Group Title: BMC Genomics
Title: Molecular cloning and characterization of the mouse Acdp gene family
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Title: Molecular cloning and characterization of the mouse Acdp gene family
Physical Description: Book
Language: English
Creator: Wang, Cong-Yi
Yang, Ping
Shi, Jing-Da
Purohit, Sharad
Guo, Dehuang
An, Haiqian
Gu, Jian-Guo
Ling, Jennifer
Dong, Zheng
She, Jin-Xiong
Publisher: BMC Genomics
Publication Date: 2004
 Notes
Abstract: BACKGROUND:We have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) in humans. To facilitate the functional study of this novel gene family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene family.RESULTS:The four Acdp genes (Acdp1, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino acids, respectively. The mouse Acdp genes showed very strong homologies (>90%) in both nucleotide and amino acid sequences to their human counterparts. In addition, both nucleotide and amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many different taxonomic species. Particularly, Acdp proteins showed very strong AA homologies to the bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux. The Acdp genes are widely expressed in all tissues tested except for Acdp1, which is only highly expressed in the brain with low levels of expression in kidney and testis. Immunostaining of Acdp1 in hippocampus neurons revealed a predominant localization on the plasma membrane.CONCLUSION:The Acdp genes are evolutionarily conserved in diverse species and ubiquitously expressed throughout development and adult tissues suggesting that Acdp may be an essential gene. Acdp showed strong homology to bacteria CorC protein and predominantly localized on the plasma membrane. These results suggest that Acdp is probably a family of proteins involved in ion transport in mammalian cells
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BMC Genomics .iol



Research article

Molecular cloning and characterization of the mouse Acdp gene
family
Cong-Yi Wang*1, Ping Yang', Jing-Da Shi2, Sharad Purohit', Dehuang Guo1,
Haiqian An', Jian-Guo Gu3, Jennifer Ling3, Zheng Dong4 and Jin-Xiong She'


Address: 1Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, PV6B 108, Augusta, GA 30912, USA,
2Center for Mammalian Genetics, College of Medicine, University of Florida, Gainesville, FL 32610, USA, 3McKnight Brain Institute of the
University of Florida & Division of Neuroscience, Department of Oral Surgery, University of Florida, Gainesville, Florida, 32610, USA and
4Department of Cellular Biology and Anatomy, Medical College of Georgia, 1120 15th Street, CB2917, Augusta, GA 30912, USA
Email: Cong-Yi Wang* cwang@mail.mcg.edu; Ping Yang pyang@mail.mcg.edu; Jing-Da Shi Shi@pathology.ufl.edu;
Sharad Purohit spurohit@mail.mcg.edu; Dehuang Guo dguo@mail.mcg.edu; Haiqian An han@mail.mcg.edu; Jian-
Guo Gu jgu@dental.ufl.edu; Jennifer Ling jling@dental.ufl.edu; Zheng Dong zdong@mail.mcg.edu; Jin-Xiong She jshe@mail.mcg.edu
* Corresponding author


Published: 15 January 2004
BMC Genomics 2004, 5:7


Received: 03 December 2003
Accepted: 15 January 2004


This article is available from: http://www.biomedcentral.com/1471-2164/5/7
2004 Wang et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.



Abstract
Background: We have recently cloned and characterized a novel gene family named ancient
conserved domain protein (ACDP) in humans. To facilitate the functional study of this novel gene
family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene
family.
Results: The four Acdp genes (Acdpl, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684
bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino
acids, respectively. The mouse Acdp genes showed very strong homologies (>90%) in both
nucleotide and amino acid sequences to their human counterparts. In addition, both nucleotide and
amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many
different taxonomic species. Particularly, Acdp proteins showed very strong AA homologies to the
bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and
cobalt efflux. The Acdp genes are widely expressed in all tissues tested except for Acdpl, which is
only highly expressed in the brain with low levels of expression in kidney and testis. Immunostaining
of Acdp I in hippocampus neurons revealed a predominant localization on the plasma membrane.
Conclusion: The Acdp genes are evolutionarily conserved in diverse species and ubiquitously
expressed throughout development and adult tissues suggesting that Acdp may be an essential
gene. Acdp showed strong homology to bacteria CorC protein and predominantly localized on the
plasma membrane. These results suggest that Acdp is probably a family of proteins involved in ion
transport in mammalian cells


