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Evolution of Cyclooxygenase in the Chordates

Permanent Link: http://ufdc.ufl.edu/UFE0022189/00001

Material Information

Title: Evolution of Cyclooxygenase in the Chordates
Physical Description: 1 online resource (131 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: cephalochordate, cyclooxygenase, gill, hagfish, lamprey, osmoregulation, phylogenetics, physiology, teleost, urochordate
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cyclooxygenase (COX) is the enzyme found in animals responsible for converting arachidonic acid into prostaglandins. These prostaglandins can then perform multiple functions including regulation of inflammation responses, changes in vascular tone, and ion transport/osmoregulation. Before this thesis, only two main forms of cyclooxygenase (COX-1 and COX-2) and one ancestral form (Ciona COX) were known from the chordates. I sequenced COX genes from several hitherto uninvestigated chordates in order to determine a more comprehensive scenario for the evolutionary history of cyclooxygenase in the chordates. After designing primers, partial COX sequences were obtained from the Sea Lamprey (Petromyzon marinus) and amphioxus (Branchiostoma lancelolatum). Complete COX sequences were obtained from the Atlantic Hagfish (Myxine glutinosa), euryhaline killifish (Fundulus heteroclitus), and longhorn sculpin (Myoxocephalus octodecemspinosus). These novel sequences along with other known COX sequences were then subjected to a variety of phylogenetic analyses using standard techniques. The results of these analyses suggest a complex history for the evolution of cyclooxygenase in the chordates. There appears to have been at least 3 evolutionary independent origins of COX in the chordates. The forms from the sea squirt (Ciona intestinalis) (COXa and COXb) represent the most ancestral forms, the forms from amphioxus represent another independent origin (COXc and COXd), and the vertebrate forms (COX-1 and COX-2) represent yet another origin, with the hagfish and lamprey as likely basal members. Furthermore, the teleosts appear to have duplicate COX-1 (COX-1a and COX-1b) or COX-2 (COX-2a and COX-2b) forms. These results agree with previous studies and reveal novel information about COX forms in the chordates. More intense sampling of other chordates could reveal other novel origins of COX or contribute to a more robust understanding of COX evolution. Finally, the function of most non-mammalian COX forms remains to be investigated.
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, 2008.
Local: Adviser: Evans, David H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022189:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022189/00001

Material Information

Title: Evolution of Cyclooxygenase in the Chordates
Physical Description: 1 online resource (131 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: cephalochordate, cyclooxygenase, gill, hagfish, lamprey, osmoregulation, phylogenetics, physiology, teleost, urochordate
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cyclooxygenase (COX) is the enzyme found in animals responsible for converting arachidonic acid into prostaglandins. These prostaglandins can then perform multiple functions including regulation of inflammation responses, changes in vascular tone, and ion transport/osmoregulation. Before this thesis, only two main forms of cyclooxygenase (COX-1 and COX-2) and one ancestral form (Ciona COX) were known from the chordates. I sequenced COX genes from several hitherto uninvestigated chordates in order to determine a more comprehensive scenario for the evolutionary history of cyclooxygenase in the chordates. After designing primers, partial COX sequences were obtained from the Sea Lamprey (Petromyzon marinus) and amphioxus (Branchiostoma lancelolatum). Complete COX sequences were obtained from the Atlantic Hagfish (Myxine glutinosa), euryhaline killifish (Fundulus heteroclitus), and longhorn sculpin (Myoxocephalus octodecemspinosus). These novel sequences along with other known COX sequences were then subjected to a variety of phylogenetic analyses using standard techniques. The results of these analyses suggest a complex history for the evolution of cyclooxygenase in the chordates. There appears to have been at least 3 evolutionary independent origins of COX in the chordates. The forms from the sea squirt (Ciona intestinalis) (COXa and COXb) represent the most ancestral forms, the forms from amphioxus represent another independent origin (COXc and COXd), and the vertebrate forms (COX-1 and COX-2) represent yet another origin, with the hagfish and lamprey as likely basal members. Furthermore, the teleosts appear to have duplicate COX-1 (COX-1a and COX-1b) or COX-2 (COX-2a and COX-2b) forms. These results agree with previous studies and reveal novel information about COX forms in the chordates. More intense sampling of other chordates could reveal other novel origins of COX or contribute to a more robust understanding of COX evolution. Finally, the function of most non-mammalian COX forms remains to be investigated.
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, 2008.
Local: Adviser: Evans, David H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022189:00001


