Protein Model : jgi_Lotgi1_176704_fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTED: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDDVNVCHGELPANAMCSGENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lottia Protein Model : jgi_Lotgi1_203811_estExt_fgenesh2_kg.C_sca_240005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 0 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTRQNPNGNRRGYECPEERDYFPYWHPTQWKDIAILAENE TLCNPLIAESFNNKPYGACLQKFPNSQVRKP SRYNNKEECEENGLDWVAFHNYLEKDKTASTQAACQAKSTNNIEYTWAIPYDSTTFTAECLVKVPSPQCQ EAPWSRSNHLGNGIGGEMNTVQWTLPHFPSGQVQNCVMRMRYNISTDDYDPFNTDSEFNGADNSPVTNN PYVDIGVGRGPLRLAINTAQFGRVFQDRTHIFQLKPRPDDIKDFRIFNVNVRGKRGNIVQVYPAVEYDFTPT DLEVTENDLVHFQW TGSNTHNNGRPGGDGQTGDAGEGKSGSDRHNILQMADRNENFPLPFEKTNMWDN AEIKWIYHGKNDVSARDLALNMASSGYYKCVSATDCPAESFKDFLVDTKDKMQVTLDNTPASYPGVVLK FKQGKYHYMCTRNNNFTNRSQKATITVAA* Lottia Protein Model : jgi_Lotgi1_204607_estExt_fgenesh2_kg.C_sca_690020 NR Annotation: ctg4a [Aedes a egypti] gb|EAT44181.1| ctg4a [Aedes aegypti] NR E value: 2.00E 45 Sequence: MAFMFFQTLLIIVSIGYLHADDSDGVQEATLCEVCRILAEELQLRLDETGKSKEVIETGHGLDTKKKKKYNF

PAGE 167

167 SELRLIEALQEPNICDRILEYSVHKEKTGIERFAKGRSQTMEALHGLVNKGVKVELGMPHEMWDKPSVEIT QMQRKCYALVEEYDEDIEDWYYNHQDQPFLDYFCRNL VLNPQNNECLDEDMRSQNEGSSSEMKGDKGK KEEL* Lottia Protein Model : jgi_Lotgi1_204654_estExt_fgenesh2_kg.C_sca_720011 NR Annotation: PREDICTED: hypothetical protein [Monodelphis domestica] NR E value: 8.00E 93 Sequence: MSLLIATGLFCVIYLLVKFLNISQPCHLPELYFKDKTSNFIQTVFTMCPILFESYIPPLLWGKSGHLQTFIYAK MGRIMSPLPNGKRIELIRPDNATMSFDVFQPHYEHPTGRDYTLAVCPGIANSSESLYIRTLVDHAQYNGFRV AVLNHLGALRKVKLTSPRIFNYGETGEYNCMIDELKRLYPNTSIIAVGCSMGANIVIKYLGECKQHENKVIG ALSFCQGYDVNDAKPFLLGWESCRRLYCYFMAINLRQLLRSHQDILFTEQAQSEYGTVELKKLYSATSLLA IDELYSVNAFK FKCCEEYYHWASCKQYIQNITKPIFILNAEDDPIVPKQLYTTPLKYQENHDNCLFIVTKHGG HLGFFEGGFIKPNTITWLDRAVVQFSNVLCELDCKERKS* Lottia Protein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematost ella vectensis] NR E value: 1.00E 10 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSCCMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMWRAVFSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI Lottia Protein Model : jgi_Lotgi1_983 30_gw1.10.599.1 NR Annotation: hypothetical protein NEMVEDRAFT_v1g147894 [Nematostella vectensis] gb|EDO28414.1| predicted protein [Nematostella vectensis] NR E value: 5.00E 06 Sequence: TVCSLGTHCLSITVCSLGTHCLSITVCSLGTHCLSITVFCSLGTHCLSITVCSLGTHCLSITVCSLGTH CLSITVCSLGTHC Lottia Protein Model: jgi_Lotgi1_154212_fgenesh2_pg.C_sca_5000252 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E Value: 2.00E 97 Sequence: MNLKLYILICVTVSGILGTPLDDYVNRPDSTYKYFEITHYVHEDHTLYILNMTSQTWLTAEISTQPIWWHYL TVTIPHKIRY PDAAFMFIEGGSNHNGPPTGTDNFVALTTAMAVSTGTIGADLKMIPNQPIMFKADPTKKQRT EDAIIAWTWKTFVETNGSNPDLLLRMPMTKASVRALDTIADFAMDKVGNKVDKFFIAGASKRGWTTWTT AAVDNRVVAMAPIVMDLLHMVKNLHHHYRAYGGWTFAFKDYYSLNFTQDLDSPWTQAMADIIDPISYN DRYANIPKLVVSTGGDEFFLPDDSHFYFKELMGPKYLRILPNAEHSCAGHEISLLFT MRAFYLSVITKTPLPQ LTWVREETPSGGRITVTCNQKPKTVNVFYARTLDGNRRDFRLLVGTPGDPSKPMPHPVVWLRDDVQDMG NNVYRAELTKPEIGWTAFFIQLTFQGFADSDLEFTTETQIIPDIFPFPDCTGQECKGTLV* Lottia Protein Model: jgi_Lotgi1_160458_fgenesh2_pg.C_sca_25000063 NR Annotation: predicted protein [Monosig a brevicollis MX1] NR E Value: 6.00E 86 Ac Annotation: no hit Sequence: MNYKMILNKYTLVLGIILVCTQPCITDVNITKVHLIFMNHLDVGFNGITPQTGFVINVINKYFTKYFPGAIEL ALQMNFLGYRERLIYTTHPWLVSLYLDCPQNLTMSGIQLMCPTEEQREGFLYAAKRGDITWHAGPMNMQ YEALDTAMVNFSLKLSEDMDARMDIKRRHRVLSQRDVPGMT RALIPIFNNHGIEGVTVGVNSVSSPPAVPK

PAGE 168

168 IFKWVFQNSSVIAMWHPGGYPIKPGSFPSMPLGLSRDNCVTFEQFDEAMCFSFRGDNQGPPTSIAEVLNSYE IVRGQFPGAIVEGSAFENFVEVVQSVKDKLPVVDQEIGDTWIQGISSDPGKTAMMRGFFRARTACLTSAPNF EDVISSWSEQRQFIDLALETLGDHPLVQAVEEEYKQLVPQAPDLSKYKQLSATTFDCQNGFKFSFAADGSL NKLQDKDGRNW ASKSQPIGQLLYKTYNDTDFDSFQKQYVLNHYYWFQVGKWNLTANCPTCESTIWNTK LQKLYAASDGSCDVVALLAMEEDRATSFYGAPPKIYVKYLSSSNTFLSIEVQLFGKKPTRLAESLSLSFQPIR QEGSKWYLHKLGQLIDPLNVVTNGSQRLHAVDEGVQYKDSNGKGMMIGSKDVGLATVHQTPTDVSVLPV PLTPIQSISGVSYNIFNNVWDCNYIFWYPFLKQDSNSKYRFSITFS* Lottia P rotein Model: jgi_Lotgi1_161657_fgenesh2_pg.C_sca_30000077 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO44809.1| predicted protein [Nematostella vectensis] NR E Value: 3.00E 17 Sequence: MTKLSEYFLICSMIFVVFMLQRGTCDDVVTEEQISYAKGSVCGYCSYCKF CKLCDKDCPCETSPSKPNCKM CKYCKYCYICSGVCDTICQPGGMIDKISAAIVNALPSFNKEEIEDDLESVKPWLDKKDEL* Lottia Protein Model: jgi_Lotgi1_164905_fgenesh2_pg.C_sca_48000037 NR Annotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSPCQKLGE S WRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCRVGKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIVVAGNAECRVGLRWK DGCKRCRCNKRGKIICKTRLCNLSDDGKKLSNKDKTHRQTSTGG LISKSTPRFQLPEKPRFRMPNLSLYNNR FQRKKRPSIKTVNCNGFKPGEKYWDKCNLCHCTMAGAPMCSSKLC* Lottia Protein Model: jgi_Lotgi1_166736_fgenesh2_pg.C_sca_60000104 NR Annotation: hypothetical protein LOC500991 [Rattus norvegicus] sp|Q5XI89|FA55D_RAT Protein FAM55D precursor gb |AAH83798.1| Hypothetical protein LOC500991 [Rattus norvegicus] NR E Value: 3.00E 25 Sequence: MALTKKLVLICCLIIATVFGLKIMHTIKYQINMRPNIEPIPFYSQNTPKRKLMFPERQSPSLEDYNMKHDDVR SEDIFNKFLSENVVYVKDTTADDYFIEGEKELLEEGEIKDLRDLPSSNYSIISTKNTVFNQYEAVTVKIQLYN GFGQLLKRGGDDVRV WITGENFQAGGEVYDLHNGTYVAEVKAFWTGKSKIHVSIAYSREELRILLRTRRE SKVLRYSVGRFLRGGDTFMTLCSPFHDIPLYPRVCNFTNFNLNPYYCGQPNSPKVACSDLRILHTINYSKQR MNNLESKILSRQNRVNELKSKSITINIGTVVNLPYCSSYKSRFTWTNTAPLGYFNNNTWHPSFCQGQITRQR LSRCIRDLHVYLVGDSTVRQIFQSFLRLTKCTNTTFIGNRPHDRVQRYCDVHDSNFR VEYGIHGLPYSMVRT PDLLNNTKAIGFHLDGLQWSQNKDKMFVMVHLYAHFLFFHTSLFEARVRSLKVPLTRLLNRYPKIKIFIKGP HAFTGADFKPIVKSDYFGKVYRSILLKELFEFRDRVYYLDYWDMSVSSGNRLLHPVNSFVDEMVKFFLNL ACDK* Lottia Protein Model: jgi_Lotgi1_168278_fgenesh2_pg.C_sca_73000118 NR Annotation: no hit Seq uence: MSISASSYSGTFKLCLFIVVLVAFSSYCGFVITREDSQFSNHLPKLTSTNVILEKLDSLRKDFLKQRTNVAGQ FKNLGSKLSELVLNKNTKPENQHIKAQDEPWRKSTKTGLTLFTSWVDVPEKDLVHGNVVRMWRKWKPLI KPLFFYSSASTGKRLEKEGWLVQPVSKTGCGVTEMPTIKDMFLDAMKNHESVLYGYANGDITFDDGMPK AIDYVVNMDVVREKPVLVLVRRTNVDFSNGPELDANSDITEMYNEGKALKDGSSDGFFTNKLFPWKYLPD IVIGRIGIGMWLASYARAMNVTVIDITKTVKAIHMTTKSGNSESHFKKNGRCNHVIYGRLKLSPTSWGCGFI TCATIDITFDKQGQTTMRRKNPKSMNPKCGNCSMDLTQILPKSLGITDRIYFGQNIEPKQVVNKRPKSPK* Lottia Protein Model: jgi_Lotgi1_173484_fgenesh2_pg.C_sca_161000006

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169 NR Annotati on: no hit Sequence: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFPWAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPGGNETFTFDFISIMLGKKQFE* Lottia Protein Model: jgi_Lotgi1_176362_fgenesh2_pg.C_sca_1085000001 NR Annotation: no hit Sequence: MVAKGTVGILIVLFNTICANIDLNYNQVPVANPLLFGRRGINPDMSSLFFGKRSGNSDHRDLRKMKDTCKA VLSSCKILFSDYEDDTVRNKVQDGFGRFK* Lottia Protein Model: jgi_Lotgi1_177026_fgenesh2_pg.C_sca_12012000001 NR A nnotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSPCQKLGE SWRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCR VXKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIV Lottia Protein Model: jgi_Lotgi1_202874_estExt_Genewise1.C_sca_21760001 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54 Seq uence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTR Lottia Protein Model: jgi_Lotgi1_202874 _estExt_Genewise1.C_sca_21760001 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMA YFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTRQNPNGNRRGYECPEERDYFPYWHPTQWKDIAILAENETLCNPLIAESFNNKPYGACLQKFPNSQVRKP SRYNNKEECEENGLDWVAFHNYLEKDKTASTQAACQAKSTNNIEYTWAIPYDSTTF TAECLVKVPSPQCQ EAPWSRSNHLGNGIGGEMNTVQWTLPHFPSGQVQNCVMRMRYNISTDDYDPFNTDSEFNGADNSPVTNN PYVDIGVGRGPLRLAINTAQFGRVFQDRTHIFQLKPRPDDIKDFRIFNVNVRGKRGNIVQVYPAVEYDFTPT DLEVTENDLVHFQWTGSNTHNNGRPGGDGQTGDAGEGKSGSDRHNILQMADRNENFPLPFEKTNMWDN AEIKWIYHGKNDVSARDLALNMASSGYYKCVSATDCPAESFKDFLVDTKDKMQVTLDNTPASYPGVVLK FKQGKYHYMCTRNNNFTNRSQKATITVAA*

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170 Object 26. P redicted signaling molecules found in L ymnaea stagnali s. Likely to be functional secreted signaling molecule Lottia Protein Model : jgi_Lotgi1_113861_e_gw1.18.363.1 NR Ann otation: PREDICTED: similar to tartan CG11280 PA [Apis mellifera] NR E value: 5.00E 27 Sequence: MVKPPTQNTRWWIIVWILLFQKYTCEPEVTCPHGCKCDVDVVNCLHSNLTSVPVGLSNGDRLQELKLGRN KITEIKLELLEYKELRLLDLISNNISTIDGSAFRTLYKLETLFLSRNFITKTDKNTFRNLTSLRFLDLSYNNLRC LKIGNFEDLQSLVN LDLSRNQISLIEAESFRCLENLEILKLPRNQLDQFPWKIFQNLTSLKMLVLDDNLLTSLP ISAFSNLINLEYLSLTGNRLHELLPSSFMMSDEGAISLEQLYLRGTDLDTIPVKLFAKLKKLTVLDLSKTQIVS VKNENLNGLSSLESFIMTSSNNLTYIEKGVFQDLHSLRKIDISGNTLLSEIQFTTSSLRNLSLFDLSANSL Lottia Protein Model : jgi_Lotgi1_157019_fgenesh2_ pg.C_sca_12000330 NR Annotation: No match found NR E value: Comment: Similar to Chordin Sequence: MFSLVLLVVTPWLVFCMPPPPPAMCTCSDGTQVPPGGTHSISDCHSCYCDENNSEAQEIIAACLIVPCVDSV KMPGQCCPTCPNGENCRAEFKDDNGVERSEIIKVSDGVVKFNSTECQCDYQNYNGEPKANCVQVLSIEPVI AEKSTP* Lott ia Protein Model : jgi_Lotgi1_159314_fgenesh2_pg.C_sca_20000054 NR Annotation: Buccalin precursor [Contains: Buccalin D; Buccalin E; Buccalin F; Buccalin G; Buccalin H; Buccalin A; Buccalin I; Buccalin J; Buccalin K; Buccalin L; Buccalin B (BUCb); Buccalin M; Buccalin gene predicted acidic peptide A (BGPAP A); Buccalin N; Buccalin O; Buccalin P; Buccalin Q; Buccalin R; Buccalin C; Buccalin S; Buccalin gene predicted acidic peptide B (BGPAP B)] gb|AAB27696.2| buccalin precursor [Aplysia californica] NR E valu e: 3.00E 66 Sequence: MAARKHELVLVLTSVLCFVSSIVGDPNVPSDSQDNSALTQDDFAKRGMDKFGFAGGVGKRGLDKFGFTG QLGKRDMDSFGFAGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGF TGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFAGQLGKRGLDQYGFTGQLGKRGLDQY GFAGQLGKRGLDHYGFAGQLGKRGLDQYG FAGQLGKRGFDQFGFAGQLGKRGLDHYGFAGQLGKRGLD QLGFTGQLGKRQMDIFGYRGQLGKRQSIDKYSFLGAGIGKRSVKNTAGIKKDDA* Lottia Protein Model : jgi_Lotgi1_164016_fgenesh2_pg.C_sca_43000014 NR Annotation: chordin [Danio rerio] sp|O57472|CHRD_BRARE Chordin precursor (Protein chordino ) gb|AAB93485.1| chordin [Danio rerio] NR E value: 1.00E 119 Sequence: MSPMDLAVCLVLLAATVSSTKFSKIPLKAARENLFGIQPNRPGCILGSKFYGIGERWTPELLPVGEMYCVKC ECVPVERKGIIDMKGQTLCKNIKNLCPRPNCLNPVLPKGKCCKVCPDEVNSFEDSFSLGSKSSAPTMISSDSK RRVRNEFVSLLVGKNVRRESVKTSAVAAVYFSIVGRDEIR YSVRYFKLDRPKFLQITDANGNVLFERPIEKK RNGDKKFCGIWTKVPSAYIQYVREERLFAVITASRHFRGLVSGRIMVNRHAKREVFSSVLASPKANGIGGL LSVVYNPRTHLVDYVINLDGVFPGSSEEYFITISKKSKVLHQSKGKASSKTRFIRGSWTLKNKRQSKQLARG RLTIRLTSTNGASVSGDIKPKVTCGVSQAVMSSGNSLEEMKLASSGTAVFELREKGSLDYKIRVTGLTSKVH RIRLEGAVN KKNRRQIIGSVHRNFKADENSFNGWCNGTFKKMTADDLHLFLNNKIFVNVATSENRISELRG

PAGE 171

171 RVIALPYHSHFEQVSADPAALGPKTITSNGHAGNAWISMGVGCSLHYDITTTTIAKHHDLTLSLVSPTDIDA YVTIEKISTTNLYEFQQASGSIAVVPESLFRDLDEGSALIQVISGDVILSGNVSVPNTCWQYSQDYDMDILVD EQTSEEVLAANRYKCVYEGKVYENGDSWLPDVNATCNTCSCNKGKIECH RLICPVVECANPIVLDGECCPT CPIETTDQTECKIDGDLRSHPVGSTWHPFLPLMGYAKCVICTCLPGGIYKCERKCPKLDCPASRRIRLNPKD CCQVCAPEEVKPEVVKPLQDVDMKGACSFKDQIYANGAKWHPRVIPFGYMKCIDCSCNNGTPSCSRPKCP KLSCSRKIREDGACCDTCADSDGEEIVEEKKSDSCIFGGKKYDDGETFQPKTSSLSSCATCTCRKGSMKCSL TCPEKCKDPKHSNDPCCKHC Lottia Protein Model : jgi_Lotgi1_164520_fgenesh2_pg.C_sca_45000185 NR Annotation: PREDICTED: similar to filaggrin 2 [Rattus norvegicus] ( molecular regulation of epidermal terminal differentiation) NR E value: 5.00E 37 Sequence: MIKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDIVQMIKLTLSILDIVQIVKVTFSILDIVQIVKLTLSILDTVQ IVKLTLSILDIVQIVKLTLSILDTVQIVKLTLSILDTVQIIKLTLSILDIVQIVKLTLSILDTVQIVKLTLSILDTVQI VKLTLSILDTVQIVKLTLSILDTVQIVKVTFSILDTVQIVKLTLSILDTVQIVKLTLSILDTVQIVKLTFSIFDTV QIVKLTLSILDIVQIVKVTFSILDTVQIVKLTLSILDIVQIVKLTFSI FDTVQIVKLTLSILDIVQIVKLTLSIHVVC Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] (Fulicin like) NR E value: 1.00E 21 Sequ ence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPM FLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNYFSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Protein Model : jgi_Lotgi1_168508_fgenesh2_pg.C_sca_76000035 NR Annotation: ependymin related protein precursor; EpenHg precursor [Holothuria glaberrima] NR E value: 3.00E 10 Sequence: MMKYCLVVAVVVVGVVIGEPSKPGCCTPSQWEGQQGSTIGIVDGGIPSVRTSMVRISYDEKNQRIAYVTSE S TPQGEKNYKIIYDYSRKFQFIVDLDTKHCTVSSLTDPFMKLCLPVDAQPKGKFYYGIDGNNLPAMAFSLN VNGTMITVVTTADNCIPIAEVLVDGQAQGVSQMHASGFVNLTSGIKDPSVFIPPGTCTQ Lottia Protein Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chai n; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica] NR E value: 4.00E 13 Sequence: MEVTCKCPLVLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYC RNSQQRISIKK* Lottia Protein Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Euprymna scolopes]

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172 NR E value: 1.00E 122 Sequence: MTVSPTVVWAAFSMALSSVVALGANIICNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTS AGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LPPFRKIGDSLKKRYKKSKIVVPYLGRRARTAVTLILKRAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK* Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37057.1| predicted protein [Nematostella vectensis] (growth factor) NR E value: 1.00E 109 Sequence: MFLEVFNISATVLCILFLIGHRKCDTIQNSKHHAELM MDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVGVNGGFFNTTTGACLGNIVSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSAVGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLEL GVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYGINCSTPCKPGYYGSNCAHQCYCLNHGTC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi1_208417_estExt_Genewise1Plus.C_sca _10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVIASSRRSRGRWYRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCP GFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSNNAFEKLEEKVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEV LKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* Predicted molecules found in Lymnaea stagnalis with unknown function or predicted to be related to the secretory pathway Lottia Protein Model: jgi_Lotgi1_103075_ e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mellifera] NR E value: 9.00E 50 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRLE NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHPNEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLEDREVYLTEEENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL*