Background
We have recently cloned and characterized a novel gene
family named ancient conserved domain protein (ACDP)
which encodes four protein members in humans [1]. We


found that this gene family is evolutionarily conserved in
diverse species ranging from bacteria, yeast, C. elegans, and
D. melanogaster to mammals. The sequence conservation
and the presence of multiple members within a species


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may imply functional importance associated with the
genes. To facilitate the functional analysis of the ACDP
gene family, we cloned and characterized Acdp, the mouse
homologue of the human ACDP gene family.

Results
Molecular cloning of the Acdp gene family
To clone the mouse Acdp genes, the human ACDP cDNA
and predicted protein sequences were used to search the
mouse EST database with the blastn and tblastn programs.
Mouse EST markers corresponding to each Acdp member
were then identified. For example, EST H3086H12-5 cor-
responds to Acdpl, W98010 for Acdp2, 603299135F1 for
Acdp3 and BG083791 for Acdp4. A modified oligo-dT with
a M13 tail was used for the RT reaction. A forward primer
from each EST marker and the M13 primer (olig-dT tail)
were used to amplify the 3' UTR sequence for each corre-
sponding Acdp gene from the RT products. To obtain 5'-
end coding sequences for the Acdp genes, we conducted a
series nested PCR with combinations of mouse and
human primers. The 5' UTR sequences were identified by
directly sequencing BAC DNA containing the correspond-
ing Acdp genes. The BAC clones were identified by screen-
ing a CITB mouse BAC DNA library (Research Genetics).
The 5' UTR sequences obtained from above were further
confirmed by RT-PCR. The Acdpl gene contains 3,631 bp
of nucleotide sequence and encodes a predicted protein
with 951 amino acids (AA). The other three Acdp genes
(Acdp2, 3 and 4) contain 3,244 bp, 2,684 bp and 2,743 bp
of cDNA sequences, and encode deduced proteins of 874
amino acids, 713 amino acids and 771 amino acids,
respectively.

Tissue distribution
Northern blot analyses of the Acdp gene family were car-
ried out using membranes purchased from Origene. A
total of 12 mouse tissues were included in the study (Fig.
1). Due to sequence homologies between each Acdp
member within the conserved domain, probes for North-
ern bolts were PCR fragments from the last exon and the
3' untranslated region sequences. The mouse Acdp mes-
sages showed almost the same tissue distributions as the
human ACDP genes. Acdpl message is highly expressed in
the brain, while kidney and testis also showed low levels
of expression. Acdp2 showed higher expressions in the
brain, kidney and liver. However, the Acdp2 transcript was
not present in the skeleton muscle and skin, and it
showed very low levels of expression in the rest of tissues.
Acdp3 and Acdp4 showed different levels of expression in
all tissues tested; the highest expressions for Acdp3 were
observed in the brain, kidney, liver and heart, and the
highest expressions for Acdp4 were observed in the kidney,
small intestine and testis. The expression levels for Acdp3
and 4 in skeleton muscle were barely detectable; however,
3-actin showed normal expression suggesting that the


results were not a consequence of bad RNA quality (data
not shown). The ubiquitous expression pattern may be
taken as another indication of the functional importance
of Acdp proteins in fundamental biological processes in
addition to the sequence conservation in evolutionarily
divergent species.

Chromosomal location
Radiation hybrid mapping indicated that the Acdpl gene
maps to chromosome 19 between markers D19Mit119
(34.3 cR proximal)and D19Mitll112 (13.6 cR distal). The
Acdp2 gene maps slightly more distal to the Acdpl on
chromosome 19 between D19Mit9 (2.4 cR proximal) and
D19Mit38 (15.1 cR distal). The Acdp3 and Acdp4 genes
map to chromosome 1 within one BAC clone (RP23-
294117), proximal to marker DlMitl71 (17.4 cR). These
regions are syntenic to the human counterparts.