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bca0482ada215f09944f21b6b46ee5698d34dfc7







EVOLUTION OF CYCLOOXYGENASE IN THE CHORDATES


By

JUSTIN CHASE HAVIRD

















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

UNIVERSITY OF FLORIDA

2008


































2008 Justin Chase Havird




























To the teachers who inspired, directed, and challenged me. My high school marine biology
teacher, Larry Joye, first introduced me to the fascinating world of life in the water and since
then my endeavors have never strayed far from it. Dr. David Evans enthusiastically welcomed
me into his lab as an undergraduate, although I had no former research experience. What started
as a part-time project soon evolved into an academic career as I was given the opportunity to
conduct original research. During this time, Dr. Keith Choe was an invaluable asset and
inspiration. Growing from this original partnership, I continued working with David on my
master's research and he has always supported my academic and research endeavors; I cannot
envision a better advisor.









ACKNOWLEDGMENTS

I most of all thank my supervisory committee chair, Dr. David H. Evans. Although

David was nearing retirement and had resolved to not accept any new students, he welcomed me

as a graduate student without any hesitation. He has guided me in developing research

strategies, implementing those strategies, and interpreting the results. He has always supported

my projects and none of them would have been accomplished without his enthusiasm.

I also thank my supervisory committee members for their commitment to my research

and their willingness to share their expertise. Dr. Michael Miyamoto contributed heavily to the

phylogenetic aspects of my project and encouraged me to be thorough in my analysis. When I

started this project I knew very little about generating meaningful evolutionary relationships.

Michael selflessly took the time necessary to make sure I understood how to develop a

biologically relevant reconstruction of evolutionary changes that took place over 600 million

years ago. He also put his own projects aside while I used his computers for months at a time.

Finally, his guidance was absolutely necessary in interpreting the conflicting results of my

analyses. Dr. Marty Cohn was a great influence on my research and prompted me to think in an

integrative and comparative framework. He generously allowed me to use his lab space. His

expertise working with evolutionarily interesting chordates was invaluable. His students also

graciously helped me learn many of the techniques used in this research.

I am also indebted to several colleagues for their assistance. Dr. Keith Choe pioneered

and taught me many of the molecular techniques now commonly used in the Evans' lab that were

critical to this research. He spent countless hours with me in multiple laboratories

troubleshooting problems, explaining theory, and developing this research. He also encouraged

me to work with the hagfish, lamprey, and amphioxus and continually helped me develop new

ideas and strategies. Dr. Kelly Hyndman was also constantly supportive of this research. She









taught me the localization techniques used here and also helped with many aspects of retrieving

and analyzing genetic data. Finally, she was always receptive to critical discussions of my

research and tackled every circumstance in a friendly, often contagious manner.

I am very thankful to my family and friends for their continued support of my education

and research. My entire family has been very interested in the research I have undertaken and

when they were not interested they feigned interest in order to make me happy. My parents,

Kurt and Dawn Havird, have encouraged my desire to be a scientist, and have been

overwhelmingly supportive during my academic career; often in the form of food, clean laundry,

or money. My friends have also been very supportive of my research interests: fellow scientists

have empathized with the typical problems associated with research and non-scientists have

given me the confidence to persevere.

I also thank the following people for providing help with this project in particular, other

related projects, or the daily support needed to become a successful student: Dr. James B.

Claiborne, Rachel Rose, Dr. GuangJun Zhang, Donovan German, Justin Catches, Andrew

Diamanduros, Sara Takeuchi, Jim Stidham, Curtis Lanier, Julia Curtis-Burnes, Oriana Galardi-

Este, Rebecca Kreh, Dr. Susan Edwards, Gini Luchini, Makesha Foster, Emily Cornwell, Dr.

Larry Page, Pete Ryschkewitsch, Frank Davis, Diana Davis, Cathy Moore, Karen Pallone, Vitrell

Sherif, Dr. David Reed, Dr. Paul Lewis, and the laboratories of Dr. Lou Guillette and Dr. David

Julian.