PAGE 173

173 Lottia Protein Model: jgi_Lotgi1_105864_e_gw1.4.718.1 NR Annotation: predi cted protein [Nematostella vectensis] gb|EDO49128.1| predicted protein [Nematostella vectensis] NR E value: 3.00E 15 Sequence: ILTCLAFSLVWHSHLSGILTCLAFSLAWHSHLPGILTCLAFSLAWHSRLPGILTCLAFSLAWHSHLPGTLACL AFSLVWHSRLPGTLACLAFSLDWHSHLPGILTCLAFSLDWHSHLTGILSCLAFSLD WHSHLTGILTCLAFSL VWHSHLTGILT* Lottia Protein Model: jgi_Lotgi1_107224_e_gw1.6.194.1 NR Annotation: No match found NR E value: Sequence: CFLSVCLCSSICFTTECCFLSACLRVCFTTECCFLSVCLLFKRSRVCFTTECCFLSPCLRVCFTTECCFLSVCLL FKRSRVCFTTECCFLSVCL* Lottia Protein Model: jgi_Lotgi1_108564_e_gw1.8.40.1 NR Annotation: PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001179636.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 2.00E 86 Sequence: MYLSCYLPVILLFIEDLKQAVVAMMLRKDEV EEKNKSLKAMLDREMEISSTLRAEIEEMKISFKLSKDKEIA KNETLQKENELLKHQLRKYINAVQLLRTEGAKDDTQGITLEDPQPIIPPAKPSIDYSHEASEYEKKLIQVAEM HGELMEFNELLHRQINCKEAVIRNLKEELTDLRGPLPYDAQSSDDSLSGDLESSLISRCLINIWIPSAFLGGSK TDSHHVYQVYVRIRDEEWNVYRRYSKFLDVHTRLKKVYPLIEKFEFPPKKTIGSKDPKVVTARRKMLQSY LRKVINHLLEKNADLSSNVSKEKLIAVLPFFK* Lottia Protein Model : jgi_Lotgi1_116422_e_gw1.24.410.1 NR Annotation: PREDICTED: similar to CG14764 PA [Apis mellifera] NR E value: 7.00E 58 Sequence: MKTKNFYILVLLISVTSVALWLVSYPSPTFRNIMSTTQNNIRKIPKHMQMSKSPDLEVDKKYLDLLGF DSES KPKNDIVVIGSALMPADTENTILFLKSAQEYVPNLKLVLFDMGLSKADKQTLTNCRNETKHCEIRKFNFDQ YPSHISDFANKSYKPICIQLLLKEFGTVIWADTSELFASSDIDNTMKQAKTEGLVAWTIEDATSSLTHPKMFD YFKTDQKKYFFHHAVQTSHIILVNTDKIHENVMLPWVKCALVEECINPMGAQNSGCNYQRKPKFRYTGCH WYDMSALNVILGLAFDFDEAYSGKDKIFWTKFDQEKAKAENMTDGAVHKVSVPHRVYF* Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Annotation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQCCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPW PLTTTTSRVPINILCNVYWFKCTKLHFSSHWECSNDDCLLEADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSNVVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFDEEPAVYHSVRIIGWGVERTIYGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWGQISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV*

PAGE 174

174 Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match fo und NR E value: Sequence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_122612_e_gw1.41.141.1 NR Annotation: predicted protein [Nematostella vecten sis] gb|EDO49127.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Sequence: MSYRRCFGISCSSMSFAVDWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILLVDVV DWCILLVDGVAWCMLLVDVVDWCILLVDGVAWCMLLVDGVEWCILMVDVVEWCILLVDVEDWCILLV DGVEWCILLVDGVEWCILMVDGVAWCM LLVDGVEWCILMVDVVEWSILLVDVEDWCILLVDGVEWCIL LVDVVDWLILFGISRFKLELLHLEI* Lottia Protein Model : jgi_Lotgi1_143169_e_gw1.194.72.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO36917.1| predicted protein [Nematostella vectensis] NR E value: 1.00 E 28 Sequence: MVKVCSIPLTVVKVCSIPLTMVKVSSIPLTISKVCSIPLTMVKVSSIPLTMVKVCSIPLTMVKVCSIPLTIVKVS SIPLTIVKVCSIPLTMVKVSSIPLTIIKVCSIPLTVVKVCSIPLTVVKVCSIPLTMVKVSSIPLTMVKVCSIPLTVV KVSSIPLTMVKVCSIPLTVVKVCS Lottia Protein Model : jgi_Lotgi1_145599_e_gw1.360.11.1 NR Annot ation: predicted protein [Nematostella vectensis] gb|EDO49883.1| predicted protein [Nematostella vectensis] NR E value: 6.00E 16 Sequence: LVVYCHCGLVVYCHWELVVYCHWGLVVYCHWDLEVYCIVYCYWGLEVYCHWGLEVQCYWCLEVYCH CDQVVYCSWELVVYCHWGLVVYCYWGLEVYCHWGLEVQCYWCLEVYCHCDQ VVYCSWELVVYCHW GLVVYCHWGLVVY* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.00E 140 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFL ALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLD ALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* Lottia Protein Model : jgi_Lotgi1_152847_fgenesh2_pg.C_sca_2000267 NR Annotation: SARP 19 precur sor [Littorina littorea] NR E value: 5.00E 08

PAGE 175

175 Ac Annotation: CNSN01 F 148317 501 Ac E Value: 2.00E 07 Sequence: MIAFICVLGFISGCLAAPCTHTNSAEHTEEQVIARVIALADHNNDGILSVTDLAAYFVINFDHNHNSEVDKN EFILQWHETFHDEKAFAEHVFTHFDTNADNVLNLSDLFALNIRLDTDGNGTVTNKELESYLQLIYNAC* L ottia Protein Model: jgi_Lotgi1_154212_fgenesh2_pg.C_sca_5000252 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E Value: 2.00E 97 Sequence: MNLKLYILICVTVSGILGTPLDDYVNRPDSTYKYFEITHYVHEDHTLYILNMTSQTWLTAEISTQPIWWHYL TVTIPHKIRYPDAAFMFIEGGSNHNGP PTGTDNFVALTTAMAVSTGTIGADLKMIPNQPIMFKADPTKKQRT EDAIIAWTWKTFVETNGSNPDLLLRMPMTKASVRALDTIADFAMDKVGNKVDKFFIAGASKRGWTTWTT AAVDNRVVAMAPIVMDLLHMVKNLHHHYRAYGGWTFAFKDYYSLNFTQDLDSPWTQAMADIIDPISYN DRYANIPKLVVSTGGDEFFLPDDSHFYFKELMGPKYLRILPNAEHSCAGHEISLLFTMRAFYLSVITKTPLPQ L TWVREETPSGGRITVTCNQKPKTVNVFYARTLDGNRRDFRLLVGTPGDPSKPMPHPVVWLRDDVQDMG NNVYRAELTKPEIGWTAFFIQLTFQGFADSDLEFTTETQIIPDIFPFPDCTGQECKGTLV* Lottia Protein Model : jgi_Lotgi1_157590_fgenesh2_pg.C_sca_14000213 NR Annotation: No match found NR E value: Sequence: MLMFT SLLFVCFASIYAGESVYPDCQNPDNNERIPNGSKFKLENSGDCIEFTCKDGGYSASSVECDNNGECV AVGRTVEENCRSKKCVYDKFVGFQVTETKCEDDEGQCHSPGEEFTRTYQGTTYSKTTCTVDGNLVKYSYK N* Lottia Protein Model : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO44886.1| predicted protein [Nematostella vectensis] NR E value: 2e 07 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNTINDCDDGSDENNCACLEKLGMEDGTI LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEG LNAFSNNSLYRSLFFPSLVTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGKFGCLSKLGSGGCKSFYLALGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVRKTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVL RTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSATNDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDNFNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWSDEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model: jgi_Lotgi1_161657_fgenesh2_pg.C_sca_30000077 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO44809.1| predicte d protein [Nematostella vectensis] NR E Value: 3.00E 17 Sequence: MTKLSEYFLICSMIFVVFMLQRGTCDDVVTEEQISYAKGSVCGYCSYCKFCKLCDKDCPCETSPSKPNCKM CKYCKYCYICSGVCDTICQPGGMIDKISAAIVNALPSFNKEEIEDDLESVKPWLDKKDEL*

PAGE 176

176 Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sc a_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus] NR E value: 1.00E 133 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHLKK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAGTPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSS DNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKGDPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALELSKELN VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAATIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KKHDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQT STKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNTWPEKEECDSH LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHPLPWYPPICVTLLTINEYDEKTYDNLKSVGHP GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGARWVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYA DVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSVAPIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKSGECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGEDCNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLH STQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN HCSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPKSWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLMLLGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTLGNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_pg.C_sca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purpuratus] NR E value: 1.00E 26 Se quence: MMKILEILLCLSFFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCSLKCSGRCRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSCTKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCNDGCKQDTGYCNKCNKGVYGNVCDKNCNKGCL DGCNIDGSCKCKSTYYGTNCSKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQCPSSCPICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEKCNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNGDCDRSTGNCGKCNEGYFGDNC NQNCSINCNG GCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICLQTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Lottia Protein Model : jgi_Lotgi1_166645_fgenesh2_pg.C_sca_60000013 NR Annotation: No match found NR E value: Sequence: MGASILLGLTILLSYVNQVHLQMFHPRDISRGGQQQEEQLRRGYLIQILKSFLNRRQEDATEKRKRSCNLNL GFHCQTDEYSSIADMYDFLQSALSPGKRKRNVKIVSIEGS* Lottia Protein Model : jgi_Lotgi1_166720_fgenesh2_pg.C_sca_60000088 NR Annotation: No mat ch found NR E value: Sequence: MMLLIKTGVLLIILFSLNTLCDSCACEFHPVLSGKWFFTGENKNINIKIHSKIIVFKVSNYLKLRYKCTERYG NIYLLRILCLFLEVDFLSDLFMKGGFTSIHVEGRGFDSFIFSPKNVTRKYANIDNTCDSEISRERKFIYRKGGC

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177 RVPRDMVGTWGFTFRQSSAITMGKRNLTIHFLNNDNLTLNCEARDRNMFLFRRSSYINSSTDAIICVQIDPV LDDPF YDYEFNRIITGNAVENLVRTVPRGKKLYINSDCDMIHSPARPEYIYTVDNIL* Lottia Protein Model : jgi_Lotgi1_166891_fgenesh2_pg.C_sca_62000015 NR Annotation: PREDICTED: similar to scavenger receptor class F, member 1 isoform 1 precursor [Canis familiaris] NR E value: 9.00E 05 Sequence: MATGLYFILSIVTCIINNCVIGLEICEDHYVSCPSQDCGIFPLEPLFRLHCMKTCGVCTDVNRASRKPTAQSSV DVNGMASHAVDGNTDLNICSKTNSSQPDTQWWCSDLQQVYEIDAIKIYSPVIGTKCNTGYYSQSSTSCSPC DTCSENSCEPTNGMCWDGCKQGFKGETCRQCEDGLFGPTCRFACNTTKCQSNCDRLTGICSECDTGYYGK NCELVCDKDCREGKCLKTDGSCTTDGKGSVVSTERAPVQHYTENINKYLVGVIGTFIGIVVTILLVLTYRCF KKRRSDTIYELPTRPPSKQELDFPLDPQYISSGVSTTSSRYTEVHPDSGSSHGSHYLARRNLSQYTHKLRPPA ELPENEKRRLQAEMRQSKISGPRGKYMEYPNDAYDRDGSDYEAVKTEI* Lottia Protein Model : jgi_Lotgi1_168040_fgenesh2_pg.C_sca_71000102 NR Annotation: No match found NR E value: Sequence: MVGTRYLQLATSLAVFLLVLFLTCTQAAPVDDFETDNEALRKALFIARLLSSSDRLKNTKPEGSNLDFSELA SIPFSNQQKRYRPPMQGRSGGMSLCLWKVCPAAPWLVSKRSEKTWDKNNMLGK* Lottia Protein Model : jgi_Lotgi1_170051_fgenesh2_pg.C_sca_92000069 NR Annotation: hypothetical prot ein TTHERM_00439050 [Tetrahymena thermophila SB210] gb|EAR97557.1| hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] NR E value: 3.00E 08 Sequence: MKMINIFIVVYCLISPCLGFWLSSKTVDPKVDISTECINKALTGDCGFFTCFEERLPCGEYGYAESYGGKYC WQFQQSHHLFTKKGVEF VEKLTRCHMNRSITSYRQNQIECFSHYDQSFVIMGDCYVESGFCDVVIDNFMSL ARILEPKDFTNYRVVREVFRAAKQCPNNISGGIISKFLSYKRGSSL* Lottia Protein Model : jgi_Lotgi1_171718_fgenesh2_pg.C_sca_119000006 NR Annotation: PREDICTED: similar to orphan G protein coupled receptor HG38 [Gallus g allus] NR E value: 1.00E 15 Sequence: MAPIYLTFIASLLLASSTFCEQLCLEGCKCEDHPSEKLGPHLMIDCSNLGSSSVPRDLNKFQNYGRPMHLKL SKNIIARVSKADFPENLPIIALDFNQNRGVMITDDAFMNIKGLRYLGLNDISMPFTDTMKYLKGMTKLRYLY LNKNYQFGEGLVPVSLFSGLELKLKDLSLHDCYLKGIENGALANVVITGSLDIGANQLDHIPEEVKTLKNLT KL DVSQNSIKSIPSNTFEGMSKLRTLSLSNNYINGQSFKTDSFNGLKLKELRLEKSELNTIPSAALSSLSSLKIL KLNNSPIKTIPKHSFNGSYCLEVLDLTNVLVEIENTHLDGLESCLQKLYLNNMQIREIPKFFKDFKVLNYVSL SGNSISEIKMDDLVGTTITELDLTENPLHRIEKGSFEEFPHAISLIISDTRLSNLSFVRDYEYGTIKTIKLGNVLP MCDCELAFAEYRVLEIHGNCNFNNTKNVAISTDE FMSVTSHCWPDGKMPQKKDVWAVVGDNSAGTFSTS ILSYFCVFLFFKVLNIV* Lottia Protein Model : jgi_Lotgi1_171757_fgenesh2_pg.C_sca_119000045 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37253.1| predicted protein [Nematostella vectensis] NR E value: 7.00E 24 Sequence: MWKIFSFHWIYFLFSLFCLLHSVTCVKKVSKTPRKSKESEVSKESTKRSASLDDDPSLWPGHLQPLGSSQQV HKVDAYKKYLKPEEFLNSYAAPYLPVLFQAVEQTSIAYEKWGNITYFTVLPQTLTNWVHISPEVGQVSFRK

PAGE 178

178 FLLNYTDKGSVFNNKVPFFMRSAKWEPYRDDVSVPKCLHCEPILNSFSTHKLWISHTDQSSPIHRRNKDTIH CTFVGSQEFTLISHIEYGDKVPVDYTT IDINKVDFTKFPTLREVEYHKVRVKAGDCLYIPTKWYWQVRSTG KTYSADIEWYHRNKTVKSSNCKTKTSISSLNSVVFKDIDDPTTIETDVLLHYFTTYLLNVPKFTLPQFERHLK KDKKFMEDLIEWTDELQHITKEVFETLDVNKDSRFSMADLESLSQPMLEDIRGLFLDRLQDYDDILKDQQE EAKESKKQSEKENKAKDAPATTGPVDDYLKNYIGQLEDVLKESIEEMSVTGHLPDIKKKFAEKQAQGK TG NADKPKQSTKPKTEREKAKEKLEQDRQRRKEKQEAENIKEKTKDDEKYLIVDDSVEEEIMADDDDDSTTG STNKRTPTADKADTKDNKSHTKDNKADTRHVDKKTGRKGQDGKEAKQKNTGSKIKTEL* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_sca_130000017 NR Annotation: predicted protein [Nematostella vectensi s] gb|EDO35011.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 26 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDAREKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPHIGNRENYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG RYTYRTTNIAQDISCCYVGRTCLHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIKSFVSYGTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPIRGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTILTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMYMFPEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTP NYPFDYPLNTECNWLFVAPEGSEVML NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEMAHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYDNFDDCEELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD GFTNWRSTYPVSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADRKSS CTRNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESF TTQCPA GQICTQNCQHFSRACRRSGEVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGPTCYFFSLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTS AGNTESSSTGASRSSPSTDAFVSEDTTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model: jgi_Lotgi1_176362_fgenesh2_pg.C_sca_1085000001 NR Annotation: no hit Sequence: MVAKGTVGILIVLFNTICANIDLNYNQVPVANPLLFGRRGINPDMSSLFFGKRSGNSDHR DLRKMKDTCKA VLSSCKILFSDYEDDTVRNKVQDGFGRFK* Lottia Protein Model : jgi_Lotgi1_176704_fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTED: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDDVNVCHGELPANAMCS GENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lottia Protein Model : jgi_Lotgi1_192261_estExt_Genewise1.C_sca_450209 NR Annotation: conserved hypothetical protein [Aedes aegypti] gb|EAT46740.1| conserved hypothetical protei n [Aedes aegypti]

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179 NR E value: 1.00E 128 Sequence: MTLYITSLVFFLFINGTLQVKNRSSKVADRCFCKLNGEIDDCSCKVETLSVLNNNKIYPRIKSLLSRNYFRYF KVNLKKKCPFWHDDSRCALKDCHVDTCKEEEIPSAIKKGSSAYRYSEEAQKEESCAEEKELSALNTTISDES IQAFKDWKEHDESQDLFCDIDDESSADSEYVDLLLNPERYTGYKGESPHRIWRSIYQENCF KPQTEYIYGPS KSSSPLCLEKRAFHRLISGLHTSINIHLCADYLYQENLGYGMKAHWKPNVAEFQKRFDPQTSNGEGPQRLK NLFFTYLVELRAIAKAAPYLQEEDFFTADSDEDKDVRQGIDDLLNIISTFPDHFDESKLFAGNPNEAKLLKDE FRNHFRNVSRIMDCVGCDKCKLWGKLQTQGMGTALKILFSGDDIGPDSTVNAQQKKHFQLTRSEIVTLFN AFGRLSKSIHSLEMFKSLLSKS* Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNAS DALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDEYNDF YKSISSDY EKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATEKEFEPLITWLKD SALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model: jgi_Lotgi1_202874_estExt_Genewise1.C_sca_21760001 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPE GTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTR Lottia Protein Model: jgi_Lotgi1_203670_estExt_fgenesh2_kg.C_sca_170029 NR Annotation: PREDICTED: hypothetical protein [Ornithorhynchus anatinus] NR E value: 1.00E 142 Sequence: MKLSVFLSLLFLTAVWADTPANCTFDDIKGTWTFFIGSNGHDNTVNCSNMGNPKSQLQVTLYFPDVAVDE FGNKGFWTIIYNQGFEVVISGRKYFAFSMYEQSGSTVSSICDKTLPGWSHDTFNRDWACYYGKKITSVPPK YYKKNKPHRLFKLNYDYINNINKQQKLWTATVYPEYEGKPMRELLNKAGGPASQVFGPIKAAPVTWEQQ RLSETIPEQFDWRNVNDNNYVSPIRNQESCGSCYAF SSMAMNEARVRIMTNNTLQPVFSPQDIVECSEYSQ GCAGGFPYLIGGKYAEDFGLVEEKCNPYKGQDHSCSTDKTCKRQYATSYRYVGGFYGGCNDALMKMEIY RHGPVVVGFNVTDDFFHYKSGIFIHTGLTDRYNPFEVTNHAVLVVGYGTEKGVKYWIVKNSWGEKWGES GYFRIRRGTDELGIESMAVSSTPIF* Lottia Protein Model : jgi_Lotgi1_204607_estExt_fgenesh2 _kg.C_sca_690020 NR Annotation: ctg4a [Aedes aegypti] gb|EAT44181.1| ctg4a [Aedes aegypti] NR E value: 2.00E 45

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180 Sequence: MAFMFFQTLLIIVSIGYLHADDSDGVQEATLCEVCRILAEELQLRLDETGKSKEVIETGHGLDTKKKKKYNF SELRLIEALQEPNICDRILEYSVHKEKTGIERFAKGRSQTMEALHGLVNKGVKVELGMPHEM WDKPSVEIT QMQRKCYALVEEYDEDIEDWYYNHQDQPFLDYFCRNLVLNPQNNECLDEDMRSQNEGSSSEMKGDKGK KEEL* Lottia Protein Model : jgi_Lotgi1_204654_estExt_fgenesh2_kg.C_sca_720011 NR Annotation: PREDICTED: hypothetical protein [Monodelphis domestica] NR E value: 8.00E 93 Sequence : MSLLIATGLFCVIYLLVKFLNISQPCHLPELYFKDKTSNFIQTVFTMCPILFESYIPPLLWGKSGHLQTFIYAK MGRIMSPLPNGKRIELIRPDNATMSFDVFQPHYEHPTGRDYTLAVCPGIANSSESLYIRTLVDHAQYNGFRV AVLNHLGALRKVKLTSPRIFNYGETGEYNCMIDELKRLYPNTSIIAVGCSMGANIVIKYLGECKQHENKVIG ALSFCQGYDVNDAKPFLLGWESCRRLYCYFMAINLR QLLRSHQDILFTEQAQSEYGTVELKKLYSATSLLA IDELYSVNAFKFKCCEEYYHWASCKQYIQNITKPIFILNAEDDPIVPKQLYTTPLKYQENHDNCLFIVTKHGG HLGFFEGGFIKPNTITWLDRAVVQFSNVLCELDCKERKS* Lottia Protein Model : jgi_Lotgi1_205030_estExt_fgenesh2_kg.C_sca_1120002 NR Annotation: PREDICTED: hypothe tical protein [Strongylocentrotus purpuratus] ref|XP_001188672.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 3.00E 20 Sequence: MKLMFLGFIGILMLSTSNNASASPWFCKGLECPKFNLLNKTKDYEVRQYGSLTWVTTSHIGQWKSDILGEL LFKNLFSYIEGNNTMKKQLDLAVP LLTKLESAGDRGSIYTMHLLIPRENIDHLPSPLNTNLTFITIPPSTYYIRS FDGVAYDRQYMKVLQELVNAINDTGAYPDDFFYTATYDHPQVKENRHNEVWLLKKEKIP* Lottia Protein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 10 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSCCMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMWRAVFSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI Lottia Protein Model : jgi_L otgi1_98330_gw1.10.599.1 NR Annotation: hypothetical protein NEMVEDRAFT_v1g147894 [Nematostella vectensis] gb|EDO28414.1| predicted protein [Nematostella vectensis] NR E value: 5.00E 06 Sequence: TVCSLGTHCLSITVCSLGTHCLSITVCSLGTHCLSITVFCSLGTHCLSITVCSLGTHCLS ITVCSLGTH CLSITVCSLGTHC