Sequence homology and molecular characteristics
The mouse Acdp genes showed very strong homologies of
both nucleotide and AA sequences to the human ACDP
genes (Table 1). The highest homologies were observed
between the human ACDP2 and the mouse Acdp2 gene
(91% of nucleotide identity, 97% of AA identity and
99.4% of AA homology). In addition, the 5' UTR nucle-
otide sequences (20 bp of nucleotides before start codon)
also showed high homologies to the human homologs,
for example, the Acdp2 5' UTR sequence showed 95%
identities to its human homolog. However, the homolo-
gies in the 3' UTR sequences (20 bp of nucleotides after
stop codon) were much lower (40-55%) for all Acdp
genes except Acdp4 (90% identity to its human homolog).
The ancient conserved domain (ACD) has 55.3% of AA
identity and 83.3% of homology between all mouse and
human ACDP proteins (Fig. 2). The ACD domain is evo-
lutionarily conserved in divergent species ranging from
bacteria, yeast, C. elegans, D. melanogaster, mouse to
human (Fig. 3). Particularly, as shown in Fig. 3, Acdp pro-
teins showed very strong AA homology to bacteria CorC
protein (35% AA identity with 55% homology), which is
involved in magnesium and cobalt efflux [7]. High AA
homology was also observed between the Acdp proteins
and the yeast Amip3 protein (35% AA identity with 56%
homology). The Amip3 is likely to be a homologous to
the bacteria CorC protein. The Amip3 mutants confer
resistance to copper toxicity (Personal communication
with Dr. V.C. Culotte, John Hopkins Bloomberg, School
of Public Health). The evolutionary relationships among
those proteins are illustrated by a phylogenetic tree con-
structed based on the AA homology of proteins (Fig. 4).

We found that all mouse Acdp members contain four dis-
tinct transmembrane domains (Fig. 5), two CBS domains
and a DUF21 domain that are found in bacteria CorC and
yeast Amip3 proteins. CBS domains are small intracellular


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Figure I
Northern blot analyses of the Acdp gene family. S. muscle represents skeletal muscle, Sm. Int. represents small intestine. Multi-
ple Choice Northern Blot filters were purchased from Origene.





Table I: Nucleotide and amino acid homologies (%) between human ACDP and mouse Acdp members.


Acdp


Acdpl


Acdp2


DNA in coding region
AA identity
AA homology
5' UTR
3' UTR


Acdp3

86
86
94
80
50


Acdp4

87
89
96
80
90


modules that are mostly found in 2 or four copies within
a protein. Pairs of CBS domains dimerise to form a stable
globular domain [8]. DUF21 (CD: pfam01959.9) is a
newly defined domain with unknown function. This
domain is a transmembrane region and found to be
located in the N-terminus of the proteins adjacent to two
intracellular CBS domains http://www.ncbi.nlm.nih.gov/
Structure/cdd/cddsrv.cgi?uid=pfamO1595. A cNMP-bind-
ing domain (cyclic nucleotide-monophosphate-binding
domain) was found in all Acdp members.

In addition, Acdp contains an Alanine-rich region (2-10:
AAAAAAAAA), a Leucine-rich region (204-257: LLRVR-


PRLYGPGGDLLPPAWLRALGALLLLALSALF
SGLRLSLLSLDPVELRVL), a Proline-rich region (78-130:
PGPPVPAAPVPAPSLA PGENGTGDWAPRLVFIEEPPGAG-
GAAPSAVPTRPPGP), and two amidation sites (917-920:
MGKK; 926-929: SGRK). Acdp2 has a glycine-rich region
(201-222: GAGGSGSASGTVGGKGGAGVAG). Acdp3
possesses a large alanine-rich region (2-261) and a large
leucine-rich region (201-299). Acdp4 contains a leucine
zipper pattern (185-206: LVMVLLVLSGIFSGLNLGLMAL)
and an amidation site (7-10: GGRR).