My research was primarily funded by National Science Foundation grants IBN-0089943

and IOB-0519579 to David Evans. A grant-in-aid of research to Justin Havird from Sigma Xi

also supported a portion of this research. Finally, initial support was provided in part by a

University Scholars award to Justin Havird.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F TA B LE S ......... ..... ............. ................................................................... 8

LIST O F FIG U RE S ................................................................. 9

A B S T R A C T ........................................... ................................................................. 1 1

CHAPTER

1 GENERAL INTRODUCTION ............................... ......................... 13

Introduction ............................ ... ............................. ...................13
Cyclooxygenase Evolution ............................................................. ...................... 16
M a m m a ls .........................................................................................................................1 7
F ish e s ................... ...................1...................8..........
O th er V erteb rates ................................................................................ 19
O th er C h o rd ate s ......................................................................................................... 19
Cyclooxygenase Function in M ammals................................. ................... 20
G gastrointestinal Tract .......................................................................... 20
T h e K id n ey ................................................................2 1
P late lets ................... ...................2...................3.........
R e p ro d u ctio n ............................................................................................................. 2 3
N ervou s Sy stem ....................................... ................................................... ............... 25
Cyclooxygenase Function in Non-Mammals ......................................... ............... 27
Fishes ................. .................................................................27
Osmoregulation .................................. ..... ....................... 27
R production .............................................................29
D differential functions of novel form s ............. .. ......................... ....................30
O their functions .................................................... .... ..................... 30
A m p h ib ia n s ................................................................................................................ 3 1
Reptiles ...... .................................. ......... ........ ....... ....... ............... 31
B ird s ...........................................................................3 2
Stu dy O v erview ................................................................33

2 CYCLOOXYGENASES IN THE ANCESTRAL CHORDATES: SEQUENCE AND
PHYLOGENETIC ANALYSES ............................ ........ 39

Introduction ............................................. ..................39
M materials an d M eth o d s ...........................................................................................................4 1
Animals and Holding Conditions ................................................41
Tissue Collection ............... .... .. ......... ... .. ................ ................ 42
Reverse Transcription, Primer Design, PCR, Cloning, and Sequencing......................43
5' and 3' Rapid Amplification of cDNA Ends (RACE)..................................................45


6










Sequence Analysis............................... ... ...... .... ............ ......45
Phylogenetic Analysis .................................... ..... .......... .............. .. 46
R esu lts .. ............ .... .... ....... .....................................................4 8
M olecular Identification of Cyclooxygenases.............................................................. 48
B ra n ch io sto m a ................................................................................................... 4 8
M y x in e ..............................................................................4 8
P e tro m y z o n ......................................................................................................... 4 8
F u n d u lu s ............................................................................................................. 4 9
Myoxocephalus ............................................. 49
Sequence Analyses ................... ......................... 50
Teleosts COX-la, COX-lb, and COX-2 .................................... .................... 50
Lamprey, hagfish, and amphioxus COX forms............................................... 52
P hylogenetic analy ses ............................................................53
D iscu ssio n ................... ...................5...................6..........
N o v el C O X F o rm s ..................................................................................................... 5 7
P hy log en etic A n aly ses............................................................................................... 5 8
S u m m ary ............................................................................... 6 2

3 SUMMARY AND FUTURE DIRECTIONS ................................................105

S u m m ary ................... ...................1...................0.........5
N ovel COX Sequences ............................................................................ 105
P hy log en etic A n aly ses............................................................................................. 10 6
F u tu re D ire ctio n s ............................................................................................................ 10 6
Completion of Novel Sequences ......................................... ...............107
Additional Phylogenetic Analysis ................................. ........................... ...107
Additional Sequences ......................................... ................... ...........108
A d d itio n al T ax a ....................................................................................................... 10 9
Function of C yclooxygenases ........................................................................ 110

L IST O F R E F E R E N C E S ....................................................................................................... 117

BIOGRAPHICAL SKETCH .........................................................................130



















7









LIST OF TABLES


Table page

2-1. Degenerate primers used in PCR ........................................................ ............. 64

2-2. Amphioxus COX specific primers used in PCR..................................... ............... 65

2-3. Hagfish COX specific primers used in PCR........................................... ............... 66

2-4. Lamprey COX specific primers used in PCR ............................................ ............ .... 67

2-5. Killifish COX specific primers used in PCR.................. ...... ...............68

2-6. Sculpin COX specific primers used in PCR ................. .......................................... 69

2-7. Sequences included in phylogenetic analyses......................................... ............... 70

2-8. LRTs between amino acid based models of COX evolution.................. .....................72

2-9. LRTs between tree topologies with altered positions of hCOX and 1COX .......................73