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181 Object 27. P redicted secreted signaling molecules found in P leurobranchaea californica. Likely to encode functional secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] (Fulicin precursor) NR E value: 1.00E 21 Sequence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKR DLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPMFLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNY FSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Protein Model : jgi_Lotgi1_168619_fgenesh2_pg.C_sca_77000042 NR Annotation: crossveinless [Saccoglossus kowalevs kii] (a new family of twisted gastrulation like modulators of bmp signaling) NR E value: 1.00E 148 Sequence: MYYTELSILAKCIALCILTCFPILIQAQLTGTVTRCYNEGGEVKIQGITTDPCISCYCMDGYVQCDKKKCDNL DGCHAILFDGPKRCCDTCKGCRLNGVKHESGKSWFDKRDRCTSYSCKAGVITKSKVQCHVPCSNPIKRKR QCCP SCKGCHFDGADRKNGEKFALASDPCVECKCRKGSVTCMKRACPVLNCPDEVIYQPKGECCPKCKG MRKIFDFPGACYFAKRVYVDGFSFEPKSETRCTCTKGTVICEKATCPPVACPVEERVQKGSCQVCEPKRNC LYDGKIHMHRSKWQPRMCTQCSCQNGVTYCQRERCNNSLSCPNGYKLQFQPGECCPKCVEHDAVCRVFG DPHYRTFDGKMYNFQGTCKYMLSQDCQGKDFTIKVKNGVRLSSGFAWTQMVVVLMGDTRISFRQNLLIKI NRRRVQLPYTIPGKFSIRREGHSVTFRADIGLKVVWDGDSFLEVTASRKYKNRLCGLCGNYNGLETDDLIG RKGKNYLRGEEFGNSWRIGSKKACKTQPKVKNLQSICDKDFKAKVRANKECSVLYSRAFSSCRRVVDVTP YVTSCVTDMCDCPHGKKCSCESIQAYAHECKRAGHKVKWEKVSNCKAPQKNCPKGAYYSLCAPACPKTC SSNKPDGSCSKKCSPGCVCQNGAVLYKNRCISPEKCPKT* Lottia Protein Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chain; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica] NR E value: 4 .00E 13 Sequence: MEVTCKCPLVLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYCRNSQQRISIKK* Lottia Protein Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Euprymna scolo pes] NR E value: 1.00E 122 Sequence: MTVSPTVVWAAFSMALSSVVALGANIICNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTSAGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LPPFRKIGDSL KKRYKKSKIVVPYLGRRARTAVTLILKRAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK*

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182 Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301 NR Annotation: predicted protein [Nematostella vectensis] gb| EDO37057.1| predicted protein [Nematostella vectensis] (growth factor) NR E value: 1.00E 109 Sequence: MFLEVFNISATVLCILFLIGHRKCDTIQNSKHHAELMMDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVG VNGGFFNTTTGACLGNIVSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSAVGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLELGVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYGINCSTPCKP GYYGSNCAHQCYCLNHGTC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi1_2084(2009). from 17_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVIASSRRSRGRW YRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSNNAFEKLEE KVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* Predicted molecules found in Pleurobranchaea californica with unknown function or predicted to be related to the secretory pathway Lottia Protein Model: jgi_Lotgi1_107224_e_gw1.6.194.1 NR Annotation: No match found NR E value: Sequence: CFLSVCLCSSICFTTECCFLSACLRV CFTTECCFLSVCLLFKRSRVCFTTECCFLSPCLRVCFTTECCFLSVCLL FKRSRVCFTTECCFLSVCL* Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Annotation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQ CCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPWPLTTTTSRVPINILCNVYWFKCTKLHFSSHWECSNDDCLLEADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSNVVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG

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183 DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFDEEPAVYHSVRIIGWGVERTIYGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWGQISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV* Lottia Protein Model: jgi_Lotgi1_ 118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Sequence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_134336_e_gw1.89.95.1 NR Ann otation: apolipoprotein D [Branchiostoma belcheri tsingtauense] NR E value: 9.00E 41 Ac Annotation: CNSN01 F 078834 501 Ac E Value: 2.00E 25 Sequence: MERILTAVLLCIGVAVYAQVPGLGPCFDAKPQSTLNVSQYLGHWYEITKLPSFFEIEQVCLTANYSVKEDG HIKVFNQGKVNGKNVSSTGDAYVPDPSEPSKLIVKFPQAAPYGPYWVLDTDYTSYSLVFSCEQLGSFAHAK FLWILSRTPSIDPAVKDRLFKRLEKDGIGASGLRATKRSDCS* Lottia Protein Model : jgi_Lotgi1_143169_e_gw1.194.72.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO36917.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Sequence: MVKVCSIPLTVVKVCSIPLTMVKVSSIPLTISKVCSIPLTMVKVSSIPLTMVKVCSIPLTMVKVCSIPLTIVKVS SIPLTIVKVCSIPLTMVKVSSIPLTIIKVCSIPLTVVKVCSIPLTVVKVCSIPLTMVKVSSIPLTMVKVCSIPLTVV KVSSIPLTMVKVCSIPLTVVKVCS Lottia Protein Model : jgi_Lotgi1_145599_e_gw1.360. 11.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO49883.1| predicted protein [Nematostella vectensis] NR E value: 6.00E 16 Sequence: LVVYCHCGLVVYCHWELVVYCHWGLVVYCHWDLEVYCIVYCYWGLEVYCHWGLEVQCYWCLEVYCH CDQVVYCSWELVVYCHWGLVVYCYWGLEVYCHWGLEVQ CYWCLEVYCHCDQVVYCSWELVVYCHW GLVVYCHWGLVVY* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.00E 140 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPD NYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAE CTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN*

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184 Lottia Protein Model : jgi_Lotgi1_153437_fgenesh2_pg.C_sca_3000340 NR Annotation: h ypothetical protein PB105991.00.0 [Plasmodium berghei strain ANKA] emb|CAI04701.1| conserved hypothetical protein [Plasmodium berghei] NR E value: 8.00E 09 Sequence: MKTMVAIIGVIIVSCVLHTSGLPLFKNILNERSVEYGGESGNGFKNERAVGYGGESGNGFLNERSVEYGGES GNGFQNERAVGYGGESGN GFLNERSVEYGGESGNGFQNERAVEYGGESGNGFLNERSVEYGGESGNGFQ NERAVGYGGESGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAIEYGGESGNGFLNERAVE YGGESGNGFLNERAIEYGGESGNGFQNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNERSVQNSGKSG NGFLNERSVQQGGKSGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAVEYGGESGN GFLNE RSVQYGGESGNGFLNERSVQYGGESGNGFLNERSVEYGGESGNGFLNERSVEYGGESGNGFLNERSVEYG GEAGNGLK* Lottia Protein Model : jgi_Lotgi1_154590_fgenesh2_pg.C_sca_6000093 NR Annotation: PREDICTED: similar to meteorin, glial cell differentiation regulator like [Tribolium cast aneum] NR E value: 3.00E 24 Comments: Secreted, involved in both glial cell differentiation and axonal network formation during neurogenesis. Sequence: MDFLQATWLLVVMVIFHIYIITADQICNQCDCTIRDSTGKGNINVKPKCQSGKITWPDPVSAVRLELKPEVV GPFKACIALKSENIVTLVSREITTETVNYRTR SYDSKVDLLPWFSVEGQGKEHCVTSSESSVMLYLEIESTTE DSGLSRTFIYYDIELIPDNKFSPDPMEECQPCSDDEILRSYCISDYVVVGRMKEVNFQSELDKTKVNIIVDQIV KTTDEDIFKRHDNLLEGALFTSVHCGVQKGPGQFLFTGRNRLGQPILTCAPFYDQWIQIQRLAQEKGTIECA YG* Lottia Protein Model : jgi_Lotgi1_158797_fgenesh2_pg.C_sca_180001 47 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E value: 5.00E 92 Sequence: MKIHILAVLFSFFSATNAATLRFVSQTPLSITINKDGSYTVNVNGEPWLESSAIYFYAEGRRVSMGDGSLKIA STFMDNGYDSVGQYGKTCWRFEIESKYIDTCFKQYMEPTPFIIFEQHYLNDTENTASIDYDYTIAGFPAFTAP PTGKLGYLHYAGSMTGDSDKSFGIWQEKAKMLTGVAGGPLVLFDKDADDCLILSPLNQFMAASSLFDGQ NYSTGVLGGVNFVPKDFVLQTVLFYGKGINHAMSDWGLLLTQYYGKDDSYRKSDLTLNYIGYWTDGGA YYYYNTEPNKTYQQTILDVRQNAKDIGMPYKFVQYDSFFYYKGPHGGVKTWVPMPDLFPDGFQYLYNQT GWPAGAHNRWWSPKTTYARQNGGNFDFIVEEAKAVPTDQAFWDYLFDSSKKWGLTLYEQDWLDNEF AG VKALLTQIDLGRTWLMQMGRAAQENGITIQYCMSNPRHIMQSVEIPVVTQARASGDYRAGKDNWKIGISSI MVDALGVAPFKDNFWSSPDQPGNPAYPDLKETHADLQVAVSTLSTAPVAPGDMLNKTNVPLVMQCCNA DGMLLQPSKPAKAIDAQILQLAFGGDVGPQGDIWTTFSNIAGFYFGIILGTDMTADYNLTPSKASFKAFLPS EVFPRFASIPKSQEFSASNPLVITTNCTKTNICLYYTSPVFSP GGTNVLIYGEENKIVPMSPKRVTDITLDRDL KLTLTGAAKESVRFGFILPDQYITITCQLGQSGSAVLSFNERSCNGY* Lottia Protein Model: jgi_Lotgi1_160458_fgenesh2_pg.C_sca_25000063 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E Value: 6.00E 86 Ac Annotation: no hit Seque nce: MNYKMILNKYTLVLGIILVCTQPCITDVNITKVHLIFMNHLDVGFNGITPQTGFVINVINKYFTKYFPGAIEL ALQMNFLGYRERLIYTTHPWLVSLYLDCPQNLTMSGIQLMCPTEEQREGFLYAAKRGDITWHAGPMNMQ YEALDTAMVNFSLKLSEDMDARMDIKRRHRVLSQRDVPGMTRALIPIFNNHGIEGVTVGVNSVSSPPAVPK IFKWVFQNSSVIAMWHPGGYPIKPGSFPSMPLGLSRDNCVTFEQFDEAMCFSFRGDNQGPPTSIAEVLNSYE IVRGQFPGAIVEGSAFENFVEVVQSVKDKLPVVDQEIGDTWIQGISSDPGKTAMMRGFFRARTACLTSAPNF EDVISSWSEQRQFIDLALETLGDHPLVQAVEEEYKQLVPQAPDLSKYKQLSATTFDCQNGFKFSFAADGSL

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185 NKLQDKDGRNWASKSQPIGQLLYKTYNDTDFDSFQKQYVLNHYYWFQVGKWNLTANCPTCESTIWNTK LQKLYAASDG SCDVVALLAMEEDRATSFYGAPPKIYVKYLSSSNTFLSIEVQLFGKKPTRLAESLSLSFQPIR QEGSKWYLHKLGQLIDPLNVVTNGSQRLHAVDEGVQYKDSNGKGMMIGSKDVGLATVHQTPTDVSVLPV PLTPIQSISGVSYNIFNNVWDCNYIFWYPFLKQDSNSKYRFSITFS* Lottia Protein Model: jgi_Lotgi1_161657_fgenesh2_pg.C_sca_30000077 NR Annot ation: predicted protein [Nematostella vectensis] gb|EDO44809.1| predicted protein [Nematostella vectensis] NR E Value: 3.00E 17 Sequence: MTKLSEYFLICSMIFVVFMLQRGTCDDVVTEEQISYAKGSVCGYCSYCKFCKLCDKDCPCETSPSKPNCKM CKYCKYCYICSGVCDTICQPGGMIDKISAAIVNALPSFNKEEIEDD LESVKPWLDKKDEL* Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sca_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus] NR E value: 1.00E 133 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHL KK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAGTPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSSDNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKGDPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALELSKELN VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAA TIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KKHDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQTSTKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNTWPEKEECDSH LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHPLPWYPPICVTLLTINEYDEKTYDNLKSVGHP GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGARWVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYADVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSVAPIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKSGECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGEDCNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLHSTQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN HCSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPKSWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLML LGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTLGNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model: jgi_Lotgi1_164905_fgenesh2_pg.C_sca_48000037 NR Annotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSPCQKLGE SW RDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCRVGKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIVVAGNAECRVGLRWK DGCKRCRCNKRGKIICKTRLCNLSDDGKKLSNKDKTHRQTSTGGL ISKSTPRFQLPEKPRFRMPNLSLYNNR FQRKKRPSIKTVNCNGFKPGEKYWDKCNLCHCTMAGAPMCSSKLC* Lottia Protein Model : jgi_Lotgi1_166720_fgenesh2_pg.C_sca_60000088 NR Annotation: No match found NR E value: Sequence: MMLLIKTGVLLIILFSLNTLCDSCACEFHPVLSGKWFFTGENKNINIKIHSKIIVFKVSNYLKLRYKCTERYG NIYLLRILCLFLEVDFLSDLFMKGGFTSIHVEGRGFDSFIFSPKNVTRKYANIDNTCDSEISRERKFIYRKGGC

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186 RVPRDMVGTWGFTFRQSSAITMGKRNLTIHFLNNDNLTLNCEARDRNMFLFRRSSYINSSTDAIICVQIDPV LDDPFYDYEFNRIITGNAVENLVRTVPRGKKLYINSDCDMIHSPARPEYIYTVDNIL* Lottia Protein Model : jgi_Lotgi1_16702 2_fgenesh2_pg.C_sca_63000026 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO43826.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 08 Sequence: MVAVRIRKYKNLLFFALLFAGVLSISTIFYSNHYQIMNTGTRSDVNTGFSYSNANSSKYVIYICDGKNSCGG YGDRQKGI VASYIISVMMNRQFGVIMNDSCDIKQMFKPNQINWIVDNNDIKSKTSKRIKALDGYSDKLRKS MIEMDLDQEYKEEVIYFSLNLEFVFYLRQNKRYEKQLKWLEHLSMSEIYNVVWQSIFKLRPYIREKLDPFL DLKKQGLKLISAQIRLGKNPTIPHDSRVVNSLNNMNVLWQFYKKYNDSSKYRIFISTDSDTVREKARSIFPD VYSDVPGKVFHVERSNKTDICNGWRKVILDQVILSLSDVLVISNSGFGRI AAFFRQNENDLYCLNLDKIRRC TTQSEIFVDKTW* Lottia Protein Model : jgi_Lotgi1_168040_fgenesh2_pg.C_sca_71000102 NR Annotation: No match found NR E value: Sequence: MVGTRYLQLATSLAVFLLVLFLTCTQAAPVDDFETDNEALRKALFIARLLSSSDRLKNTKPEGSNLDFSELA SIPFSNQQKRYRPPMQGRSGGMSLCLW KVCPAAPWLVSKRSEKTWDKNNMLGK* Lottia Protein Model : jgi_Lotgi1_168805_fgenesh2_pg.C_sca_79000073 NR Annotation: PREDICTED: similar to plexin A CG11081 PA, isoform A [Apis mellifera] (a neuronal semaphorin receptor that controls axon guidance) NR E value: 1. 00E 121 Sequence: MEGGIRLQLFLLVSLICHTQCIKILHSFRDPGGNILQKLVIDDQTGNVFLGALNRLHKLDPDLNIVQSASTGP RLDNVECPPPLLPCDKPKTQLNSQIKGLVIDSASNSLILCSSLFHGSCQTLALNNITNVKKFFHKPLVPNDHH SCYMFLAPSINNTQALYIATAYSDLGDAAYRDLVPSISSRSLDNLDFIHRDTEGSTKLEILKEYRESFIVRQLY GFQNGGFVYFITLQRENP GSQKIVSRINRLCQQDRYFRSFVEIPIKCQKSDSDSYDVVQNAYFTNGKLYVIFS NTVSSAGAKKSTFCEYDIQDIDLEFNETVRNCYKAVGNFGPEHYKKIEPCPPLIFGSVDFCGESGEWKDYSA IEGSKPISATPLHFFTSDKPTAIFVHPEGNHRYAYIGTQDGHVIKLRFENGQVNQLFKITHSVGDIILQITNHPQ SNLLYILGSHKLSVLSLNHCDDLTSCDECMNDGDETCGWCVMDNRCTTKELCQS SVITPPWLSRTDQSCAS ISSLQPSSLSYKKFINGGDSRQIKFQVNKLKFESNRNLNCLFINGEKHDNTMAILDQKENTITCPLPRQLPTIP QGKDHEDLELQFHVEGKMIVKRDVSVFDCQGNNNCTSCSTSQFNCHWCPVSHSCVEQSESCPQGDSIKVN DRCPRLETAASDTDILVHSGELKTISVQVRALEANQRSNLKCHFDLSGQVQTVSAKISSSTLTCETIKLAYA DDIPYIVAGFKVTWGQQNHPLDNPQRMNVRIYKCASMVTNCGKCLSLDVEYECGWCEDQCSLQKKCQKS WLDQSETCPNPQILRYSPSKGPVKGKTEIQVNGINLGKEISDILGQVTVAGLPCDVMQEHYEPSSSFKCEVK GVTSAKSGTVKVVVNNKYTAESDTVFEFVDPVLNHVDPKQGLMSGGTRVLIKGNNLDVGSDTQVIVGGN SVPVIK* Lottia Protein Model : jgi_Lotgi1_170051_fgenesh2_pg.C_sca_92 000069 NR Annotation: hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] gb|EAR97557.1| hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] NR E value: 3.00E 08 Sequence: MKMINIFIVVYCLISPCLGFWLSSKTVDPKVDISTECINKALTGDCGFFT CFEERLPCGEYGYAESYGGKYC WQFQQSHHLFTKKGVEFVEKLTRCHMNRSITSYRQNQIECFSHYDQSFVIMGDCYVESGFCDVVIDNFMSL ARILEPKDFTNYRVVREVFRAAKQCPNNISGGIISKFLSYKRGSSL*

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187 Lottia Protein Model : jgi_Lotgi1_171450_fgenesh2_pg.C_sca_115000014 NR Annotation: No match found NR E value: Seq uence: MKRFTLLALLLVAILYISQSESKCQGVTKCMVNLFTCKREQISVNIAADCCKEYFECFNECKPGSRPPCKNT APGKRGSWNKRYSSRYGSDILGDIISGY* Lottia Protein Model : jgi_Lotgi1_171757_fgenesh2_pg.C_sca_119000045 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37253.1| predi cted protein [Nematostella vectensis] NR E value: 7.00E 24 Sequence: MWKIFSFHWIYFLFSLFCLLHSVTCVKKVSKTPRKSKESEVSKESTKRSASLDDDPSLWPGHLQPLGSSQQV HKVDAYKKYLKPEEFLNSYAAPYLPVLFQAVEQTSIAYEKWGNITYFTVLPQTLTNWVHISPEVGQVSFRK FLLNYTDKGSVFNNKVPFFMRSAKWEPYRDDVSVPKCLHCEPIL NSFSTHKLWISHTDQSSPIHRRNKDTIH CTFVGSQEFTLISHIEYGDKVPVDYTTIDINKVDFTKFPTLREVEYHKVRVKAGDCLYIPTKWYWQVRSTG KTYSADIEWYHRNKTVKSSNCKTKTSISSLNSVVFKDIDDPTTIETDVLLHYFTTYLLNVPKFTLPQFERHLK KDKKFMEDLIEWTDELQHITKEVFETLDVNKDSRFSMADLESLSQPMLEDIRGLFLDRLQDYDDILKDQQE EAKESKKQSEKENKAKDAPATTGPVDDYLKNYIGQLEDVLKESIEEMSVTGHLPDIKKKFAEKQAQGKTG NADKPKQSTKPKTEREKAKEKLEQDRQRRKEKQEAENIKEKTKDDEKYLIVDDSVEEEIMADDDDDSTTG STNKRTPTADKADTKDNKSHTKDNKADTRHVDKKTGRKGQDGKEAKQKNTGSKIKTEL* Lottia Protein Model: jgi_Lotgi1_173484_fgenesh2_pg.C_sca_161000006 NR Annotation: no hit Sequence: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFPWAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPGGNETFTFDFISIMLGKKQFE* Lottia Prot ein Model: jgi_Lotgi1_176362_fgenesh2_pg.C_sca_1085000001 NR Annotation: no hit Sequence: MVAKGTVGILIVLFNTICANIDLNYNQVPVANPLLFGRRGINPDMSSLFFGKRSGNSDHRDLRKMKDTCKA VLSSCKILFSDYEDDTVRNKVQDGFGRFK* Lottia Protein Model: jgi_Lotgi1_177026_fgenesh2_pg.C_sca_12012 000001 NR Annotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSPCQKLGE SWRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKV DGFKIRSKKCRVXKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIV Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEA VQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT

PAGE 188

188 EEVEEPIEEEEEEKKEEEK KEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDEYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGY EVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATEKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model: jg i_Lotgi1_202874_estExt_Genewise1.C_sca_21760001 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVT SAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTR Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_kg.C_sca_820009 NR Annotation: unnamed protein prod uct [Tetraodon nigroviridis] NR E value: 3.00E 35 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SYIQSEFVAACVAILVWWLAFSAKSKLSP* Lottia Protein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 10 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSCCMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMWRAV FSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI Lottia Protein Model : jgi_Lotgi1_173484_fgenesh2_pg.C_sca_161000006 NR Annotation: No match found NR E value: Sequence: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFPWAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPGGNETFTFDFISIMLGKKQFE*

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189 Object 28. P redicted secreted signaling molecules found in T ritonia diomedea. Likely to encode functional secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_119019_e_gw1.30.87.1 NR Annotation: PREDICTED: similar to seleno protein N, 1 isoform 2 precursor [Canis familiaris] NR E value: 2.00E 85 Sequence: MVLFTLLCCLFIIGRH DPTQIPEEIVINIGDDGVLLFQQHDRDNDGYLSIQEFEPLVYRLLEINVSGPVYDVPIS TDDEMITLKSYFIPIVKESMSKDLNDSVSIGLLRTMNSLHGLEKWQNVNLQWMNFGASHFSGFLPKDVDSI MLGSSYFIINVEKGLFNAALSSNRYYPPKVTTNESIIVHRLLTLFHPRPFVVSRFPAQSSVACVRAYNDKYLD IVFRIHAEFQLNEPPYHPFWFTPAQFTGNLIISKDGKHIQYFNLYVPNNKRLN IDMEWLNGPNESENMEVDI GFMPLMQLNSTQSSVPVKNLEEYELLEPLPERPQHKTEGENIEWMATIDLEDAKSSLEKALYPFKKVPYYN FTEAFKKAEDNKKLVHSILLWGALDDQSC* Lottia Protein Model : jgi_Lotgi1_164016_fgenesh2_pg.C_sca_43000014 NR Annotation: chordin [Danio rerio] sp|O57472|CHRD_BRARE Chordin p recursor (Protein chordino) gb|AAB93485.1| chordin [Danio rerio] NR E value: 1.00E 119 Sequence: MSPMDLAVCLVLLAATVSSTKFSKIPLKAARENLFGIQPNRPGCILGSKFYGIGERWTPELLPVGEMYCVKC ECVPVERKGIIDMKGQTLCKNIKNLCPRPNCLNPVLPKGKCCKVCPDEVNSFEDSFSLGSKSSAPTMISSDSK RRVRNEFVSLLVGK NVRRESVKTSAVAAVYFSIVGRDEIRYSVRYFKLDRPKFLQITDANGNVLFERPIEKK RNGDKKFCGIWTKVPSAYIQYVREERLFAVITASRHFRGLVSGRIMVNRHAKREVFSSVLASPKANGIGGL LSVVYNPRTHLVDYVINLDGVFPGSSEEYFITISKKSKVLHQSKGKASSKTRFIRGSWTLKNKRQSKQLARG RLTIRLTSTNGASVSGDIKPKVTCGVSQAVMSSGNSLEEMKLASSGTAVFELREK GSLDYKIRVTGLTSKVH RIRLEGAVNKKNRRQIIGSVHRNFKADENSFNGWCNGTFKKMTADDLHLFLNNKIFVNVATSENRISELRG RVIALPYHSHFEQVSADPAALGPKTITSNGHAGNAWISMGVGCSLHYDITTTTIAKHHDLTLSLVSPTDIDA YVTIEKISTTNLYEFQQASGSIAVVPESLFRDLDEGSALIQVISGDVILSGNVSVPNTCWQYSQDYDMDILVD EQTSEEVLAANRYKCVYEGKVYE NGDSWLPDVNATCNTCSCNKGKIECHRLICPVVECANPIVLDGECCPT CPIETTDQTECKIDGDLRSHPVGSTWHPFLPLMGYAKCVICTCLPGGIYKCERKCPKLDCPASRRIRLNPKD CCQVCAPEEVKPEVVKPLQDVDMKGACSFKDQIYANGAKWHPRVIPFGYMKCIDCSCNNGTPSCSRPKCP KLSCSRKIREDGACCDTCADSDGEEIVEEKKSDSCIFGGKKYDDGETFQPKTSSLSSCATCTCRKG SMKCSL TCPEKCKDPKHSNDPCCKHC* Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] (Fulicin like) NR E value: 1.00E 21 Sequence : MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPMFLGK RAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNYFSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Prote in Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chain; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica]

PAGE 190

190 NR E value: 4.00E 13 Sequence: MEVTCKCPL VLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYCRNSQQRISIKK* Lottia Protein Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Euprymna scolopes] NR E value: 1.00E 122 Sequence: MTVSPTVVWAAFSMALSSVVALGANIICNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTSAGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LPPFRKIGDSLKKRYKKSKIVVPYLGRRARTAVTLILK RAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK* Lottia Protein Model : jgi_Lotgi1_208417_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVI ASSRRSRGRWYRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIP SNNAFEKLEEKVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* Predicted molecules found in Tritonia diomedea with unknown function or predicted to be related to the secretory pathway Lottia Protein Model: jgi_Lotgi1_103075_e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mellifera] NR E value: 9.00E 50 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRLE NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHPNEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLE DREVYLTEEENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL* Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Sequence:

PAGE 191

191 CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVK LYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_132441_e_gw1.79.137.1 NR Annotation: PREDICTED: similar to CG11081 PA [Nasonia vitripennis] NR E value: 4.00E 72 Sequence: MAWKIYVLFVCVLCLRFILAARENYIKATFEGAKDRGI FNKLVVNRETNELYLAAVNHLYKLSPNLEVIQE VVTGPHLDDPSCTSLTNLTCDPDLKSCACKLRPYYTKALVVDYVNKKLITCSTLYHGHCDKLDLDNITNKQ MFPFMPMVANNATASTVMFIAPGPAVVKDEPQRQVLYVAASYTRTGLKAYREQVPAFCSRSLEDFNLLLK NAFGSSSIEIESQHRDTFPVRYIYGFGSDNFSYVLSIQKANVQTDRYVTKISRICQSDDDYYSYAEVGLSCTM QNTEFNLLQ AAYVGKSGTKLARSLGIPTTEDVLYTVFSIGDSNSANPSATSAMCVYSLRDIRKKFTENIQEC FSGIGNTGPDHMIQSSKCLKVVSSHFFVIFFLESFESADLSVL* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.00E 140 Sequence : MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECLKLDPDNKQCFPHY KKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* Lottia Protein M odel : jgi_Lotgi1_160631_fgenesh2_pg.C_sca_25000236 NR Annotation: Substrate specific endoprotease Tex31 precursor emb|CAD36507.1| substrate specific endoprotease [Conus textile] NR E value: 4.00E 29 Sequence: MNRLCFLLVVWQIALSQSKVVSSVEGFNGNLLRIVRSANNAAPQGNL NYNSNCNEAYKNISSSHSMCLVD VCTAVALSQEEIDAAVAAHNDYRKNVSPKAAAMQKMVWDDELAEIATKWAQQGRAGHDTKDARKTISL KGTYIGQNAAAGQANLIAAIKAWHDEVVDFKYGVGSINGKDVGHYTQVVHHQSTRVGCGKAHCPDSTY KSYYVCNYAIGQMGLTHPYKSGSSSCSDCPSSCSDNLCDCNGKICFNGGTLDINTCTCSCPSLYSGDQCQTL QCTKTEKSWCRRSYTVADCTKYSNF PIDCNMMCGVCPYPCDGKQCENGGTLDPNTCQCTCKDPYTGPTC ADKSCPGQEEWWCQKFFTAADCTKYSNFPTDCNIMCGVCPACKITA* Lottia Protein Model: jgi_Lotgi1_164905_fgenesh2_pg.C_sca_48000037 NR Annotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDN SPCQKLGE SWRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCRVGKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIVVAGNAECRVGLRWK DGCKRCRCNKRGKIICKTRLCNLSDDGKKLSNKDK THRQTSTGGLISKSTPRFQLPEKPRFRMPNLSLYNNR FQRKKRPSIKTVNCNGFKPGEKYWDKCNLCHCTMAGAPMCSSKLC* Lottia Protein Model : jgi_Lotgi1_165259_fgenesh2_pg.C_sca_50000100 NR Annotation: hypothetical protein CHLREDRAFT_144736 [Chlamydomonas reinhardtii] gb|EDP05543.1| predict ed protein [Chlamydomonas reinhardtii]

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192 NR E value: 6.00E 23 Sequence: MKVLLVLDICCLIGLVSVGEAVCPDNCNYNGVCDVKHSRCNCYEGFTGSDCKTDCRCNGHGTCQSGSVC KCDEGWKYSGGQCVWDCHCLNGAKCIGPGECGCVHNCKMGNCRNGQCQCWNGYKGSDCSEYDPTM* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_ pg.C_sca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purpuratus] NR E value: 1.00E 26 Sequence: MMKILEILLCLSFFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCS LKCSGRCRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSCTKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCNDGCKQDTGYCNKCNKGVYGNVCDKNCNKGCLDGCNIDGSCKCKSTYYGTNCSKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQ CPSSCPICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEKCNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNGDCDRSTGNCGKCNEGYFGDNC NQNCSINCNGGCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICLQTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Lottia Protein Model : jgi_Lotgi1_166645_fgenesh2_pg.C_sca_60000013 NR Annotation: No match found NR E value: Sequence: MGASIL LGLTILLSYVNQVHLQMFHPRDISRGGQQQEEQLRRGYLIQILKSFLNRRQEDATEKRKRSCNLNL GFHCQTDEYSSIADMYDFLQSALSPGKRKRNVKIVSIEGS* Lottia Protein Model: jgi_Lotgi1_166736_fgenesh2_pg.C_sca_60000104 NR Annotation: hypothetical protein LOC500991 [Rattus norvegicus] sp|Q5XI89|FA55 D_RAT Protein FAM55D precursor gb|AAH83798.1| Hypothetical protein LOC500991 [Rattus norvegicus] NR E Value: 3.00E 25 Sequence: MALTKKLVLICCLIIATVFGLKIMHTIKYQINMRPNIEPIPFYSQNTPKRKLMFPERQSPSLEDYNMKHDDVR SEDIFNKFLSENVVYVKDTTADDYFIEGEKELLEEGEIKDLRDLPSSNYSIISTK NTVFNQYEAVTVKIQLYN GFGQLLKRGGDDVRVWITGENFQAGGEVYDLHNGTYVAEVKAFWTGKSKIHVSIAYSREELRILLRTRRE SKVLRYSVGRFLRGGDTFMTLCSPFHDIPLYPRVCNFTNFNLNPYYCGQPNSPKVACSDLRILHTINYSKQR MNNLESKILSRQNRVNELKSKSITINIGTVVNLPYCSSYKSRFTWTNTAPLGYFNNNTWHPSFCQGQITRQR LSRCIRDLHVYLVGDSTVRQIFQS FLRLTKCTNTTFIGNRPHDRVQRYCDVHDSNFRVEYGIHGLPYSMVRT PDLLNNTKAIGFHLDGLQWSQNKDKMFVMVHLYAHFLFFHTSLFEARVRSLKVPLTRLLNRYPKIKIFIKGP HAFTGADFKPIVKSDYFGKVYRSILLKELFEFRDRVYYLDYWDMSVSSGNRLLHPVNSFVDEMVKFFLNL ACDK* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_sca_1 30000017 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO35011.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 26 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDAREKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPHIGNRE NYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG

PAGE 193

193 RYTYRTTNIAQDISCCYVGRTCLHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIKSFVSY GTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPIRGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTILTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMYMFPEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTPNYPFDYPLNTECNWLFVAPEGSEVML NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEMAHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYDNFDDC EELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD GFTNWRSTYPVSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADRKSS CT RNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESFTTQCPA GQICTQNCQHFSRACRRSGEVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGPTCYFF SLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTSAGNTESSSTGASRSSPSTDAFVSEDTTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model: jgi_Lotgi1_173484_fgenesh2_pg.C_sca_161000006 NR Annotation: no hit Sequenc e: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFPWAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPGGNETFTFDFISIMLGKKQFE* Lottia Protein Model : jgi_Lotgi1_176704_ fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTED: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDDVNVCHGELPANAMCSGENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lot tia Protein Model: jgi_Lotgi1_177026_fgenesh2_pg.C_sca_12012000001 NR Annotation: no hit Sequence: MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSPCQKLGE SWRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENT LRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCRVXKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIV Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 S equence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGT MITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDEYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNR T RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY

PAGE 194

194 CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATEKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEP EEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model : jgi_Lotgi1_201804_estExt_Genewise1.C_sca_1950010 NR Annotation: DICTED: similar to putative porin precursor [Strongylocentrotus purpuratus] ref|XP_001192936.1| PREDICTED: similar to putative porin p recursor [Strongylocentrotus purpuratus] NR E value: 8.00E 19 Sequence: MRLLIFTLSLLVAVVRSVDASQCPEPPSGFGPCVITDANCLTDSECSNGQICCPHLCGKICKDKVSLTVVKPG TCPSNAAISCFVGSVADTCSSDSDCPGNQKCCNLCGKNCSPPQGSAPQLPPIGLPAPPPVCPPVCAIACEYGN VLDQNGCPTCSCKKTPCKGEAPLTLDRNGNTINCGRGGVRCPLNSECKIHPADAYALCCPIEPDDPKPGVCP KLPNPLPRCLIFQNDCKQDSECPGDQKCCSVTCGIGCIDPVKLCPVVDCLQYCEFGYRLDSNGCQTCQCKK SPCEDEAPMTLDSTGNTINCGRGGVRCPSNSECKIHPADRYAVCCPKVCPPVCEIYCEFGNVLDENGCKTC KCKKSPCEDGAPMTLDSNGNTINCGRGGVRCPLNSECKIHPADRYAVCCPKVCPPVCAIYCQYGNVLDEN GCATCTCKKT PCKGEAPLTLDRNGNTINCGRGGVRCPLNSDCKIHPADAYAVCCPKEPSPVCKFGSCPVPP SSFLLRCASIDRCKYDSECEGTKKCCKHPCGRQCRDIVKSKPGVCPKKADPTLVLCKRRGDDCYEDATCPG SQKCCQGACGNACMDPRLTSTDHAGTCPALDPKIQSLILCASLDTECELDSDCKVDQKCCKDYCGIKCVKS VPKPIIKKGTCPRLRYRCRRKRRDFCRSDGDCPGDRKCCRLPCGYKCRRPKRI PIIRKECPAVNTHPIYKLFC SPPLNPPCQVDRDCPQGKICCPGVCANRCLKAPTKPVCDPNPCGDAQIKCQYGQTKDENGCLLCQCKSYDF KMS* Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_kg.C_sca_820009 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Se quence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SYIQSEFVAACVAILVWWLAFSAKSKLSP*

PAGE 195

195 Object 29. P redicted secreted signaling molecules found in M elibe le onine Likely to encode function al secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_159314_fgenesh2_pg.C_sca_20000054 NR Annotation: Buccalin precursor [Contains: Buccalin D; Buccalin E; Buccalin F; Buccalin G; Buccalin H; Buccalin A; Buccalin I; Buccalin J; Buccalin K; Buccalin L; Buccalin B (BUCb); Buccalin M; Buccalin gene predicted acidic peptide A (BGPAP A); Buccalin N; Buccalin O; Buccalin P; Buccalin Q; Buccalin R; Buccalin C; Buccalin S; Buccalin gene predicted acidic peptide B (BGPAP B )] gb|AAB27696.2| buccalin precursor [Aplysia californica] NR E value: 3.00E 66 Sequence: MAARKHELVLVLTSVLCFVSSIVGDPNVPSDSQDNSALTQDDFAKRGMDKFGFAGGVGKRGLDKFGFTG QLGKRDMDSFGFAGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGFTGQLGKRGLDQYGF TGQLGKRGLDQYGFTGQLGKRGLDQYGFT GQLGKRGLDQYGFAGQLGKRGLDQYGFTGQLGKRGLDQY GFAGQLGKRGLDHYGFAGQLGKRGLDQYGFAGQLGKRGFDQFGFAGQLGKRGLDHYGFAGQLGKRGLD QLGFTGQLGKRQMDIFGYRGQLGKRQSIDKYSFLGAGIGKRSVKNTAGIKKDDA* Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] (Fulicin like) NR E value: 1.00E 21 Sequence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDL EDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFIGRRGMDVPAFVGKREFQTPMFLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNYFSLNILDDVQTKILL PLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQKRYLEFIG R* Lottia Protein Model : jgi_Lotgi1_171718_fgenesh2_pg.C_sca_119000006 NR Annotation: PREDICTED: similar to orphan G protein coupled rec eptor HG38 [Gallus gallus] NR E value: 1.00E 15 Sequence: MAPIYLTFIASLLLASSTFCEQLCLEGCKCEDHPSEKLGPHLMIDCSNLGSSSVPRDLNKFQNYGRPMHLKL SKNIIARVSKADFPENLPIIALDFNQNRGVMITDDAFMNIKGLRYLGLNDISMPFTDTMKYLKGMTKLRYLY LNKNYQFGEGLVPVSLFSGLELKLKDLSLHDCYLKGIENGALANVVITGSLDIGANQLDHIPEEVKTLKNLT KLDVSQNSIKSIPSNTFEGMSKLRTLSLSNNYINGQSFKTDSFNGLKLKELRLEKSELNTIPSAALSSLSSLKIL KLNNSPIKTIPKHSFNGSYCLEVLDLTNVLVEIENTHLDGLESCLQKLYLNNMQIREIPKFFKDFKVLNYVSL SGNSISEIKMDDLVGTTITELDLTENPLHRIEKGSFEEFPHAISLIISDTRLSNLSFVRDYEYGTIKTIKLGNVLP MCDCELAFAEYRVL EIHGNCNFNNTKNVAISTDEFMSVTSHCWPDGKMPQKKDVWAVVGDNSAGTFSTS ILSYFCVFLFFKVLNIV* Predicted molecules found in Melibe leonina with unknown function or predicted to be related to the secretory pathway Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_8000 0004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.00E 140

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196 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVL GGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRECLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK E KAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* Lottia Protein Model : jgi_Lotgi1_153437_fgenesh2_pg.C_sca_3000340 NR Annotation: hypothetical protein PB105991.00.0 [Plasmodium berghei strain ANKA] emb|CAI04701.1| conserved hypothetical protein [Plasmodium berghei] NR E value: 8.00E 09 Sequence: MKTMVAIIGVIIVSCVLHTSGLPLFKNILNERSVEYGGESGNGFKNERAVGYGGESGNGFLNERSVEYGGES GNGFQNERAVGYGGESGNGFLNERSVEYGGESGNGFQNERAVEYGGESGNGFLNERSVEYGGESGNGFQ NERAVGYGGESGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAIE YGGESGNGFLNERAVE YGGESGNGFLNERAIEYGGESGNGFQNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNERSVQNSGKSG NGFLNERSVQQGGKSGNGFLNERSVEYGGESGNSFLNKRAVEYGGESGNGFLNERAVEYGGESGNGFLNE RSVQYGGESGNGFLNERSVQYGGESGNGFLNERSVEYGGESGNGFLNERSVEYGGESGNGFLNERSVEYG GEAGNGLK* Lottia Protein Mode l : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO44886.1| predicted protein [Nematostella vectensis] NR E value: 2e 07 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNTINDCDDGSDENNCACLEK LGMEDGTI LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEGLNAFSNNSLYRSLFFPSLVTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGKFGCLSKLGSGGCKSFYLA LGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVRKTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVLRTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSATNDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDNFNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWSDEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model : jgi_Lotgi1_163475_fgenesh2_pg. C_sca_40000029 NR Annotation: Myc homolog [Crassostrea virginica] NR E value: 8.00E 08 Sequence: MDLSYLYYGMFWFSCLLVSTVSAEFSYEEVTYRIPPTPTGFYDQNCTDKISDCEEFGIASCFKPYESWAREN CAKTCEFCQGKPEKPPTCTDVEKGCDLYTTSICTDPSFISWAKAKCRRFCRMCPEQVLAELDLMTTTLTPV DCFDKVVCERYGKKAC TGDFASWATDNCQKYCGFCSTGVTKSKRLCVDKLPNCPQYDSRICTDKKYYSW TDENCRSTCKFCQDKLVPFLIPTPQAVRTPKPPTT* Lottia Protein Model: jgi_Lotgi1_164905_fgenesh2_pg.C_sca_48000037 NR Annotation: no hit Sequence:

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197 MMTKMLTIIFSVLIVLCSSSPECGPNYFEGDKWFDGCNWCTCTDNKEKCTNATCTEYQDELDNSP CQKLGE SWRDGCYHCRCLPDGIRCRANKRCVHALKSDDLNCPVDGLCVCGLDGVPSCNEQIMSRFSFKNVPELGPD TSSDKKLILSSKGENTLRLNPDEAETDLGDKDLHLIPTDDKLCHLGPRWYGGGLRCYCDPQNGVACGNPRF IARKVDGFKIRSKKCRVGKIWEERCRICTCKKSGLSLCRIDRECTKNKNIRHSVKSIVVAGNAECRVGLRWK DGCKRCRCNKRGKIICKTRLCNLSDDGKKLSNKDKTH RQTSTGGLISKSTPRFQLPEKPRFRMPNLSLYNNR FQRKKRPSIKTVNCNGFKPGEKYWDKCNLCHCTMAGAPMCSSKLC* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_pg.C_sca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purpuratus] NR E value: 1.00E 26 Sequence: MMKILEILLCLSFFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCSLKCSGRCRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSC TKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCNDGCKQDTGYCNKCNKGVYGNVCDKNCNKGCLDGCNIDGSCKCKSTYYGTNCSKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQCPSSCPICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEK CNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNGDCDRSTGNCGKCNEGYFGDNC NQNCSINCNGGCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICLQTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDE YNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATE KEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_kg.C_sca_820009 N R Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SYIQSEFVAACVAILVWW LAFSAKSKLSP* Lottia Protein Model: jgi_Lotgi1_202874_estExt_Genewise1.C_sca_21760001 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54