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&Cd3.2


6U
LcMZ 1. -, A U
I~L1'J MA. 11
JL3L" NA:1
'jc. IF4
-dpq 9 Li


Figure 2
Amino acid sequence homology alignment for all of the ACDP and Acdp genes within the ACD domain. The sequence data for
the Acdp genes have been deposited in GenBank under accession number AF202994 (Acdp I), AF216961 (Acdp2), AF216964
(Acdp3) and AF216963 (Acdp4). Identical amino acids or amino acids with very strong homologies among all proteins were
shaded black. Identical amino acids or amino acids with very strong homologies in most of the proteins were shaded grey. Dot
lines represent gaps for the alignment.


Antibody production, Western results and subcellular
localization
Peptides from Acdpl N- (TSFLLRVYFQPGPPATAAPVP-
SPT) and C- (TQQLTLSPAAVPTR) terminuses, conserved
peptide from ACD domain of Acdpl (HNIVDILFVKD-
LAFVDPDDCTPLLTVTRF) were commercially synthesized
(Sigma Genosys). These antigenic sites were predicted by
software from Sigma Genosys and polyclonal antibodies
for each peptide were produced by immunizing rabbits.
To test the specificity of the antibodies, we conducted
Western-blot analysis of mouse brain tissue extracts. As
shown in Fig. 6A, the antibody produced by C-terminal
peptide specifically detected Acdp i (lane 3). The antibody
generated by N-terminal peptide recognized Acdp4 in
addition to Acdpl, although the reactivity to latter was
significantly higher (Fig. 6A, lane 2). As expected, the anti-
body produced by the conserved sequence peptide
detected all Acdp proteins (Fig. 6A, lane 1). To further
determine the specificity of the antibody against the
Acdpl C-terminus, we analyzed extracts of HEK293, 3T3
and PC12 cells. The results are shown in Fig. 6B. Appar-
ently, this antibody detected a specific band of Acdpl in
all cell lysates. Of note, shown in Fig. 6B are signals of 10
1ig extracts of HEK293 cell lysates, 100 |ig extracts of 3T3
and PC12 cell lysates. Thus, the expression levels ofAcdpl
in these cell types vary a lot, with the highest expression in
HEK293 cells. Nevertheless, these immunoblot results


support our analysis of brain tissue extracts that the anti-
body against Acdpl C-terminus specifically recognizes
Acdp-1.

The specificity of the Acdp C-terminus antibody suggests
the possibility of using it to localize Acdp-1 within cells.
Since Northern blot revealed almost exclusive expression
of Acdp in the brain, we examined its subcellular locali-
zation in hippocampus neurons isolated from mouse
embryos. The neurons were cultured on glass coverslips
coated with a confluent monolayer of mouse cortical
astrocytes in dishes. Immunostaining was using the
Acdpl C-terminus specific antibody. Confocal imaging
revealed that Acdpl is predominantly localized on the
plasma membrane. A series of sections of a cell at the
thickness of 0.5 micrometer clearly showed membrane
location of Acdpl-immunoreactivity (Fig. 7), which is
consistent with the observation of transmembrane
domains within the Acdp proteins.

Discussion
Although the exact functions of the ACDP gene family are
not yet elucidated, several lines of evidence suggest that
ACDP genes may play an important role in biological
processes. First, these genes are evolutionarily conserved
in many phylogenetically divergent species; Second, mul-
tiple genes are present in a species; Third, these genes are


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16U UU zU U ZUM _24U 26U 2 UU
AvgC -- -



Drp ;