LIST OF FIGURES


Figure page

1-1. Cascade of reactions depicting the conversion of arachidonic acid to the primary
prostaglandins via catalyzation with either cyclooxygenase (COX) form ......................35

1-2. General phylogeny representing the different COX forms in the fishes .........................36

1-3. Accepted evolution of ancestral chordates (Pough, et. al, 2005)...................................37

1-4. Evolution of COX forms in the chordates, based on Jarving et al. (2004).....................38

2-1. Amino acid alignment of the forms of COX sequenced from the gills of the
euryhaline killifish (Fundulus heteroclitus)..................... .... ......................... 74

2-2. Amino acid alignment of the three forms of COX sequenced from the gills of the
longhorn sculpin (Myoxocephalus octodecemspinosus): COX-la, COX-lb, and
C O X -2 ................... ........................................................... ................ 7 6

2-3. Amino acid alignment of the forms of COX sequenced from the gills of the lamprey
(Petromyzon marinus), hagfish (Myxine glutinosa), and amphioxus (Branchiostoma
lancelolatum), along with sCOX from the dogfish (Squalus acanthias, Accession
#A AL37727) ............................................................... .... ...... ......... 78

2-4. Amino acid alignment of 1COX sequenced from the gills of the lamprey (Petromyzon
marinus), with COX-1 and COX-2 forms from vertebrates (Accession # NP_032995,
NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942).............................80

2-5. Amino acid alignment of 1COX sequenced from the gills of the lamprey (Petromyzon
marinus), with COX-1 and COX-2 forms from vertebrates (Accession # NP_032995,
NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942).............................82

2-6. Amino acid alignment of COXc and COXd sequences from the lancelet
(Branchiostoma lancelolatum) with COXa and COXb sequences from the sea squirt
(C io n a in testing a lis)............................................................................................... .. 8 5

2-7. Evolutionary history of COX sequences inferred using the minimum evolution
method with a distance optimality criterion ............. ............................................. 87

2-8. Evolutionary history of COX sequences excluding hagfish (hCOX) and lamprey
(1COX) sequences inferred using the minimum evolution method with a distance
optim ality criterion ........ ......................................................................... ....... .. .... 89

2-9. Evolutionary history of COX sequences inferred using a maximum likelihood
optim ality criterion ........ ......................................................................... ....... .. .... 90









2-10. Evolutionary history of COX sequences excluding sequences from the hagfish
(hCOX) and lamprey (1COX) inferred using a maximum likelihood optimality
criteria n ....... ......... ......................................................................... 9 2

2-11. Alternate likelihood ratio test (aLRT) scores for branch support of COX sequences.......94

2-12. Alternate likelihood ratio test (aLRT) scores for branch support of COX sequences
excluding hagfish (hCOX) and lamprey (1COX) sequences..............................96

2-13. Evolutionary history of COX sequences inferred using a Bayesian optimality
c rite rio n ................... .......................................................... ................ 9 7

2-14. Evolutionary history of COX sequences excluding hagfish (hCOX) and lamprey
(1COX) sequences inferred using a Bayesian optimality criterion ..................................99

2-15. The 21 alternate tree topologies generated to test the effects of relocating the hagfish
(hCOX) and lamprey (1COX) sequences on the likelihood score (Ln L) ......................100

2-16. Unrooted phylogeny showing the most likely scenario for COX evolution in the
chordates based on several other analyses ............................................ ...............104

3-1. Euryhaline killifish (Fundulus heteroclitus) COX-lb mRNA expression following
procedures outlined in Choe et al. (2006) ............................ .................................... 113

3-2. Longhorn sculpin (Myoxocephalus octodecemspinosus) COX-la and COX-2 mRNA
expression following procedures outlined in Choe et al. (2006)...................................114

3-3. Atlantic hagfish (Myxine glutinosa) COX (hCOX) mRNA expression following
procedures outlined in Choe et al. (2006) .................................................................... 115

3-4. Sea lamprey (Petromyzon marinus) COX (1COX) mRNA expression in various
tissues following procedures outlined in Choe et al. (2006) .................................. 116









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EVOLUTION OF CYCLOOXYGENASE IN THE CHORDATES

By

Justin Chase Havird

May 2008

Chair: David Evans
Major: Zoology

Cyclooxygenase (COX) is the enzyme found in animals responsible for converting

arachidonic acid into prostaglandins. These prostaglandins can then perform multiple functions

including regulation of inflammation responses, changes in vascular tone, and ion

transport/osmoregulation. Before this thesis, only two main forms of cyclooxygenase (COX-1

and COX-2) and one ancestral form (Ciona COX) were known from the chordates. I sequenced

COX genes from several hitherto uninvestigated chordates in order to determine a more

comprehensive scenario for the evolutionary history of cyclooxygenase in the chordates.