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198 Sequence: MMKLFVILALLAGQSLADIYLHN PRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTR Lottia Protein Model : jgi_Lotgi1_203811_estExt_fgenesh2_kg.C_sca_2400 05 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 0 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQKQNCRL VLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTRQNPNGNRRGYECPEERDYFPYWHPTQWKDIAILAENETLCNPLIAESFNNKPYGACLQKFPNSQVRKP SRYNNKEECEENGLDWVAFHNYLEKDKTASTQAACQAKSTNNIEYTWAIPYDSTTFTAECLVKVPSPQCQ EAPWSRSNHLGNGIGGEMNTVQW TLPHFPSGQVQNCVMRMRYNISTDDYDPFNTDSEFNGADNSPVTNN PYVDIGVGRGPLRLAINTAQFGRVFQDRTHIFQLKPRPDDIKDFRIFNVNVRGKRGNIVQVYPAVEYDFTPT DLEVTENDLVHFQWTGSNTHNNGRPGGDGQTGDAGEGKSGSDRHNILQMADRNENFPLPFEKTNMWDN AEIKWIYHGKNDVSARDLALNMASSGYYKCVSATDCPAESFKDFLVDTKDKMQVTLDNTPASYPGVVLK F KQGKYHYMCTRNNNFTNRSQKATITVAA* Lottia Protein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 10 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSC CMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMWRAVFSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI

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199 Object 210. P redicted secreted signaling molecules found in C lione limacine Likely to encode function al secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_163440_fgenesh2_pg.C_sca_39000174 NR Annotation: No match found NR E value: Comment: Bel 2 like protein Sequence: MDLYGVVWLLAILQEVTSCFLTEKLPSLEGKEWFTKIRSGGSNDVIAYFDRNSVTFNDVKNTTNGETKWR CFMKIRGRYFLKNVRQGGGYLYKCMGIIVRSESAIQLEWSHVSRLADPALCAEENINLDPWLLISYKTVVN DFTSCPVSGGFDIKHAGYYSRDVRDSRGYDIGCNLMNIPMRLEFDCIAGEGAIFNFRSKNCVPDIHFNIYQN TICVAKWSNKRHHFVLLRTKTGLEFWCARFPVGIENQNETELYLYSDVACLEGEYAEEFVKFMKFDLQRV MYTTICADEYPQCKEGTCNVFSKLECQKTCGLCNPHRPPGICTFPKRMQGTY LLHSKHGNQNVTLEGRTLN IENVGQFDCIVFEDSPLRSTKTYTTMSIFQNGCRPRYTCVRLKRLGPSALRYSLAQNLVWPIKKERIGANICN EDNFRADGDPIRDTFRSYKKTGKPVIKIHPKPRFINCGMNTSYTIRAELPDGSICHGGFYQHCKNETKLRFDF HTCNSFIKPKEDFNCAGIFRTNYWERAVLIQNVDNLNDIRCLIFNSLRPYQALIVVADECDKMASGLVDSNI RIPIMRLHIRSDVYPCKHI QLPLKDDVITNVVTTTEPIKMNVTLKESKNVNSFSVDLELSSRNSSGSRPFENKL NSLLLLFLCYVVFVKNL* Predicted molecules found in Clione limacina with unknown function or predicted to be related to the secretory pathway Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Anno tation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQCCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPWPLTTTTSRVPINILCNVYWFKCTKLHFSSHWECSNDDCLLEADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSN VVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFDEEPAVYHSVRIIGWGVERTIYGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWG QISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV* Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Sequence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_143169_e_gw1.194.72.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO36917.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 28 Sequence:

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200 MVKVCSIPLTVVKVCSIPLTMVKVSSIPLTISKVCSIPLTMVKVSSIPLTMVKVCSIPLTMVKVCSIPLTIVKVS SIPLTIVKVCSIPLTMVKVSSIPLTIIKVCSIPLTVVKVCSIPLTVVKVCSIPLTMVKVSSIPLTMVKVCSIPLTVV KVSSIPLTMVKVCSIPLTVVKVCS Lottia Protein Model: jgi_Lotgi1_166736_fgenesh2_pg.C_sca_60000104 N R Annotation: hypothetical protein LOC500991 [Rattus norvegicus] sp|Q5XI89|FA55D_RAT Protein FAM55D precursor gb|AAH83798.1| Hypothetical protein LOC500991 [Rattus norvegicus] NR E Value: 3.00E 25 Sequence: MALTKKLVLICCLIIATVFGLKIMHTIKYQINMRPNIEPIPFYSQNTPK RKLMFPERQSPSLEDYNMKHDDVR SEDIFNKFLSENVVYVKDTTADDYFIEGEKELLEEGEIKDLRDLPSSNYSIISTKNTVFNQYEAVTVKIQLYN GFGQLLKRGGDDVRVWITGENFQAGGEVYDLHNGTYVAEVKAFWTGKSKIHVSIAYSREELRILLRTRRE SKVLRYSVGRFLRGGDTFMTLCSPFHDIPLYPRVCNFTNFNLNPYYCGQPNSPKVACSDLRILHTINYSKQR MNNLESKILSRQNRVNE LKSKSITINIGTVVNLPYCSSYKSRFTWTNTAPLGYFNNNTWHPSFCQGQITRQR LSRCIRDLHVYLVGDSTVRQIFQSFLRLTKCTNTTFIGNRPHDRVQRYCDVHDSNFRVEYGIHGLPYSMVRT PDLLNNTKAIGFHLDGLQWSQNKDKMFVMVHLYAHFLFFHTSLFEARVRSLKVPLTRLLNRYPKIKIFIKGP HAFTGADFKPIVKSDYFGKVYRSILLKELFEFRDRVYYLDYWDMSVSSGNRLLHPVNSFVDEMVKFFLNL ACDK* Lottia Protein Model : jgi_Lotgi1_173456_fgenesh2_pg.C_sca_159000027 NR Annotation: No match found NR E value: Sequence: MWSGVWCGVVYGVMWSGVWCGVWSGVERSVVWRGVWSDVDGVMWKECGVVYGVVWCMKWCGV WSDVERSVVWCMKWCGVWSDVERSVVWCMEWCGVWSGV Lottia Prote in Model : jgi_Lotgi1_175543_fgenesh2_pg.C_sca_302000002 NR Annotation: No match found NR E value: Sequence: MNTMFLLVVFLLIPATSAFDSTQTNRTIVNSTITTKINGSSIGLGTCSEFTPQSCSGVCGQFFELWCSCDEYCQ VYRRCCFDYEAVCKEESEKSKQDHENLMNVTVECDSNMSGVHVPQQRISPASG* Lottia Protein Model : jgi_Lotgi1_67930_gw1.39.124.1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO32094.1| predicted protein [Nematostella vectensis] NR E value: 1.00E 10 Sequence: VGHFVLSCCMWRAVFSCCMWRVVFSCCMWRAVFSCCMWRAVFSCCMWRAIFSCCMWRAVFSCCMWR AVFSCCMW RAVFSCCMWRAVFSCCMWRAVFSCCMWQHVASCIQLLHVASCIQLLHVASCTQLKHVASCI QLLHVASCTQLKHVASCIQLLHVASCIQLLHVASCI Lottia Protein Model : jgi_Lotgi1_98330_gw1.10.599.1 NR Annotation: hypothetical protein NEMVEDRAFT_v1g147894 [Nematostella vectensis] gb|EDO28414.1| predicted protein [Nematostella vectensis] NR E value: 5.00E 06 Sequence: TVCSLGTHCLSITVCSLGTHCLSITVCSLGTHCLSITVFCSLGTHCLSITVCSLGTHCLSITVCSLGTH CLSITVCSLGTHC

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201 Object 211. P redicted secreted signaling molecules found in O ctopus vulgaris Likely to encode funct ion al secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_121665_e_gw1.37.7.1 NR Annotation: hedgehog [Patella vulgata] NR E value: 1.00E 171 Sequence: MKLFFSFQSVCSWTFIFLFITSLTHACGPGRGSGSRRRPRKRTPLVFKQHVPNVSENTLGASGISDGKIRRNS EKFKNLVKNENPDIVFKNEEG DGSDYLMSRRCQDKLNSLAVSVMNNWKDVRLRVTEAWDDSPNSHAKD SLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESRGHIHCSVKSDSSVAIKIGGCFPPTGSVQTL HGWKTMGQLTVGDKVLSINSAGQLEYSPVIAFIDRNDLEFEKYLTLHTEDDTDITLTSKHLIYASGTNSSNF ESYDVVYADDIMEGDHVLITSSEKGAISPTRVVTISEKTLQGVYAPLTVNGNIVVDGVVVSCYAVVSDANL AHAVFAPMRGLHYLSQYVPWFLHSTQQENAPQNGVHWYAKMLYNIGSTFLNEKTLYIA* Lottia Protein Model : jgi_Lotgi1_168377_fgenesh2_pg.C_sca_74000087 NR Annotation: TBL 1 NR E value: 2.00E 07 Sequence: MLPHITQCVILFITFLCDISVLLPSRDGGRGLTYAQLVPHRTSNDVYMDPCKAAGFMGDIALSEEEFKRER K YLQQMNNTMFELQVEDPRTTYIGRKPSKYSGQPENTLKKLPMSELDVKKKKYLRKILKEKKQNLKSLCKK GFQTVVYTRQVPVGFKFISPFLYLLDLNIL* Lottia Protein Model : jgi_Lotgi1_168619_fgenesh2_pg.C_sca_77000042 NR Annotation: crossveinless [Saccoglossus kowalevskii] (a new family of twisted gast rulation like modulators of bmp signaling) NR E value: 1.00E 148 Sequence: MYYTELSILAKCIALCILTCFPILIQAQLTGTVTRCYNEGGEVKIQGITTDPCISCYCMDGYVQCDKKKCDNL DGCHAILFDGPKRCCDTCKGCRLNGVKHESGKSWFDKRDRCTSYSCKAGVITKSKVQCHVPCSNPIKRKR QCCPSCKGCHFDGADRKNGEKFALASDPCVECKCRKGS VTCMKRACPVLNCPDEVIYQPKGECCPKCKG MRKIFDFPGACYFAKRVYVDGFSFEPKSETRCTCTKGTVICEKATCPPVACPVEERVQKGSCQVCEPKRNC LYDGKIHMHRSKWQPRMCTQCSCQNGVTYCQRERCNNSLSCPNGYKLQFQPGECCPKCVEHDAVCRVFG DPHYRTFDGKMYNFQGTCKYMLSQDCQGKDFTIKVKNGVRLSSGFAWTQMVVVLMGDTRISFRQNLLIKI NRRRVQLPYTIPGKF SIRREGHSVTFRADIGLKVVWDGDSFLEVTASRKYKNRLCGLCGNYNGLETDDLIG RKGKNYLRGEEFGNSWRIGSKKACKTQPKVKNLQSICDKDFKAKVRANKECSVLYSRAFSSCRRVVDVTP YVTSCVTDMCDCPHGKKCSCESIQAYAHECKRAGHKVKWEKVSNCKAPQKNCPKGAYYSLCAPACPKTC SSNKPDGSCSKKCSPGCVCQNGAVLYKNRCISPEKCPKT* Lottia Protein Mode l : jgi_Lotgi1_205842_estExt_fgenesh2_pm.C_sca_70027 NR Annotation: BMP2/4 [Patella vulgata] NR E value: 1.00E 159 Sequence: MIADFYRSVVLLLLVLVVNYTSSLSTNTLHPRKHGNEKVLAAVESNLLGLFGLKSRPVPGRRTPIPDYMLD LYKRLDSEPDFISPFAESKGKGIVEANTVRSFYHSDINFVKDCDERTCARIWFNISTIPFA EIMAASELRVFKD VYNHFKLTDANSLSGDRIKASLFKHRLEIHEIMRVSPDDSECISRLIDTKIVDTRNSSWESFDVHPAVLKWRR RPQYNYGLEVRIVSKSPWISTNSHVRLRRSAHMDEKNWQIQRPILVTYTHDGRNPNSRIKRASARNRRRKS RKRRRRKKKGNKNECKRHALYVDFGDVGWNDWIVAPTGYNAYFCRGECPFPMGQHLNSTHHAVMQTL VHSVNPSAVPKACCVPSELSAISMLYLDEWDK VVLKNYQDMVVEGCGCR* Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301

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202 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37057.1| predicted protein [Nematostella vectensis] (growth factor) NR E value: 1.00E 109 Sequence: MF LEVFNISATVLCILFLIGHRKCDTIQNSKHHAELMMDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVGVNGGFFNTTTGACLGNIVSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSA VGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLELGVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYGINCSTPCKPGYYGSNCAHQCYCLNHGTC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi 1_208417_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVIASSRRSRGRWYRGRSGRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSF KNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYDTLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLE PSIRDRLIKGEKSCLEKVLKNHLLTSVIC STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSNNAFEKLEEKVSLEELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* Predicted molecules found in Octopus vulgaris with unknown function or predicted to be related to the secretory pathway Lottia P rotein Model: jgi_Lotgi1_103075_e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mellifera] NR E value: 9.00E 50 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRLE NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHPNEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLEDREVYLTEEENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL* Lottia Protein Model: jgi_Lotgi1_108564_e_ gw1.8.40.1 NR Annotation: PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001179636.1| PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] NR E value: 2.00E 86 Sequence: MYLSCYLPVILLFIEDLKQAVVAMMLRKDEVEEKNKSLKAMLDREMEISST LRAEIEEMKISFKLSKDKEIA KNETLQKENELLKHQLRKYINAVQLLRTEGAKDDTQGITLEDPQPIIPPAKPSIDYSHEASEYEKKLIQVAEM HGELMEFNELLHRQINCKEAVIRNLKEELTDLRGPLPYDAQSSDDSLSGDLESSLISRCLINIWIPSAFLGGSK TDSHHVYQVYVRIRDEEWNVYRRYSKFLDVHTRLKKVYPLIEKFEFPPKKTIGSKDPKVVTARRKMLQSY LRKVINHLLEKNADLSSNVSKEKLIAVLPFFK*

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203 Lottia Protein Model : jgi_Lotgi1_116422_e_gw1.24.410.1 NR Annotation: PREDICTED: similar to CG14764 PA [Apis mellifera] NR E value: 7.00E 58 Sequence: MKTKNFYILVLLISVTSVALWLVSYPSPTFRNIMSTTQNNIRKIPKHMQMSKSPDLEVDKKYLDLLGFDSES KPKNDIVVIGSALMPA DTENTILFLKSAQEYVPNLKLVLFDMGLSKADKQTLTNCRNETKHCEIRKFNFDQ YPSHISDFANKSYKPICIQLLLKEFGTVIWADTSELFASSDIDNTMKQAKTEGLVAWTIEDATSSLTHPKMFD YFKTDQKKYFFHHAVQTSHIILVNTDKIHENVMLPWVKCALVEECINPMGAQNSGCNYQRKPKFRYTGCH WYDMSALNVILGLAFDFDEAYSGKDKIFWTKFDQEKAKAENMTDGAVHKVSVPHRVYF Lottia Protein Model : jgi_Lotgi1_118077_e_gw1.28.72.1 NR Annotation: LOC100124858 protein [Xenopus tropicalis] NR E value: 1.00E 108 Sequence: MGSTLNILVILGCILVPFSFSNFLEWGPDLEGPWCATRPIEQCCPGRDDECTVPILGTKCYCDIFCNETAEDC CPDFWNLCLGVTRPTPWPLTTTTSRVPINILCNVYWF KCTKLHFSSHWECSNDDCLLEADHITQINNGPYS WVASNYSDFWGLTLEDGVKYRLGTFPLGSNVVQMTPLRVKLTDVLPESFDARTKWPSYIKPIRDQGNCGA SWAFSTTAVASDRLAIESLGEIKDELSAQHMLSCNVRKQKGCEGGNLDRAWWFLRKTGVVTEECYPYESG DTTDKGDCLVSRRKSNTTCPSRILYKEKRRYKATPPYRIAPKEREIMKEIMDNGPVQATFMVKSDFFMYKN GVYRYSNISFD EEPAVYHSVRIIGWGVERTIYGDILKYWICANSWGTQWGENGYFRIIRGRDECEIESYIVG VWGQISGDASLRTLLTRGRRRRLFAERSLRNLRVNSMAKGRKSRSRQVGRKKVKRGRKSREERRRLRQEH RNKRKLRRQSRKQDSDSQNTV* Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Seq uence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Protein Model : jgi_Lotgi1_132441_e_gw1.79.137.1 NR Annotation: PREDICTED: similar to CG11081 PA [Nasonia vitripennis] NR E value: 4.00E 72 Sequence: MAWKIYVLFVCVLCLRFILAARENYIKATFEGAKDRGIFNKLVVNRETNELYLAAVNHLYKLSPNLEVIQE VVTGPHLDDPSCTSLTNLTCDPDLKSCACKLRPYYTKALVVDYVNKKLITCSTLYHGHCDKLDLDNITNKQ MFPFMPMVANNATASTVMFIAPGPAVVKDEPQRQVLYVAASYTRTGLKAYREQVPAFCSRSLEDFNLLLK NAFGSSSIEIESQHRDTFPVRYIYGFGSDNFSYVLSIQKANVQTDRYVTKISRICQSDDDYYSYAEVGLSCTM QNTEFNLLQAAYVGKSGTKLARSLGIPTTEDVLYTVFSIGDSNSANPSATSAMCVYSLRDIRKKFTENIQEC FSGIGNTGPDHMIQSSKCLKVVSSHFFVIFFLESFESADLSVL* Lottia Protein Model : jgi_Lotgi1_149784_fgenesh2_kg.C_sca_15000101 NR Annotation: PREDICTED: similar to chromosome 19 open reading frame 10 [Canis familiaris] NR E value: 9.00E 20 Sequence: MLISNDFKCLFMLILGFIQWVSSYEMTRDYEEASHFDVMPGGAVLSHEKQLDGFTCKFTYACQGGTKEGW QLTINTNTDKTKYLCSVQRPDGKSYLFFQSFKLEFTGLEVKEGYAMGANFKPLSPSEYQVDFQKHSISDVA GKFGSQ LEKVELYGEKGKAEL* Lottia Protein Model: jgi_Lotgi1_150026_fgenesh2_kg.C_sca_22000005

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204 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO30774.1| predicted protein [Nematostella vectensis] NR E value: 3.00E 25 Sequence: MLVYLLLAALLTDFSSAGVFYKDC GSVDGTISNIEVSSCASEPCQFQKNTNISVTIDFTSKTVVSSATTVVHG IIAGIPVPFTSVQPDACKDMSCPLASGTKYTYKNQVAVLPIYPTIQLVVQWEVKDQDGKDLLCFKIPVRITN* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laevis] NR E value: 1.0 0E 140 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKLSSLHYEMGEADESLVQIRE CLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNPFGQGFSGGSFKFHFN* L ottia Protein Model : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO44886.1| predicted protein [Nematostella vectensis] NR E value: 2e 07 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNT INDCDDGSDENNCACLEKLGMEDGTI LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEGLNAFSNNSLYRSLFFPSLVTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGK FGCLSKLGSGGCKSFYLALGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVRKTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVLRTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSATNDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDNFNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWSDEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model: jgi_Lotgi1_1 61657_fgenesh2_pg.C_sca_30000077 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO44809.1| predicted protein [Nematostella vectensis] NR E Value: 3.00E 17 Sequence: MTKLSEYFLICSMIFVVFMLQRGTCDDVVTEEQISYAKGSVCGYCSYCKFCKLCDKDCPCETSPSKPNCKM CKYCK YCYICSGVCDTICQPGGMIDKISAAIVNALPSFNKEEIEDDLESVKPWLDKKDEL* Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sca_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus] NR E value: 1.00E 133 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHLKK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAGTPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSSDNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKGDPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALEL SKELN

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205 VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAATIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KKHDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQTSTKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNTWPEKEECDSH LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHP LPWYPPICVTLLTINEYDEKTYDNLKSVGHP GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGARWVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYADVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSVAPIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKS GECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGEDCNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLHSTQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN HCSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPK SWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLMLLGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTLGNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model : jgi_Lotgi1_165259_fgenesh2_pg.C_sca_50000100 NR Annotation: hypothetical protein CHLREDRAFT_144736 [Chlamydomo nas reinhardtii] gb|EDP05543.1| predicted protein [Chlamydomonas reinhardtii] NR E value: 6.00E 23 Sequence: MKVLLVLDICCLIGLVSVGEAVCPDNCNYNGVCDVKHSRCNCYEGFTGSDCKTDCRCNGHGTCQSGSVC KCDEGWKYSGGQCVWDCHCLNGAKCIGPGECGCVHNCKMGNCRNGQCQCWNGYKGSDCSEYDPTM* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_pg.C_sca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purpuratus] NR E value: 1.00E 26 Sequence: MMKILEILLCLS FFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCSLKCSGRCRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSCTKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCNDGCKQDTGYCNKCNKGVYGNVCDKNCNKGCLDGCNIDGSCKCKSTYYGTNC SKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQCPSSCPICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEKCNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNGDCDRSTGNCGKCNEGYFGDNC NQNCSINCNGGCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICLQTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Lottia Protein Model : jgi_Lotgi1_167022_fgenesh2_pg.C_sca_63000026 NR Annotation: predi cted protein [Nematostella vectensis] gb|EDO43826.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 08 Sequence: MVAVRIRKYKNLLFFALLFAGVLSISTIFYSNHYQIMNTGTRSDVNTGFSYSNANSSKYVIYICDGKNSCGG YGDRQKGIVASYIISVMMNRQFGVIMNDSCDIKQMFKPNQINWIVDNNDIKSKTSKR IKALDGYSDKLRKS MIEMDLDQEYKEEVIYFSLNLEFVFYLRQNKRYEKQLKWLEHLSMSEIYNVVWQSIFKLRPYIREKLDPFL DLKKQGLKLISAQIRLGKNPTIPHDSRVVNSLNNMNVLWQFYKKYNDSSKYRIFISTDSDTVREKARSIFPD VYSDVPGKVFHVERSNKTDICNGWRKVILDQVILSLSDVLVISNSGFGRIAAFFRQNENDLYCLNLDKIRRC TTQSEIFVDKTW* Lottia Prote in Model: jgi_Lotgi1_168278_fgenesh2_pg.C_sca_73000118 NR Annotation: no hit Sequence:

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206 MSISASSYSGTFKLCLFIVVLVAFSSYCGFVITREDSQFSNHLPKLTSTNVILEKLDSLRKDFLKQRTNVAGQ FKNLGSKLSELVLNKNTKPENQHIKAQDEPWRKSTKTGLTLFTSWVDVPEKDLVHGNVVRMWRKWKPLI KPLFFYSSASTGKRLEKEGWLVQPVSKTGCGVTEMPTIKDMFLDAMKNHESVLYGYANGDITFDDGMPK AIDYVVNMDVVREKPVLVLVRRTNVDFSNGPELDANSDITEMYNEGKALKDGSSDGFFTNKLFPWKYLPD IVIGRIGIGMWLASYARAMNVTVIDITKTVKAIHMTTKSGNSESHFKKNGRCNHVIYGRLKLSPTSWGCGFI TCATIDITFDKQGQTTMRRKNPKSMNPKCGNCSMDLTQILPKSLGITDRIYFGQNIEPKQVVNKRPKSPK Lottia Protein Model : jgi_Lotgi1_170051_fgenesh2_pg.C_sca_92000069 NR Annotation: hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] gb|EAR97557.1| hypothetical protein TTHERM_00439050 [Tetrahymena thermophila SB210] NR E value: 3.00E 08 Sequence: MKMINIFIVVYCLISPCLGFWLSSKTVDPKVDISTECINKALTGDCGFFTCFEERLPCGEYGYAESYGGKYC WQFQQSHHLFTKKGVEFVEKLTRCHMNRSITSYRQNQIECFSHYDQSFVIMGDCYVESGFCDVVIDNFMSL ARILEPKDFTNYRVVREVFRAAKQCPNNISGGIISKFLSYKRGSSL* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_ sca_130000017 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO35011.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 26 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDAREKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPH IGNRENYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG RYTYRTTNIAQDISCCYVGRTCLHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIK SFVSYGTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPIRGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTILTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMY MFPEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTPNYPFDYPLNTECNWLFVAPEGSEVML NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEMAHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYD NFDDCEELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD GFTNWRSTYPVSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADR KSS CTRNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESFTTQCPA GQICTQNCQHFSRACRRSGEVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGP TCYFFSLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTSAGNTESSSTGASRSSPSTDAFVSEDTTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model : jgi_Lotgi1_176704_fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTE D: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDDVNVCHGELPANAMCSGENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lottia Protein Model : jgi_Lotgi1_186451_estExt_Genewise 1.C_sca_160091

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207 NR Annotation: PREDICTED: similar to out at first [Nasonia vitripennis] (Vital for proper neuronal development and hatching) NR E value: 7.00E 69 Sequence: MSMSLIPSLSYSIFVCVLIKICHTQLVVNVKNGGGDILVESIQSNVSSDTITLEYPSSDGSLITQFIDFKSEAQIF RAYVPGEEE RGQPPGSVQVLCFITRFSKNDFISSDAMSKLRQKNPTAIRTPEEERPQEVHSFDLSVDLNKSHV ISPHVFNICREAKESTFFNEDDLKTISRSLSKDYNTMMSAMIKLAPTKYGKCSDTSDIFKPCTCRYDACVGW YPCGLKYCRGKDSSGKVVSYRCGIKTCKRCLAFDHFVKQKFLCLWN Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFL SKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEITDDEYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FI RGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKEATEKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPR HPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model: jgi_Lotgi1_203670_estExt_fgenesh2_kg.C_sca_170029 NR Annotation: PREDICTED: hypothetical protein [Ornithorhynchus anatinus] NR E value: 1.00E 142 Sequence: MKLSVFLSLLFLTAVWADTPANCTFDDIKGTWTFFIGSNGHDNTVNCSNMGNPKSQLQVTLYFPDVAVDE FGNKGFWTIIYNQGFEVVISGRKYFAFSMYEQSGSTVSSICDKTLPGWSHDTFNRDWACYYGKKITSVPPK YYKKNKPHRLFKLNYDYINNINKQQKLWTATVYPEYEGKPMRELLNKAGGPASQVFGPIKAAPVTWEQQ RLSETIPEQFDWRNVNDNNYVSPIRNQESCGSCYAFSSMAMNEARVRIMTNNTLQPVFSPQDIVECSEYSQ GCAGGFPYLIGGKYAEDFGLVEEKCNPYKGQDHSCSTDKTCKRQYATSYRYVGGFYGGCNDALMKMEIY RHGPVVVGFNVTDDFFHYKSGIFIHTGLTDRYNPFEVTNHAVLVVGYGTEKGVKYWIVKNSWGEKWGES GYFRIRRGTDELGIESMAVSSTPIF* Lottia Protein Model : jgi_Lotgi1_20477 0_estExt_fgenesh2_kg.C_sca_820009 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNS VTPLFSINISHSGLWQG SYIQSEFVAACVAILVWWLAFSAKSKLSP* Lottia Protein Model : jgi_Lotgi1_205030_estExt_fgenesh2_kg.C_sca_1120002 NR Annotation: PREDICTED: hypothetical protein [Strongylocentrotus purpuratus] ref|XP_001188672.1| PREDICTED: hypothetical protein [St rongylocentrotus purpuratus] NR E value: 3.00E 20 Sequence: MKLMFLGFIGILMLSTSNNASASPWFCKGLECPKFNLLNKTKDYEVRQYGSLTWVTTSHIGQWKSDILGEL

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208 LFKNLFSYIEGNNTMKKQLDLAVPLLTKLESAGDRGSIYTMHLLIPRENIDHLPSPLNTNLTFITIPPSTYYIRS FDGVAYDRQYMKVLQELVNAINDTGAYPDDFFYTATYDHPQVKENRHNEV WLLKKEKIP* Lottia Protein Model : jgi_Lotgi1_211937_estExt_Genewise1Plus.C_sca_110124 NR Annotation: PREDICTED: similar to SPFH domain family, member 1 [Monodelphis domestica] NR E value: 1.00E 124 Sequence: MANPALALLSVSAVALAIFLNFSIHKIEEGHVGVYYRGGALLSSTSEP GYHFMLPFLTTFRSVQTTLQTDEV KNVPCGTSGGVIIYFERIEVVNKLDRAIVYETVRNYTADYDKTLIYNKIHHELNQFCSVHNLQEVYIDLFDQI DENLKKALQLDLNVMAPGLMVQAVRVTKPKIPESIRKNYEAMEGEKTKLLIAIQKQKLVEKEAETERKKA VIEAEKVALVAKIQWEQKVMEKESEKRMSEIEDSANTARAKAKADAEFYKTQKEIESNKLKLTKEFLEFTK YESISKNTKIYFGNSIPGIFSDKVEPQEIAKVVGERK*

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209 Object 212. P redicted secreted signaling molecules found in N autilus pompilius Likely to encode function al secreted signaling molecules Lottia Protein Model : jgi_Lotgi1_110650_e_gw1.12.27.1 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 1.00E 32 Sequence: MKIILVLLCFCHLIYSAKTCPSNCQCHLTDKAVNCYKRELQFVPANIPLDTEILDLSSNDIANIDPTMFHNLT KLTDLVLNDNKISKLDPRTFQNLHKLEVIKLSQNFLSTIPEGLFDNLPIMVPKLMELNLSQNKIVKIENYVFR PLSMLSTVSLNDNPLKNADRLLSKNRRLSYIDMSECQLTTVPRGLPDSIEHLK LIKNNITKIKPKDFRNKNYL KILALDENEIEFIAPNSFKSLQKLIQLWLNGNRLRKFPSPISPSVTNVYLAKNNITKLSAADF Lottia Protein Model : jgi_Lotgi1_119019_e_gw1.30.87.1 NR Annotation: PREDICTED: similar to seleno protein N, 1 isoform 2 precursor [Canis familiaris] NR E value: 2.00E 85 Sequence: MVLFTLLCCLFIIGRHDPTQIPEEIVINIGDDGVLLFQQHDRDNDGYLSIQEFEPLVYRLLEINVSGPVYDVPIS TDDEMITLKSYFIPIVKESMSKDLNDSVSIGLLRTMNSLHGLEKWQNVNLQWMNFGASHFSGFLPKDVDSI MLGSSYFIINVEKGLFNAALSSNRYYPPKVTTNESIIVHRLLTLFHPRPFVVSRFPAQSSVACVRAYNDKYLD IVFRIHAEFQLNEPPYHPFWFTPAQFT GNLIISKDGKHIQYFNLYVPNNKRLNIDMEWLNGPNESENMEVDI GFMPLMQLNSTQSSVPVKNLEEYELLEPLPERPQHKTEGENIEWMATIDLEDAKSSLEKALYPFKKVPYYN FTEAFKKAEDNKKLVHSILLWGALDDQSC* Lottia Protein Model : jgi_Lotgi1_121665_e_gw1.37.7.1 NR Annotation: hedgehog [Patella vulgata] NR E value: 1 .00E 171 Sequence: MKLFFSFQSVCSWTFIFLFITSLTHACGPGRGSGSRRRPRKRTPLVFKQHVPNVSENTLGASGISDGKIRRNS EKFKNLVKNENPDIVFKNEEGDGSDYLMSRRCQDKLNSLAVSVMNNWKDVRLRVTEAWDDSPNSHAKD SLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESRGHIHCSVKSDSSVAIKIGGCFPPTGSVQTL HGWKTMGQLTVGDKVLSINSAGQLE YSPVIAFIDRNDLEFEKYLTLHTEDDTDITLTSKHLIYASGTNSSNF ESYDVVYADDIMEGDHVLITSSEKGAISPTRVVTISEKTLQGVYAPLTVNGNIVVDGVVVSCYAVVSDANL AHAVFAPMRGLHYLSQYVPWFLHSTQQENAPQNGVHWYAKMLYNIGSTFLNEKTLYIA* Lottia Protein Model : jgi_Lotgi1_137329_e_gw1.113.40.1 NR Annotation: insulin like growth factor binding protein, acid labile subunit [Sus scrofa] gb|ABE73450.1| acid labile subunit [Sus scrofa] gb|ABE73451.1| acid labile subunit [Sus scrofa] NR E value: 1.00E 21 Sequence: MSFVQLLPFFLFLTISQSSSQRWPLSGTCYPCSCDINQKGFGRVKYVNCSNLALTRIPR NLPLDLHTLDFSNN RILPQEIQQLCTFNSMQYVALSYNSLDSIPPEIFKDLENLHTLVLHGISSIPTTNIFEDLFNLEYLDINDCHVNQ IPDNWFRKLYSLKTLKLRQTGIRSIEPGVLDGLLNLQELDLSHNVMRTAQTESFKPIVQSIRRINFRGNMFKT IGDHLFEKMYNLVELDLSDNKLESIHKRSFLDLRQLINLDIRDNKLSNLPGGLFKNLRNLQTLNLAMNTFNN FPETLFKIGYLLKLDLSYNRIR RLPDSFVHSFPYLEFLNVDKNPLHCDCKSLDVKFYQPNLVII Lottia Protein Model : jgi_Lotgi1_152641_fgenesh2_pg.C_sca_2000061 NR Annotation: interleukin 17 [Crassostrea gigas] NR E value: 5.00E 07 Sequence:

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210 MVLMKVFICALLVNMVYSFTLIKNIEHILERERRSDECAVPNNIDELFTNLNSHVNRVEFLIAVNATHQPNA QGLIPPAAGDQTDTECPKEYTGKDTSNSGIWQRSTCPWFYSERNYGAEYYPSKVYNAACKCVDCLYSKSN SETGCSRIYRNINILKKTGCSNGFYTYEQRSISVSVGCTCAKLQANIG* Lottia Protein Model : jgi_Lotgi1_163440_fgenesh2_pg.C_sca_39000174 NR Annotation: No match found NR E value: Sequence: MDLYGVVW LLAILQEVTSCFLTEKLPSLEGKEWFTKIRSGGSNDVIAYFDRNSVTFNDVKNTTNGETKWR CFMKIRGRYFLKNVRQGGGYLYKCMGIIVRSESAIQLEWSHVSRLADPALCAEENINLDPWLLISYKTVVN DFTSCPVSGGFDIKHAGYYSRDVRDSRGYDIGCNLMNIPMRLEFDCIAGEGAIFNFRSKNCVPDIHFNIYQN TICVAKWSNKRHHFVLLRTKTGLEFWCARFPVGIENQNETELYLYSDVACL EGEYAEEFVKFMKFDLQRV MYTTICADEYPQCKEGTCNVFSKLECQKTCGLCNPHRPPGICTFPKRMQGTYLLHSKHGNQNVTLEGRTLN IENVGQFDCIVFEDSPLRSTKTYTTMSIFQNGCRPRYTCVRLKRLGPSALRYSLAQNLVWPIKKERIGANICN EDNFRADGDPIRDTFRSYKKTGKPVIKIHPKPRFINCGMNTSYTIRAELPDGSICHGGFYQHCKNETKLRFDF HTCNSFIKPKEDFNCAGIFR TNYWERAVLIQNVDNLNDIRCLIFNSLRPYQALIVVADECDKMASGLVDSNI RIPIMRLHIRSDVYPCKHIQLPLKDDVITNVVTTTEPIKMNVTLKESKNVNSFSVDLELSSRNSSGSRPFENKL NSLLLLFLCYVVFVKNL* Lottia Protein Model : jgi_Lotgi1_164016_fgenesh2_pg.C_sca_43000014 NR Annotation: chordin [Danio rerio] sp|O57 472|CHRD_BRARE Chordin precursor (Protein chordino) gb|AAB93485.1| chordin [Danio rerio] NR E value: 1.00E 119 Sequence: MSPMDLAVCLVLLAATVSSTKFSKIPLKAARENLFGIQPNRPGCILGSKFYGIGERWTPELLPVGEMYCVKC ECVPVERKGIIDMKGQTLCKNIKNLCPRPNCLNPVLPKGKCCKVCPDEVNSFEDSFSLGSKSS APTMISSDSK RRVRNEFVSLLVGKNVRRESVKTSAVAAVYFSIVGRDEIRYSVRYFKLDRPKFLQITDANGNVLFERPIEKK RNGDKKFCGIWTKVPSAYIQYVREERLFAVITASRHFRGLVSGRIMVNRHAKREVFSSVLASPKANGIGGL LSVVYNPRTHLVDYVINLDGVFPGSSEEYFITISKKSKVLHQSKGKASSKTRFIRGSWTLKNKRQSKQLARG RLTIRLTSTNGASVSGDIKPKVTCGVSQAVM SSGNSLEEMKLASSGTAVFELREKGSLDYKIRVTGLTSKVH RIRLEGAVNKKNRRQIIGSVHRNFKADENSFNGWCNGTFKKMTADDLHLFLNNKIFVNVATSENRISELRG RVIALPYHSHFEQVSADPAALGPKTITSNGHAGNAWISMGVGCSLHYDITTTTIAKHHDLTLSLVSPTDIDA YVTIEKISTTNLYEFQQASGSIAVVPESLFRDLDEGSALIQVISGDVILSGNVSVPNTCWQYSQDYDMDILVD EQTSEEVLAANRYKCVYEGKVYENGDSWLPDVNATCNTCSCNKGKIECHRLICPVVECANPIVLDGECCPT CPIETTDQTECKIDGDLRSHPVGSTWHPFLPLMGYAKCVICTCLPGGIYKCERKCPKLDCPASRRIRLNPKD CCQVCAPEEVKPEVVKPLQDVDMKGACSFKDQIYANGAKWHPRVIPFGYMKCIDCSCNNGTPSCSRPKCP KLSCSRKIREDGACCDTCADSDGEEIVEEKKSDSCIFGGKKYDDGETFQPKTSSLSSCATCTCRKGSMKCSL TCPEKCKDPKHSNDPCCKHC* Lottia Protein Model : jgi_Lotgi1_167943_fgenesh2_pg.C_sca_71000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO34356.1| predicted protein [Nematostella vectensis] (Fulicin like) NR E value: 1.00E 21 Sequence: MKSILVTSVVVVAGTLLTLVQALGADKFAEPATQKHSKIPTAALSHSGDTLLNTDAHQTNPSNIWKSKSKF DLDALPVVNRQDVQSHYAQHRRSYDTPMFLGKRDLKNLSLRFKRATDLEDLDATEFSDDFDKRGVDIPQF IGRRRYDTPQFIGRRGYGVPAFVGKRAFRAPAFIGKKAFGAPAFIGKKAFDAPSFVGKRKLTAPSFIGKRGL DVPAFVGRRAYAPPQFI GRRGMDVPAFVGKREFQTPMFLGKRAFSAPSFVGKRGLDAPTFIGKRKEESFRD LLATLQAYREYRRLMQTDKRFDAPLFVGKRSDYENDNLAAETLSRRFQCIPQWVFRMRLNVVDLNMSFIG GYKQNYFSLNILDDVQTKILLPLDDLHKVDSSKMDVPTVQLWDADMPRPNGARFLKLDPAHALGFLGQE ASPVSKRYLEFVGKRDNDKRYAEFVGKRAGFGEKRYSRGSLSEYLSKRLRIRRPEFVGKRSNQ KRYLEFIG R*

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211 Lottia Protein Model: jgi_Lotgi1_168278_fgenesh2_pg.C_sca_73000118 NR Annotation: no hit Sequence: MSISASSYSGTFKLCLFIVVLVAFSSYCGFVITREDSQFSNHLPKLTSTNVILEKLDSLRKDFLKQRTNVAGQ FKNLGSKLSELVLNKNTKPENQHIKAQDEPWRKSTKTGLTLFTSWVDVPEKDLVHGNVVRMWRKWKPLI KPL FFYSSASTGKRLEKEGWLVQPVSKTGCGVTEMPTIKDMFLDAMKNHESVLYGYANGDITFDDGMPK AIDYVVNMDVVREKPVLVLVRRTNVDFSNGPELDANSDITEMYNEGKALKDGSSDGFFTNKLFPWKYLPD IVIGRIGIGMWLASYARAMNVTVIDITKTVKAIHMTTKSGNSESHFKKNGRCNHVIYGRLKLSPTSWGCGFI TCATIDITFDKQGQTTMRRKNPKSMNPKCGNCSMDLTQILPKSLGITDRIYFGQNIEPKQVVNKRPKSPK Lottia Protein Model : jgi_Lotgi1_169325_fgenesh2_pg.C_sca_84000054 NR Annotation: Insulin precursor [Contains: Insulin B chain; Insulin B chain'; Insulin A chain] gb|AAF80383.1|AF160192_1 insulin precursor [Aplysia californica] NR E value: 4.00E 13 Sequence: MEVTCKCPLVLLGVLFLNFGTVLTHLEWTCTLETKRESPRGVCGQRLPEVLSMVCKRYGGYRDTWFRKR NGEGTNSRLGNIILGKRDAFSYLGKRGQSYGEQGITCECCYHSCSFRELRQYCRNSQQRISIKK* Lottia Protein Model : jgi_Lotgi1_179503_fgenesh2_pm.C_sca_71000002 NR Annotation: Wnt7 [Eupry mna scolopes] NR E value: 1.00E 122 Sequence: MTVSPTVVWAAFSMALSSVVALGANIICNKIPGMAPKQRAICRSRPDAIVSIGEGAKLGLTECQYQFRFMR WNCSTLDSNDSMFGYESLGGTKEAAFIYAMTSAGVSYAITQSCGLGSLPNCGCDKDKGDGKLAPQGWKW GGCSADIKHGLRLARKFMDAREIAQNARSLMNLHNNRAGRKAVKDNMGTDCKCHGVSGSCTMKTCWTT LP PFRKIGDSLKKRYKKSKIVVPYLGRRARTAVTLILKRAKRPHRKPRRSHLVYLDKSPNYCDFDGKTGSL GTVGRKCNRTTKDTDGCDLMCCGRGYNTHQYTRTWQCNCKFHWCCYVNCNKCSERTEEYTCK* Lottia Protein Model : jgi_Lotgi1_188920_estExt_Genewise1.C_sca_260301 NR Annotation: predicted protein [Nematostella vecte nsis] gb|EDO37057.1| predicted protein [Nematostella vectensis] (growth factor) NR E value: 1.00E 109 Sequence: MFLEVFNISATVLCILFLIGHRKCDTIQNSKHHAELMMDLLQPYKNYHGQRLKKSNRHIRECQEIKYGNVT HSKVKVKPDTNISYPLVEVKHLTKEIGSYYYKNPVTIRYQRVDFPLNTLSVLEPGEPGTCREGHGTRSITSKT ALSGNCIVGVNGGFFNTTTGACLGNIVSNGRLVQDSQGLQNAHFGITEDGYIYVGYLSELDLITQQFKQLLG GVLWLIRDGEKYIDKSKQMECPDVEETGTIETFIEVQSARSAVGHDKDGRVIMVQVDGKTNARGVNLHEF TDILLELGVVNAINLDGGGSTTTVINHTLVNYPSDKCADGTFSCEREISTILCVHEPECQPNDCNQHGHCVL GKCVCDGYWVGSDCSSLSCPFSCSHHGTCTKDGCICDDGWYG INCSTPCKPGYYGSNCAHQCYCLNHGTC NSQTGHCVCSPGFTGKYCESLCEFGYYGDKCNNLCTCDNGCFCHHVTGSCNLS Lottia Protein Model : jgi_Lotgi1_208417_estExt_Genewise1Plus.C_sca_10710 NR Annotation: fasciclin like protein NR E value: 5e 178 Sequence: MSRNLWIFLLILLSIIVIASSRRSRGRWYRGRS GRRSLADRLRDRGTKPSNHRPRQRHGWGRWGSGWGAG QDHRTRVEEELDDLFRRIKDRRYGFNFEDKFWLDFGWNSRNEPKPWWEGPNVCKDEKESIENTSISDTGPI SRHFSFKNQVCDQSETSYKCTTKTGIFMKQKTVTEVYECCPGFTRKDEMFGCPTEFKLDGLVKTARNLGLT EFLRATESVSLTDDLEKGNFTVFAPVNKAFEDEKNILPELETILPKDMPSVVMVSKPLTDLVIADMSNVLLG HLVYD TLKTSGLNDEQLIETASPFDSLLRVNFFSRPEKLITVNCARILSADNQATNGIIHTVDRIIKPVTDSIVD IISQNPNLSYLKTALARADLVKALRGTGQYTLFAPTDAAFEKLEPSIRDRLIKGEKSCLEKVLKNHLLTSVIC