KyrD -- - 0 E - -- -




Figure 3
Amino acid sequence alignment showing the conservation of ACD domain in various species. Amip3 is a protein from Saccha-
romyces cerevisiae (NP_014581). CanG is a protein from Candida glabrata (AAF33 142). NeuC (EAA31204) is a hypothetical pro-
tein from Neurospora crassa. DroM is a gene product from D. melanogaster. The accession number for this gene is CG40084 in
BDGP (Berkeley Drosophila Genome Project). AnoG represents a protein from the anopheles gambiae str. (EAA01004). CaeE
(AAK77203) is a hypothetical protein from the Caenohabditis elegans. CorC represents bacteria magnesium and cobalt efflux
protein from the Shewanella oneidensis. XyFD is a hypothetical protein from the Xylella fastidiosa Dixon (ZP_00038107).
















r1--. ACDP3
- A dpa
-Acdpi
DrOM
CaeE
-Amip3
core








Figure 4
Phylogenetic tree showing relationships among proteins containing the ACD domain from figure 2 and 3. The phylogenetic
tree was constructed according to the calculation of the best match for the selected sequences. Abbreviations for each protein
are the same as presented in figure 3.




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0 100 200 300 400 500 600 700

transmembrane -- inside -- outside -




Figure 5
Four transmembrane domains within Acdp4 protein. Transmembrane domains were predicted by the TMHMM program http:/
/www.cbs.dtu.dk/services/TMHMM-2.0/. The plot shows the posterior probabilities of inside /outside/TM helix. At the top of
the plot (between I and 1.2) the N-best prediction is shown. The plot is obtained by calculating the total probability that a res-
idue sits in helix, inside, or outside summed over all possible paths through the model.


ubiquitously expressed throughout development and
adult tissues (unpublished data). The cloning and charac-
terization of the mouse Acdp gene family are a very impor-
tant step towards the elucidation of the functions of this
multigene family.

Sequence homology analyses revealed that Acdp proteins
shared very strong AA homologies to the bacteria CorC
protein and yeast Amip3 protein. CorA, B, C and D belong
to a protein family involved in both influx and efflux of
magnesium and cobalt. It has been shown that CorA
mutants confer resistance to cobalt toxicity [9]. Amip3
appears to be a homologous to CorC protein which is
involved in efflux of magnesium and cobalt in bacteria.
Amip3 mutants confer resistant to copper toxicity. Acdp
proteins also possess the domains that are found in bacte-
ria CorC and yeast Amip3 proteins, such as the CBS
domains, DUF21 domain and transmembrane domains.
In addition, a cNMP-binding domain was found in all
Acdp proteins, which is usually present in ion channels
and cNMP-dependent kinases [10-131. Using antibody


produced by Acdp I C-terminal peptide, we have shown
that Acdpl is predominantly localized on the plasma
membrane in hippocampus neurons. In our previous
study, we found human ACDP proteins are predomi-
nantly localized in the nucleus in HeLa cells [ 1]. The dis-
crepancy for Acdp localization in neuronal cells could be
caused by non-specificity for previous antibody which
was produced by the recombinant ACD domain, or differ-
ent cells used. In current study, the Acdpl C-terminus
antibody only recognizes Acdpl in brain tissue extracts as
well as in HEK293, 3T3 and PC12 cell lysates, suggesting
the specificity of the antibody. Our observations sug-
gested that Acdp might be involved in ion transport in
mammalian cells. However, more detailed functional
studies are needed to demonstrate the real functions of
these proteins.

Conclusions
Our previous work has described human ACDP genes [ 1 ].
The new information of the current work includes: 1). It is
the first time to report the existence of multiple Acdp


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kD
200

150


100


188 HEK 3T3


!W ~.. .5


75 Of



6A M 1 2 3


49

6B kD


PC12



ACDP1











3


Figure 6
Fig. 6A: Immunoblot analysis of Acdp proteins in brain tissue extracts. Immunoblotting were carried out using a Western blot-
ting detection system (ECL) (PIERCE). Lane I, probed with antibody generated by conserved peptide. From top to bottom,
each band corresponding to Acdp I (I 15 kD), Acdp2 (100 kD), Acdp4 (90 kD) and Acdp3 (80 kD). Lane 2 and 3, probed with
the Acdp I antibodies generated by N-terminal and C-terminal peptides, respectively. Fig. 6B: Immunoblot analysis of Acdp I in
HEK293, 3T3 and PC 12 cells. The blots were probed with the antibody against the C-terminus of Acdp-1. Lane 1, 10 pIg of
HEK293 cell lysates. Lane 2 and 3, 100 pg of 3T3 and PC 12 cell lysates.