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212 STVIQGTVTTTNLLNKYLNLTRTEDDKLFVEGSQMVGRDIMATNGVIHVIEDVLVPDQALGLIDVAEKNNL TKFVELLKETGMFKGITKMENLTLFIPSNNAFEKLEEKVSLE ELKKDKKKVEDIVMYHVVPEVVTCSRLHN NYKLSTENERNIRMNRYSTFPFRHHSIQTAQCAPIIKENVPACNGVINIVNEVLLPPVGNVVDNLASKKKFST LVRLMKKSGLADMLQEEGPFTMFAPTNEAFDDLYEEDLEKIEDNPEVLKKVLQNHIVQEFVCCASISMVRS WFDVASVRTLSGVRFPVKERHGTIQFNHAVVTECDNTATNGVVHAIDKVTLSPERPFWKRDFYHW* Lottia Protein Model : jgi_Lotgi1_168619_fgenesh2_pg.C_sca_77000042 NR Annotation: crossveinless [Saccoglossus kowalevskii] (a new family of twisted gastrulation like modulators of bmp signaling) NR E value: 1.00E 148 Sequence: MYYTELSILAKCIALCILTCFPILIQAQLTGTVTRCYNEGGEVK IQGITTDPCISCYCMDGYVQCDKKKCDNL DGCHAILFDGPKRCCDTCKGCRLNGVKHESGKSWFDKRDRCTSYSCKAGVITKSKVQCHVPCSNPIKRKR QCCPSCKGCHFDGADRKNGEKFALASDPCVECKCRKGSVTCMKRACPVLNCPDEVIYQPKGECCPKCKG MRKIFDFPGACYFAKRVYVDGFSFEPKSETRCTCTKGTVICEKATCPPVACPVEERVQKGSCQVCEPKRNC LYDGKIHMHRSKWQPRM CTQCSCQNGVTYCQRERCNNSLSCPNGYKLQFQPGECCPKCVEHDAVCRVFG DPHYRTFDGKMYNFQGTCKYMLSQDCQGKDFTIKVKNGVRLSSGFAWTQMVVVLMGDTRISFRQNLLIKI NRRRVQLPYTIPGKFSIRREGHSVTFRADIGLKVVWDGDSFLEVTASRKYKNRLCGLCGNYNGLETDDLIG RKGKNYLRGEEFGNSWRIGSKKACKTQPKVKNLQSICDKDFKAKVRANKECSVLYSRAFSSCR RVVDVTP YVTSCVTDMCDCPHGKKCSCESIQAYAHECKRAGHKVKWEKVSNCKAPQKNCPKGAYYSLCAPACPKTC SSNKPDGSCSKKCSPGCVCQNGAVLYKNRCISPEKCPKT* Lottia Protein Model : jgi_Lotgi1_205842_estExt_fgenesh2_pm.C_sca_70027 NR Annotation: BMP2/4 [Patella vulgata] NR E value: 1.00E 159 Seque nce: MIADFYRSVVLLLLVLVVNYTSSLSTNTLHPRKHGNEKVLAAVESNLLGLFGLKSRPVPGRRTPIPDYMLD LYKRLDSEPDFISPFAESKGKGIVEANTVRSFYHSDINFVKDCDERTCARIWFNISTIPFAEIMAASELRVFKD VYNHFKLTDANSLSGDRIKASLFKHRLEIHEIMRVSPDDSECISRLIDTKIVDTRNSSWESFDVHPAVLKWRR RPQYNYGLEVRIVSKSPWISTNSHVRLRRSAHM DEKNWQIQRPILVTYTHDGRNPNSRIKRASARNRRRKS RKRRRRKKKGNKNECKRHALYVDFGDVGWNDWIVAPTGYNAYFCRGECPFPMGQHLNSTHHAVMQTL VHSVNPSAVPKACCVPSELSAISMLYLDEWDKVVLKNYQDMVVEGCGCR* Predicted molecules found in Nautilus pompilius with unknown function or predicted to be related t o the secretory pathway Lottia Protein Model: jgi_Lotgi1_103075_e_gw1.1.1164.1 NR Annotation: PREDICTED: similar to CG31650 PC, isoform C [Apis mellifera] NR E value: 9.00E 50 Sequence: MNVYYNILILCLGVYCTLGEKEKVPNEPVDQKHTKDGEHNPHYDHEAVIGSKQLEHEFDELPPAEAIQRL E NLVKHHDLNADNIITHDELKKWILASYMSLDEEESDEKFQENDVDGNGNVSWRELVKKEFGYDLEDIEDF KKNTDKQEPDTKEFLLMLDEEEKRFMTADTDNNGNLEKQEFRAYYHPNEYPHMHGVEIERALRTHDKNK DNQVSKAEFIGGLLFLEDREVYLTEEENFNSFDLDGNGKLSQDEVRAWVIQDNNQAAEDEVLHLISQADD NKNNELTLAEILEHYDDFVGSATDYGHKLSDEL* Lottia Protein Model : jgi_Lotgi1_132441_e_gw1.79.137.1 NR Annotation: PREDICTED: similar to CG11081 PA [Nasonia vitripennis] NR E value: 4.00E 72 Sequence:

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213 MAWKIYVLFVCVLCLRFILAARENYIKATFEGAKDRGIFNKLVVNRETNELYLAAVNHLYKLSPNLEVIQE VVTGPHLDDPSCTSLTNLTCDPDLKSCACKLRPYYTKAL VVDYVNKKLITCSTLYHGHCDKLDLDNITNKQ MFPFMPMVANNATASTVMFIAPGPAVVKDEPQRQVLYVAASYTRTGLKAYREQVPAFCSRSLEDFNLLLK NAFGSSSIEIESQHRDTFPVRYIYGFGSDNFSYVLSIQKANVQTDRYVTKISRICQSDDDYYSYAEVGLSCTM QNTEFNLLQAAYVGKSGTKLARSLGIPTTEDVLYTVFSIGDSNSANPSATSAMCVYSLRDIRKKFTENIQEC FSGIGNTGP DHMIQSSKCLKVVSSHFFVIFFLESFESADLSVL* Lottia Protein Model: jgi_Lotgi1_150026_fgenesh2_kg.C_sca_22000005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO30774.1| predicted protein [Nematostella vectensis] NR E value: 3.00E 25 Sequence: MLVYL LLAALLTDFSSAGVFYKDCGSVDGTISNIEVSSCASEPCQFQKNTNISVTIDFTSKTVVSSATTVVHG IIAGIPVPFTSVQPDACKDMSCPLASGTKYTYKNQVAVLPIYPTIQLVVQWEVKDQDGKDLLCFKIPVRITN* Lottia Protein Model : jgi_Lotgi1_151060_fgenesh2_kg.C_sca_80000004 NR Annotation: LOC431836 protein [Xenopus laev is] NR E value: 1.00E 140 Sequence: MDFARLFQSLCICLLTLDIQLFGLTSGANPEVEQHLEMGKKLLAAGQLADALSHYHAAVEGDPDNYMTYF KRATVFLALGKSRSALPDLAKVVELRPDFTAARIQRGSVLLKQGKYKEAKNDFETVLKTSPNNEEAKRNID KIETIPPDIELAYRLVEEGNYGDAIQVLGGIIDTCPWDPTFHEIRSECYEAQGDLFKAASDIRQSTKLRADNTR GYFKL SSLHYEMGEADESLVQIRECLKLDPDNKQCFPHYKKVKKLVKLMTSAQESINNEEYDKCVKKAEE MLKVESLVHHYVSRAGQHKCKCYSKGGKVKEALAECTHLLKIDPNNLDALIDRAEAHIFNEDYEAAINDY QEASNIDGENRRVKEGLNKANKLLKQSKKRDYYKILGVKRNAKKKAIMKAYRKLAVLWHPDKFTTDEEK EKAQKKFLDIAAAKEVLTDPEKRAQFDNGEDPLDPEEQSGHGGNPFQQGFNP FGQGFSGGSFKFHFN* Lottia Protein Model: jgi_Lotgi1_118302_e_gw1.28.365.1 NR Annotation: No match found NR E value: Sequence: CVMLYMLCKCSSLDCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHCVKLYVLCECSICHC VKLYCSICHCVKLYVICECSICHCVKLYMFCECNICHCVKLYVLCEV* Lottia Pr otein Model: jgi_Lotgi1_154212_fgenesh2_pg.C_sca_5000252 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E Value: 2.00E 97 Sequence: MNLKLYILICVTVSGILGTPLDDYVNRPDSTYKYFEITHYVHEDHTLYILNMTSQTWLTAEISTQPIWWHYL TVTIPHKIRYPDAAFMFIEGGSNHNGPPTGTDNFV ALTTAMAVSTGTIGADLKMIPNQPIMFKADPTKKQRT EDAIIAWTWKTFVETNGSNPDLLLRMPMTKASVRALDTIADFAMDKVGNKVDKFFIAGASKRGWTTWTT AAVDNRVVAMAPIVMDLLHMVKNLHHHYRAYGGWTFAFKDYYSLNFTQDLDSPWTQAMADIIDPISYN DRYANIPKLVVSTGGDEFFLPDDSHFYFKELMGPKYLRILPNAEHSCAGHEISLLFTMRAFYLSVITKTPLPQ LTWVREETP SGGRITVTCNQKPKTVNVFYARTLDGNRRDFRLLVGTPGDPSKPMPHPVVWLRDDVQDMG NNVYRAELTKPEIGWTAFFIQLTFQGFADSDLEFTTETQIIPDIFPFPDCTGQECKGTLV* Lottia Protein Model : jgi_Lotgi1_159188_fgenesh2_pg.C_sca_19000225 NR Annotation: predicted protein [ Nematostella vectensis] gb|EDO4 4886.1| predicted protein [Nematostella vectensis] NR E value: 2e 07 Sequence: MRGPTWSVILLLINLIVTLQACDDVTEFQCADNNQCISKTDTCNTINDCDDGSDENNCACLEKLGMEDGTI

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214 LDSSLIASSSKTGSSPSYARASNDKGNWCPKSDDSKPFFKVKFDTPRAISAIVFTLKQSVVDPLNYLSTWTLK AVLPGSDEEVTLYEGLNAFSNNSLYRSLFFPSL VTDTFIIAPTTWQNEPCLSFELYGCNIDNLCPTGCQNNGK CSGNDICACADGYFGTKCQFQKTTIPFSSYIFKSLESGSLVTSSETFTVRTSTTIRTSKIIEIQGRKVLSLPVAD YFTAPKDGKFGCLSKLGSGGCKSFYLALGCELEELKDTVQLFTTGSDSTYIGAEATLKSDNLVVTFRTSELI WTLNVRKTNMFSSSIKYVLEFDWDISTGIRIYVNKKLIGQQRQATRTTQVLRTNTLATSGVEFFKSKSVNVK LFSLKSSSVNRISLIKTSLISLTPTEECDQEKEHKCANSDKCIDKSNVCDSVEDCDDGSDEQNCPTTTCDVKV YKKLVTKSASLSLDLLEVDDAQRVAKCTLPVISYVVDGILLDFLSKNQYGDQLSATNDLGANQNHNNNNK DDNNNNKKSSQDEFKEDVFNWADEPWTPCDDVEVPDWARMDDPTFDNFNSELIWAEDPWTQLPLVGLPS WMLLPGIDYPVEAKDPVVYTPVNDEDDAAFLASGIDIVNWS DEPYFQLPLDVPRWALLPGIDYPVRHGYN SPYWTYKLNQYNSSRREFGKKMFARQFPTLREERVLYNNNGQSKLCIPGNFQRRNHRF* Lottia Protein Model: jgi_Lotgi1_160458_fgenesh2_pg.C_sca_25000063 NR Annotation: predicted protein [Monosiga brevicollis MX1] NR E Value: 6.00E 86 Ac Annotation: no hit Sequence: MNYKMILNKYTLVLGIILVCTQPCITDVNITKVHLIFMNHLDVGFNGITPQTGFVINVINKYFTKYFPGAIEL ALQMNFLGYRERLIYTTHPWLVSLYLDCPQNLTMSGIQLMCPTEEQREGFLYAAKRGDITWHAGPMNMQ YEALDTAMVNFSLKLSEDMDARMDIKRRHRVLSQRDVPGMTRALIPIFNNHGIEGVTVGVNSVSSPPAVPK IFKWVFQNSSVIAMWHPGGYPIKPGSFP SMPLGLSRDNCVTFEQFDEAMCFSFRGDNQGPPTSIAEVLNSYE IVRGQFPGAIVEGSAFENFVEVVQSVKDKLPVVDQEIGDTWIQGISSDPGKTAMMRGFFRARTACLTSAPNF EDVISSWSEQRQFIDLALETLGDHPLVQAVEEEYKQLVPQAPDLSKYKQLSATTFDCQNGFKFSFAADGSL NKLQDKDGRNWASKSQPIGQLLYKTYNDTDFDSFQKQYVLNHYYWFQVGKWNLTANCPTCESTIWNTK L QKLYAASDGSCDVVALLAMEEDRATSFYGAPPKIYVKYLSSSNTFLSIEVQLFGKKPTRLAESLSLSFQPIR QEGSKWYLHKLGQLIDPLNVVTNGSQRLHAVDEGVQYKDSNGKGMMIGSKDVGLATVHQTPTDVSVLPV PLTPIQSISGVSYNIFNNVWDCNYIFWYPFLKQDSNSKYRFSITFS* Lottia Protein Model : jgi_Lotgi1_160696_fgenesh2_pg.C_sca_25000301 NR Annotation: No match found NR E value: Sequence: MKLLLLLTVLIPLCVSVPTKPGCCVPPVWEGIQGMFSGDGSGPAPELIQQSALISYDEKRQMININITQSIHGL TSDVRILTDYKKGVQYLINSKSRSCTTSKVPQTFQRFCIPDYFYYKGSFNVAFTLRASTEQFIVPLIWSKCKS NSSNFFKMRKACFSRDIQLVVGTCDCLFN* Lottia Protein Model: j gi_Lotgi1_161657_fgenesh2_pg.C_sca_30000077 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO44809.1| predicted protein [Nematostella vectensis] NR E Value: 3.00E 17 Sequence: MTKLSEYFLICSMIFVVFMLQRGTCDDVVTEEQISYAKGSVCGYCSYCKFCKLCDKDCPCETSPS KPNCKM CKYCKYCYICSGVCDTICQPGGMIDKISAAIVNALPSFNKEEIEDDLESVKPWLDKKDEL* Lottia Protein Model : jgi_Lotgi1_161950_fgenesh2_pg.C_sca_31000192 NR Annotation: RE49668p, putative [Brugia malayi] NR E value: 3.00E 08 Sequence: MKLGGDYGILCVLFTILTPVISIDCYACTSLGGNNEGCE DSFERTLKTVHFIARDCYYGYFRGTHCYKLKGE RADGSSIMVRHCSDSHWGSHCGDIKYTNEHNVEEKIKGCLETCDHDGCNTATQKQFNFSLVIMAVLMTSA LLL* Lottia Protein Model : jgi_Lotgi1_163300_fgenesh2_pg.C_sca_39000034 NR Annotation: PREDICTED: similar to TTC17 protein, partial [Bos taurus]

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215 NR E va lue: 1.00E 133 Sequence: MASSVVVIVLLHFLFEQCLLTRGSMHWVVTEDGRIQSQADSVYNLRRPYDLVAFMHQEDRATVLNHLKK ELLNRKGEIDKSEDRDTTGLEQKFYKSDYDCLQAGTPLPEIDLYISTILPIEHKHISDELSIEIQKAAQSSDNKI GTIPPEPDCTKVLQLDFTPHGFEHLEGVQERMNLTGSAELGLTHAIPFIPDKTEIPDFGHVIAKELKKNSTSW VLYNMAAFYWRVKG DPWMTVECLRRALHFSPRENKDIALISLGNILHRARYSNEAAIVVHSALELSKELN VNHFTLGNIYAVLGEYNKSIICYENTLRIQPDFEAATIRKHAVLCHQKLEAALEAQHRSLQKTLRDLKDYQ KKHDLWHIQNDRMSVEQVSQEEKVSQNIAFEFNKAKQTSTKDIGEYCNMVEREGKQVLLCTWNRQAPTL EMLDQFALEEQKKESERNRLKLNKYEKKAIDYNLPVRAPLYVKHDRQAVPRFNELELDNT WPEKEECDSH LLTSPDPFNLSTVYLSPENKGFEVKALLTEAQNLKDGAEHPLPWYPPICVTLLTINEYDEKTYDNLKSVGHP GRTKVPLKMYDPSMRKTLLSHVNGGTVTEEEVGQRILSAIKQEVGARWVLFNLAGLYWRIVGNNYHGIEC IRRSLALAPDEYADVPLVNMANILYKWGRYDDAIVLMKDALKINDAEPDSNFLMANLLWVTKNYSGAISH YRTVLDIIPEHQEAFDSLRAIKCLQKFLIKEQSV APIEVPSQPVGNSCQQKGVANKNQQTESRVICKTENGEE KCIIETRMRGKSGECNGHCTQTCTITPIKVESSCGGPDLAVDTSNPVQCHQKGSQCGEAGEELLFTNDFTNK LDEVSDHYEKKGICNGEDCNTLRVQCLIPMKTHSGLVAHVITPPKLFVRPLSLHSTQCMTEQKKPHIKLEFI DGVLHQKLIFVQDANDIHVEDNECIIFNDGIKSPGCDQAQYRSYSEELARSNINVEFRYVGDESPVEKDKKN H CSKTPNSPGVPSDEIPYSDISDGILQSLNGDVEPSEVVGHRIALTLQQNPKSWVAASAAGLYWRVEGDFTK ALDCYRLSLSYAPLEMADLMLLGYANILVQGGYYSNAIMVEDLALDIQSTLPIHYFTLGNIYAALGKWRTA VSFYEATRFYQEDFKPAVDRLRAIHCENIA* Lottia Protein Model : jgi_Lotgi1_165259_fgenesh2_pg.C_sca_50000100 NR Annotation: hypothetical protein CHLREDRAFT_144736 [Chlamydomonas reinhardtii] gb|EDP05543.1| predicted protein [Chlamydomonas reinhardtii] NR E value: 6.00E 23 Sequence: MKVLLVLDICCLIGLVSVGEAVCPDNCNYNGVCDVKHSRCNCYEGFTGSDCKTDCRCNGHGTCQSGSVC KCDEGWKYSGGQCVWDCHCLNGAKCIG PGECGCVHNCKMGNCRNGQCQCWNGYKGSDCSEYDPTM* Lottia Protein Model: jgi_Lotgi1_166436_fgenesh2_pg.C_sca_58000065 NR Annotation: PREDICTED: similar to MEGF6 [Strongylocentrotus purpuratus] ref|XP_001187830.1| PREDICTED: similar to M EGF6 [Strongylocentrotus purp uratus] NR E value: 1.00E 26 Sequence: MMKILEILLCLSFFRATVQQFDCSVCKVCDNSTCIECIEGRYGISCGSFCSLKCSGRCRQSDGYCTSGCDEGY WGPGGLCDQTCSNCNPGGCVMNSGLCIAIGCRTGWYGTTCNNKCNSNCDGNCNQSNGNCNQCIPGKYGT DCQLDCSNCKDTCNKNNGVCNNGCKEGLWGTSCTKTCPNCQSSGCDIDNGNCNNPGCRIGWYGDSCNDK CNERCN DGCKQDTGYCNKCNKGVYGNVCDKNCNKGCLDGCNIDGSCKCKSTYYGTNCSKQCSSNCVDS LCNEISVGYNSCIRGCVAGYKGLYCDKQCPSSCPICNRFDGSCTECSKGFYGTECQLKCPEQCDKCKIKEGT CESCNPKYHGPVCSKRCSDKCLNGECNGVGGRCIDCIPGTFGDNCERSCSNGCIGDTSSTTCDRNGSCISGCI NRIYGEKCNLNCSKNCYGSCNRSNGYCNSCLSGYFSDNCNQKCSIHCNG DCDRSTGNCGKCNEGYFGDNC NQNCSINCNGGCQKSSGSCLECNAGYYGDYCKQQCNDKCINNECLRNGQCRFGCMNGKTGSQCDGNCNS NCTICLQTDGNICKECKPGLYGISCDKLCNNTCKKVNSKPACNITTGKCTTGCIDGYKGYYCQDTCLDNCIS CNNNQCTSCNTGWYGNQCQTKCNSNCINRCDQATGDCLDGCIDGKSGKHCKQEIG* Lottia Protein Model : jgi_Lotgi1_168 805_fgenesh2_pg.C_sca_79000073 NR Annotation: PREDICTED: similar to plexin A CG11081 PA, isoform A [Apis mellifera] (a neuronal semaphorin receptor that controls axon guidance) NR E value: 1.00E 121 Sequence: MEGGIRLQLFLLVSLICHTQCIKILHSFRDPGGNILQKLVIDDQTGN VFLGALNRLHKLDPDLNIVQSASTGP RLDNVECPPPLLPCDKPKTQLNSQIKGLVIDSASNSLILCSSLFHGSCQTLALNNITNVKKFFHKPLVPNDHH SCYMFLAPSINNTQALYIATAYSDLGDAAYRDLVPSISSRSLDNLDFIHRDTEGSTKLEILKEYRESFIVRQLY