genes in other mammals in addition to human, while
Acdp appears to be a single copy gene in lower organisms
such as in C. elegans, yeasts and bacteria. We have also
suggested the evolutionary relationships of Acdp genes in
different species phylogeneticc tree); 2). Molecular clon-
ing and characterization of murine Acdp gene family are
essential for study of this novel gene family in animal
model, e.g. for generation of knockout or transgenic mod-
els; 3). We have demonstrated both DNA and amino acid
conservation between mouse and human for each Acdp
gene and the whole Acdp gene family, which provide
important information for the possibility of functional
redundancy or overlap between Acdp members; 4). We
have generated antibodies specific for Acdp 1 and all Acdp
proteins. The Acdpl C-terminus antibody appears to spe-
cifically recognize Acdpl. Using this antibody, we have
demonstrated that Acdpl is predominantly localized on
the plasma membrane in hippocampus neurons. These
results represent an important step towards the character-
ization of functions for the Acdp gene family.


Methods
cDNA cloning
cDNA cloning of the Acdp gene family was performed
based on human homologue sequences as previously
reported [2]. Briefly, the human ACDP cDNAs and pre-
dicted AA sequences were used to search the mouse EST
database for EST markers corresponding to each Acdp
member. A forward primer within the human ACDP 5'
cDNA coding region (after start codon) and a mouse
reverse primer from the mouse EST marker were used to
amplify the homologue sequence from mouse cDNA at
very low annealing temperature (45-500 C). A nested PCR
using an inside reverse primer from the mouse EST
sequence and the same human forward primer was then
carried out to amplify the specific mouse gene from the
first round PCR products at high annealing temperature
(62C). The expected PCR products were directly excised
from agarose gel and sequenced by an ABI377 automatic
sequencer. The sequence was further confirmed using a
forward primer from newly identified sequence and a


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Figure 7
Subcellular localization of AcdpI in hippocampus neurons. A series of confocal images from a cultured neuron stained with an
anti-Acdp I antibody. The step of each imaging section is 0.5 rIm, from the surface of the neuron (0 rIm) to the middle plan (4.5
Im).


reverse primer from known sequence. Once most of the
coding sequences were identified, partial sequence of
exon 1 and the 5' UTR sequences were obtained by BAC
DNA sequencing.

Northern blot analyses
Multiple Choice Northern Blot filters containing 12 differ-
ent mouse tissues were purchased from Origene. The fil-
ters were probed for each Acdp gene with a PCR fragment
(around 350 bp) from last exon and the 3' untranslated
region labeled with a-32P dCTP using the random primer
extension system (Life Technologies). Hybridizations
were carried out overnight. The filters were washed twice
with washing buffer I (2xSSC, 0.1% SDS) at 42C for 15-
min, and then washed twice with washing buffer II
(0.25xSSC, 0.1% SDS) at 65 0C for 15-min. Washed filters
were exposed to X-ray films for overnight or longer [3,4].

Antibody production and Western blot analyses
Peptides linked to KLH (keyhole limpet hemacyanin)
from Acdpl N- and C-terminals and the conserved
domain (ACD) were used for generation of antibodies
specifically for Acdp and all Acdp members as reported,
respectively [5]. Western blots were carried out Using ECL
(PIERCE) as described previously [1]. The membranes
were washed extensively after incubation with primary
and secondary antibodies and were then developed with
X-ray films with optimal exposure time.


Chromosome localization
The T31 mouse radiation hybrid panel from Research
Genetics was used to map the chromosome location of
each Acdp member. Primers from 3' UTR of each Acdp
member were used to amplify the 100 radiation hybrid
clones representing the mouse genome. The data were
submitted to the Jackson Laboratory Mouse Radiation
Hybrid Database for analysis.

Sequence analyses
Sequence assembly was performed with program
Sequencher (Gene Codes Corp). Protein and DNA
homology searches were carried out with tblastn, tblastx,
blastp and blastn programs htp://
www.ncbi.nlm.nih.gov/BLAST!. Multiple sequence align-
ments were performed with GeneDoc and pairwise
sequence alignment http://pir.georgetown.edu/pirwww/
search/pairwise.html. Multiple programs including BCM
Search Launcher http://searchlauncher.bcm.tmc.edu/,
ProfileScan http://hits.ish-sib.ch/cgi.hbin/PFSCAN,
sequence motif search http://motif.genome. ad jp/
ExPASy http://www.expasy.ch/ and 3Dpssm hhttpj4
www.bmm.icnet.uk/~33dpssm/ were used for searching
sequence features of known protein. Phylogenetic tree
was constructed by Clustalw program (version 1.81) using
UPGMA (Unweighted Pair Group Method using Arithme-
tic averages) algorithm [14].






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Neuronal cell preparation and immunostanining
Hippocampal neuron cultures were prepared as previ-
ously reported [6]. In brief, the hippocampuses were dis-
sected out from mouse embryos at 16 days in utero. The
tissues were then incubated for 20 min at 37C in MEM
(minimum essential medium) modified for suspension
culture (Life Technologies) plus 0.25 % trypsin (Life Tech-
nologies). The dissociated hippocampal neurons were
plated on glass coverslips coated with a confluent monol-
ayer of mouse cortical astrocytes obtained as described
below. The neurons were maintained at 37C in a
humidified atmosphere with 5% CO2. Cortical astrocytes
dissociated from newborn mouse cortices were grown in
culture flasks at 37C in a humidified atmosphere with
5% CO2 until confluent. The cells were exposed to 10-5 M
cytosine arabinoside (Sigma) and cultured for additional
12-24 hrs at 37C. After remove of the media with cellu-
lar debris, the cells were used for coating coverslips.

For immunostaining, neuronal cells on the coverslips
were first fixed in PBS containing 4% paraformaldehyde
(PFA) for 12 hrs at 4 C and then incubated in a solution
containing 4% PFA and 0.4% Triton X- 100 at 4 0 C for 1 hr.
After washing with PBS three times, the cells were incu-
bated with a blocking solution containing 1:30 normal
goat serum, and subsequently incubated with a rabbit
polyclonal anti-ACDP antibody (1:3000) overnight at
4C. After extensive washing with 1% goat serum PBS
solution, the cells were incubated with an Alex 488 conju-
gated secondary antibody (1:100 in 1% goat serum PBS
solution, Molecular Probes) for 3 hrs at room tempera-
ture. Following final washes with 1% goat serum PBS
solution, the neuronal cells on the coverslips were cover-
slipped with a glycerol-based anti-photobleach medium.
The cells were viewed under a confocal microscope (Carl
Zeiss). Images were captured with a CCD camera and
acquired by the Scion Image software (Scion Corporation,
Frederick, MD).

Gene bank accession number
The cDNA sequences for the Acdp gene family have
already been deposited in gene bank with accession num-
bers AF202994 (Acdpl), AF216961 (Acdp2), AF216964
(Acdp3) and AF216963 (Acdp4).

Authors' contributions
This study was designed by CYW and JXS. The study was
performed as follows: CYW, PY, JDS and HA contributed
to the molecular cloning part of the manuscript. CYW and
SP were responsible for the Acdp antibody production.
JGG and JLG did immunostaining for Acdp 1 localization
in neuronal cells. CYW, DG and ZD carried out the West-
ern analyses.


Acknowledgements
This work was partly supported by a CIGP grant to C.Y.W.

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