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216 GFQNGGFVYFITLQRENPGSQKIVSRINRLCQQDRYFRSFVEIPIKCQKSDSDSYDVVQNAYFTNGKLYVIFS NTVSSAGAKK STFCEYDIQDIDLEFNETVRNCYKAVGNFGPEHYKKIEPCPPLIFGSVDFCGESGEWKDYSA IEGSKPISATPLHFFTSDKPTAIFVHPEGNHRYAYIGTQDGHVIKLRFENGQVNQLFKITHSVGDIILQITNHPQ SNLLYILGSHKLSVLSLNHCDDLTSCDECMNDGDETCGWCVMDNRCTTKELCQSSVITPPWLSRTDQSCAS ISSLQPSSLSYKKFINGGDSRQIKFQVNKLKFESNRNLNCLFINGEKHDNTMAILDQKENTITCPLPRQLPTIP QGKDHEDLELQFHVEGKMIVKRDVSVFDCQGNNNCTSCSTSQFNCHWCPVSHSCVEQSESCPQGDSIKVN DRCPRLETAASDTDILVHSGELKTISVQVRALEANQRSNLKCHFDLSGQVQTVSAKISSSTLTCETIKLAYA DDIPYIVAGFKVTWGQQNHPLDNPQRMNVRIYKCASMVTNCGKCLSLDVEYECGWCEDQCSLQKKCQKS WLDQSETCPNPQILRYSPS KGPVKGKTEIQVNGINLGKEISDILGQVTVAGLPCDVMQEHYEPSSSFKCEVK GVTSAKSGTVKVVVNNKYTAESDTVFEFVDPVLNHVDPKQGLMSGGTRVLIKGNNLDVGSDTQVIVGGN SVPVIK* Lottia Protein Model : jgi_Lotgi1_171167_fgenesh2_pg.C_sca_110000033 NR Annotation: No match found NR E value: Sequence: MAIS HDWSFTLLWLIWIFTLLFSDSYGKRTQNYYMEGLDCGDSKHIGGATVYSNFRGDVSTVYGNDIECQ MTFKAENKDWRLMLRIIELDIPDRTSTGLCNDALYVYDESSIYARAMEEANGNTGLCGNILPPTLYSTGQY LTVHFSVSLQNQKDIV* Lottia Protein Model : jgi_Lotgi1_171718_fgenesh2_pg.C_sca_119000006 NR Annotation: PREDICTED: simila r to orphan G protein coupled receptor HG38 [Gallus gallus] NR E value: 1.00E 15 Sequence: MAPIYLTFIASLLLASSTFCEQLCLEGCKCEDHPSEKLGPHLMIDCSNLGSSSVPRDLNKFQNYGRPMHLKL SKNIIARVSKADFPENLPIIALDFNQNRGVMITDDAFMNIKGLRYLGLNDISMPFTDTMKYLKGMTKLRYLY LNKNYQFGEGLVPVSLFSGLE LKLKDLSLHDCYLKGIENGALANVVITGSLDIGANQLDHIPEEVKTLKNLT KLDVSQNSIKSIPSNTFEGMSKLRTLSLSNNYINGQSFKTDSFNGLKLKELRLEKSELNTIPSAALSSLSSLKIL KLNNSPIKTIPKHSFNGSYCLEVLDLTNVLVEIENTHLDGLESCLQKLYLNNMQIREIPKFFKDFKVLNYVSL SGNSISEIKMDDLVGTTITELDLTENPLHRIEKGSFEEFPHAISLIISDTRLSNLSF VRDYEYGTIKTIKLGNVLP MCDCELAFAEYRVLEIHGNCNFNNTKNVAISTDEFMSVTSHCWPDGKMPQKKDVWAVVGDNSAGTFSTS ILSYFCVFLFFKVLNIV* Lottia Protein Model : jgi_Lotgi1_172278_fgenesh2_pg.C_sca_130000017 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO35011.1| predict ed protein [Nematostella vectensis] NR E value: 2.00E 26 Sequence: MGVKLLFLGICLIYLVLDLHDAEVTAVSDRQAKLKEKDAREKLQQELSKNYAVKQRRRKKKKFRDPRTR GKSSKVVNRGGAPKVQNGDDGGDDPHIGNRENYLQKYFKGKMWGIRDKKKAPKGNWRKKLRKQKHKK KEKRQNMKKKEKSKRTKVKKFKKKRKKHKKRRRGKRSTDENEDDPGSDQEHTHRTKRYAITDQERMWD YGVVPYSFSSDVTDVMARTYRGVFDALEKRTCISFVEWDGTESFYSNRSLNHMVHLNFVDQGGCWAYLG RYTYRTTNIAQDISCCYVGRTCLHEIMHSLSMEHEHTSPLRDGWLRVNWDNLNEDGQFQFSVIDPLMLGRE SWGFDIKSFVSYGTTEFTKNGMMGFQPLMPDFYPTGNSYWQPFKEVQMNYDCFGIKCSGMEEPVCENDG YPGYYEGECGCVCPPGLDPATNCASIKPTDPI RGWPPSSFGLPEPIEGCPAGFQLGSLTQPLAGDSEVSSSSHI NAGFENSTAEFGFCLKDTASQTEGGLTEWPRGQYCIHSYSNACPPGFQEGLITYDDSVNATRTILTESPLPD GSFVNNTELWYCCRSDGMVQLDIKLPNTESFILYRHGTAECQNVEGMYMFPEYYSLMIEGETIRSISVYQTL GSVPYNVLDGNELFVYYCYYSPLNKDCGEVIHLGPENPTHDIMTPNYPFDYPLNTECNWLFVAPEGSEVM L NFNDFDVEGDDFDCFDYVTIKRGLPGIPGVSYCGTEFKAAIKSIDRYLMITFTSGSEITYTGFHATGRLLDVA DLCYNPDDFGRTYNGIHNYAEDFTECLPWVEMAHCDTNLFNTEDAFDNLDGNYCRNPGARYDSPWCYTE KENCTIKYCDVCQERTIYDNFDDCEELIAEHPNFCLDGDHHHYGCLATCIEYGHITIPEKSNRAQDVSCPVP DDIVDADPIVHDVTRYNVGDSLAYVCSNGTATKMRRCLTDGTWTDIEYVCGGCGDEMKSDTRGTCYAYV AEEVRFVEAEEYCIEKDWDVASAQTEDAFNFLVQYRSDMGSDGKQVWLDLYEDPVGVWSWGDESLVQD

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217 GFTNWRSTYPVSGPIETDGNNWYRCATMVGTGQSSVWNQVACDNKWYKAHYICQTMESEREICADRKSS CTRNLIDIPLLCSQYQSYAHEVCPYTCGICDVENADSCTVTADFVVGLNLVSGGSTPFSLNPGESFTTQCPA GQICTQNCQHFSRACRRSG EVTGATPVCQDADSVPTLVNNIERIPRLDTSEARRIYQGDNSYMNITRSGEITK WMTLCENDGIVHLMAFKYWSQDRYFKVMGVNQVECKAGRRMTWEIPEGERIQVDPSMNIGLMDWNGG CIPLYECFASEYPDLNKIYHTMAESQDEVALNKLVWMRAGPTCYFFSLNAEISPVGYIAPATTQLTTPSTTT EEPEPEPEATSTSSEDTSESTDMTTSSGDVSTSSTSTSAGNTESSSTGASRSSPSTDAFVSED TTSSVWQRTTT HSVVITTPRRTINEDFIKSFIKKKKKKRKWWKNRGKKTDKTRKRKNKRRKKAQKGR* Lottia Protein Model : jgi_Lotgi1_172798_fgenesh2_pg.C_sca_142000011 NR Annotation: gb|AAA30047.1| calcium binding protein [Strongylocentrotus purpuratus] NR E value: 7.00E 07 Sequence: MRADSIQVRGLKALVLILLVLYEGHTKETCRTGVKTDKNLRGVNLDKTSGLPFYWFDLHRASDGYHSEYT YTFRLHSSSEASPFSDAVLQVETANNCGTGDLVVNTTFLYQPRNCPKMIMTEYKKPMTNVPFKWRAPKCG CIKFRATVIRNGVTYFYDDENISDGNLVKTVCPHGVRVAEQLVEVDEKTTDSTTKTLSTVTSKMDRAALLD LLCKITSTLNNLELLKRPEFIQRRKLQNSSLVKLLNIELALEQRK ADISSCCHKKGEDKTICFDNVRRYRIDQ LCGDGQPRIPFVEQKRNYMRKRESECCWRIGQPRYQCFSKSSEQLTEYSVDYSLDESDPANDIADYPKEVE RYEMASNTKFFSPPPTLKPDNQNIETLTKLEETTTPTSVTKSVTLPPIRQPSARPHRQTTPTSVLTHHRQRPVP AVKTKLRQSRTVGQGDMGYNLRKQAKYTRKTTNLRHSASSVEITVNTLKQKLWKSSLKLECCQQGRIFAA KTGRTDVWDRCRS AAKDFRSSVVRGKKICSNTFQKCCVEQFITATAKASTIPKPNTPKGSPSTKQLVTTTSL RTNIKKENYRRAPLDVDSSREADEKYASMESKAEDDLMEQDDKASAEDAGEHVDDDGDDVSDDSDNDD QGEDGDQDEDDDQDDDDKDGDVDDDKVDEINNDEKNRKRKFIDYPREDPSADTFHNLQTKRKSTPNTDI AHRDSNHRNLKIESAKRKRNRRRHNRKLRRRKHKSD* Lottia Protein Model : jgi_Lotgi1_173484_fgenesh2_pg.C_sca_161000006 NR Annotation: No match found NR E value: Sequence: MRLHYLVTVLVVLILIDVGEGQDNISKCLELPSVKEIVTRYICPKPDPCATVDCNCNGKCVDGKCQCKNGF FGEFCTALTSLIPKVAGFVSNNKSLISSEAQTIGSIAKAGVSKKSKELKELQQIRKLRNRRLEEKKGKDFKDI QKSLGSLLGFP WAKYPGEKHLQDHNYTGPGTRLDLRLDENDKPKPGGNETFTFDFISIMLGKKQFE* Lottia Protein Model : jgi_Lotgi1_176704_fgenesh2_pg.C_sca_2718000001 NR Annotation: PREDICTED: similar to rCG46800 [Nasonia vitripennis] NR E value: 6.00E 09 Sequence: MLTIRVLGVFAVIVLAFVLVESDVELCEDD VNVCHGELPANAMCSGENNKTKRCQCKKGYKMSLDIDCTS NGSVFSCQDIDECATNNGSCEYKCQNEPGSYNCSCPEGSELQGNGLNCGPSGP Lottia Protein Model : jgi_Lotgi1_192261_estExt_Genewise1.C_sca_450209 NR Annotation: conserved hypothetical protein [Aedes aegypti] gb|EAT46740.1| conserved hypo thetical protein [Aedes aegypti] NR E value: 1.00E 128 Sequence: MTLYITSLVFFLFINGTLQVKNRSSKVADRCFCKLNGEIDDCSCKVETLSVLNNNKIYPRIKSLLSRNYFRYF KVNLKKKCPFWHDDSRCALKDCHVDTCKEEEIPSAIKKGSSAYRYSEEAQKEESCAEEKELSALNTTISDES IQAFKDWKEHDESQDLFCDIDDESSADSEYVDLLLNPERYTGYKGE SPHRIWRSIYQENCFKPQTEYIYGPS KSSSPLCLEKRAFHRLISGLHTSINIHLCADYLYQENLGYGMKAHWKPNVAEFQKRFDPQTSNGEGPQRLK NLFFTYLVELRAIAKAAPYLQEEDFFTADSDEDKDVRQGIDDLLNIISTFPDHFDESKLFAGNPNEAKLLKDE FRNHFRNVSRIMDCVGCDKCKLWGKLQTQGMGTALKILFSGDDIGPDSTVNAQQKKHFQLTRSEIVTLFN AFGRLSKSIHSLEMFK SLLSKS*

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218 Lottia Protein Model : jgi_Lotgi1_197084_estExt_Genewise1.C_sca_820083 NR Annotation: glucose regulated protein 94 [Crassostre NR E value: 0 Sequence: MKRFIVLGILGFLFIAGAGLLNVQAEEVVTEPDVVEDIGKSREASRTDDEAVQREEEAIKLDGLNVAQMKE LREKAEKFAFEAEVNRMMKLIINSLY KNKEIFLRELISNASDALDKIRFVSLTDKSAMAATDELSIKIKADKA SHVIQITDTGIGMTKDDLVKNLGTIAKSGTSDFLSKLGESKDQAQMSDLIGQFGVGFYSAFLVADRIVVTSK HNDDDQYVWESDSSSFSVAKDPRGNTLGRGTMITLHLKEEAHDYLEESTIRSLVKKYSQFINFDIFLWASKT EEVEEPIEEEEEEKKEEEKKEDEDAEVEEEKEEKPKTKKVSKTIWDWELINNVKPIWTRKPSEIT DDEYNDF YKSISSDYEKPMTKIHFTAEGEVTFKSILYVPNSSPSDTFQNYGKQMEQIKMYVRRVFITDDFEDMMPKYLS FIRGVVDSDDLPLNVSRETLQQHKLLKVIKKKLVRKTLDMIKKIDKDTYEKFWKEFSTNIKLGVIEDTTNRT RLAKLLRFSSSNSDTELTSLAEYLERMKEKQEHIYFVAGHNRAEVEKSPFVERLLKKGYEVLYLTEPVDEY CIQSLPEFESKKFQNVAKEGLKLDDSEKAKERKE ATEKEFEPLITWLKDSALNDKIEKATISERLASSPCALV ASQYGWSGNMERIMQAQAYAKAKDPSQEFYATQKKTLEINPRHPLIKELKKRVEANKEDETAKDLAVVM FETATVRSGYRITDTAGFAQRVENMLRLSLEVPLDAKIEEEPEEEPEEAEEEKTEEVEAEAEEEAEEKKSEHE EL* Lottia Protein Model: jgi_Lotgi1_202874_estExt_Genewise1.C_sca_2176000 1 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 2.00E 54 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADAETILTVEWYNQHGCGGNEDDNPQ KQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTR Lottia Protein Model: jgi_Lotgi1_203670_estExt_fgenesh2_kg.C_sca_170029 NR Annotation: PREDICTED: hypothetical protein [Ornithorhynchus anatinus] NR E va lue: 1.00E 142 Sequence: MKLSVFLSLLFLTAVWADTPANCTFDDIKGTWTFFIGSNGHDNTVNCSNMGNPKSQLQVTLYFPDVAVDE FGNKGFWTIIYNQGFEVVISGRKYFAFSMYEQSGSTVSSICDKTLPGWSHDTFNRDWACYYGKKITSVPPK YYKKNKPHRLFKLNYDYINNINKQQKLWTATVYPEYEGKPMRELLNKAGGPASQVFGPIKAAPVTWEQQ RLSETIPEQFDWRNVNDNNYV SPIRNQESCGSCYAFSSMAMNEARVRIMTNNTLQPVFSPQDIVECSEYSQ GCAGGFPYLIGGKYAEDFGLVEEKCNPYKGQDHSCSTDKTCKRQYATSYRYVGGFYGGCNDALMKMEIY RHGPVVVGFNVTDDFFHYKSGIFIHTGLTDRYNPFEVTNHAVLVVGYGTEKGVKYWIVKNSWGEKWGES GYFRIRRGTDELGIESMAVSSTPIF* Lottia Protein Model : jgi_Lotgi1_203811_ estExt_fgenesh2_kg.C_sca_240005 NR Annotation: predicted protein [Nematostella vectensis] gb|EDO37140.1| predicted protein [Nematostella vectensis] NR E value: 0 Sequence: MMKLFVILALLAGQSLADIYLHNPRGSNDRLNEKSAQRANANRLFDSQNNNRGGYNVGDVTSAPHGKDA SKQYKMAYFQSEADA ETILTVEWYNQHGCGGNEDDNPQKQNCRLVLQYMCQPEGTTEDVLRNGVVTNT QDYNRPPNSNYNLANRNSRKNNNVKADRGLQESWDWYEECFVRERNKGLFTADQNLRGNNGLGYSSAI YTRQNPNGNRRGYECPEERDYFPYWHPTQWKDIAILAENETLCNPLIAESFNNKPYGACLQKFPNSQVRKP SRYNNKEECEENGLDWVAFHNYLEKDKTASTQAACQAKSTNNIEYTWAIPYDSTTFTAECLVKV PSPQCQ EAPWSRSNHLGNGIGGEMNTVQWTLPHFPSGQVQNCVMRMRYNISTDDYDPFNTDSEFNGADNSPVTNN PYVDIGVGRGPLRLAINTAQFGRVFQDRTHIFQLKPRPDDIKDFRIFNVNVRGKRGNIVQVYPAVEYDFTPT DLEVTENDLVHFQWTGSNTHNNGRPGGDGQTGDAGEGKSGSDRHNILQMADRNENFPLPFEKTNMWDN

PAGE 219

219 AEIKWIYHGKNDVSARDLALNMASSGYYKCVSATDCPAESF KDFLVDTKDKMQVTLDNTPASYPGVVLK FKQGKYHYMCTRNNNFTNRSQKATITVAA* Lottia Protein Model : jgi_Lotgi1_204607_estExt_fgenesh2_kg.C_sca_690020 NR Annotation: ctg4a [Aedes aegypti] gb|EAT44181.1| ctg4a [Aedes aegypti] NR E value: 2.00E 45 Sequence: MAFMFFQTLLIIVSIGYLH ADDSDGVQEATLCEVCRILAEELQLRLDETGKSKEVIETGHGLDTKKKKKYNF SELRLIEALQEPNICDRILEYSVHKEKTGIERFAKGRSQTMEALHGLVNKGVKVELGMPHEMWDKPSVEIT QMQRKCYALVEEYDEDIEDWYYNHQDQPFLDYFCRNLVLNPQNNECLDEDMRSQNEGSSSEMKGDKGK KEEL* Lottia Protein Model : jgi_Lotgi1_204770_estExt_fgenesh2_k g.C_sca_820009 NR Annotation: unnamed protein product [Tetraodon nigroviridis] NR E value: 3.00E 35 Sequence: MAATTHKMFLVACLVFLPIFASADTCLGPSVSAETYTTSEVTVSTETVFIAQFTLTCKNGLQNVNLYAEVA GRMIPAVKTSKPNEYQISITDEYKALRSGTYEAKFYDEESFSALKKAKRNNEDSNSVTPLFSINISHSGLWQG SY IQSEFVAACVAILVWWLAFSAKSKLSP* Lottia Protein Model : jgi_Lotgi1_98330_gw1.10.599.1 NR Annotation: hypothetical protein NEMVEDRAFT_v1g147894 [Nematostella vectensis] gb|EDO28414.1| predicted protein [Nematostella vectensis] NR E value: 5.00E 06 Sequence: TVC SLGTHCLSITVCSLGTHCLSITVCSLGTHCLSITVFCSLGTHCLSITVCSLGTHCLSITVCSLGTH CLSITVCSLGTHC

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220 APPENDIX A MODERN VIEW OF ANIMAL PHYLOGENY A. A conservative consensus of the current state of all animal phy logeny. Phyla for which a large amount of data (genome or EST pr ojects) is available are ind icated in red. The dashed lines indicate controversial relationships. [ Image taken from Philippe, H., and Telford, M.J. (2006). Large scale sequencing and the new animal phylogeny. Trends Ecol Evol 21, 614620.]

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221 B. Recent work h as improved the resolution of the animal tree of life by sampling from 29 animals belonging to 21 phyla. Phylogeny is based on collected ESTs and a maximum likelihood analysis. [Image taken from Dunn, C.W., Hejnol, A., Matus, D.Q., Pang, K., Browne, W.E. Smith, S.A., Seaver, E., Rouse, G.W., Obst, M., Edgecombe, G.D ., et al (2008). Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452, 745749.]

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222 APPENDIX B MOLLUSCAN CLASSES The t opology of the molluscan phylogeny is still highly debated. There are two pr evailing models, A) Aculiferan and B) T estarian models. Recent work (Sigwart and Sutton, 2007) incorporating both Paleozoic and extant molluscs supports the monophylyl of Aculifera. [Image taken from Sigwart, J.D., and Sutton, M.D. (2007). Deep molluscan phylogeny: synthesis of palaeontological and neontological data. Proc Biol Sci 274, 24132419.] A B

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229 BIOGRAPHICAL SKETCH Jinnie was born in Tampa, Florida in 1984. She g raduated from Land O Lakes Hi gh School in 2002. From there she went to the University of Central Florida and graduated with a Bachelor of Science degree in micro and m olecu lar b iology in 2006. Following graduation Jinnie be gan her graduate work at the University of Florida with a focus in neuroscience from a bioinformatic and molecular biology perspective. She completed a Master of Science in medical scienes in 2009.