After designing primers, partial COX sequences were obtained from the Sea Lamprey

(Petromyzon marinus) and amphioxus (Branchiostoma lancelolatum). Complete COX

sequences were obtained from the Atlantic Hagfish (Myxine glutinosa), euryhaline killifish

(Fundulus heteroclitus), and longhorn sculpin (Myoxocephalus octodecemspinosus). These novel

sequences along with other known COX sequences were then subjected to a variety of

phylogenetic analyses using standard techniques. The results of these analyses suggest a

complex history for the evolution of cyclooxygenase in the chordates.

There appears to have been at least 3 evolutionary independent origins of COX in the

chordates. The forms from the sea squirt (Ciona intestinalis) (COXa and COXb) represent the









most ancestral forms, the forms from amphioxus represent another independent origin (COXc

and COXd), and the vertebrate forms (COX-1 and COX-2) represent yet another origin, with the

hagfish and lamprey as likely basal members. Furthermore, the teleosts appear to have duplicate

COX-1 (COX-la and COX-lb) or COX-2 (COX-2a and COX-2b) forms. These results agree

with previous studies and reveal novel information about COX forms in the chordates. More

intense sampling of other chordates could reveal other novel origins of COX or contribute to a

more robust understanding of COX evolution. Finally, the function of most non-mammalian

COX forms remains to be investigated.









CHAPTER 1
GENERAL INTRODUCTION

Introduction

Cyclooxygenase (COX) is the enzyme responsible for the rate limiting step that converts

arachidonic acid to Prostaglandin G2 and Prostaglandin H2. Arachidonic acid is an essential fatty

acid required by most mammals and can be obtained by the conversion of linoleic acid found in

plants (occurring in herbivores). Some animals cannot convert linoleic acid into arachidonic acid

and therefore must obtain it from other animals (obligate carnivores). COX oxidizes arachidonic

acid to make the hydroperoxy endoperoxide Prostaglandin G2, which is then reduced to form the

hydroxy endoperoxide Prostaglandin H2. Prostaglandin H2 can then be converted to the primary

prostanoids (Prostaglandin 12, Prostaglandin D2, Prostaglandin E2, Prostaglandin F2u, and

Thromboxane A2) by a variety of enzymatic and non-enzymatic pathways. Therefore, the

expression of COX can directly influence the amount of prostaglandins that are synthesized due

to the arachidonic acid cascade (Vane et al., 1998, Fig. 1-1).

Prostaglandins are found in vertebrates, some invertebrates, and possibly even in plants

(Rowley et al., 2005; Ryan, 2000). In mammals, they are expressed in virtually all tissues and

have a myriad of physiological functions, but they mainly regulate vascular tone in smooth

muscle by acting as vasodilators and vasoconstrictors. In addition, prostaglandins are key

regulators of such diverse processes as salt and water homeostasis in the mammalian kidney

(Harris and Breyer, 2001), protection of stomach lining (Kargman et al., 1996), platelet

aggregation in blood (F6rster and Parratt, 1996), birth (Challis et al., 2002), and central nervous

system function (Hopkins, 2007). They also may play prominent roles in pathological states

such as fever, inflammation, and cancer (Vane et al., 1998).









Prostaglandins were first extracted from semen, prostate (hence the name prostaglandin),

and seminal vesicles by Goldblatt and von Euler in the 1930s (von Euler, 1936) and were shown

to affect blood pressure. The reaction that catalyzes Prostaglandin G2 synthesis was first

characterized in the 1970s (Hamberg and Samuelsson, 1973; Hamberg et al., 1974), but it wasn't

until 1988 that cyclooxygenase (COX) was first described as the enzyme responsible for

endoperoxide synthesis by three separate groups (DeWitt and Smith, 1988; Merlie et al., 1988;

Yokoyama et al., 1988), all of which described a COX from sheep seminal vesicles which was

later named COX-1. However, before COX-1 was isolated there was early evidence that

multiple COX forms existed, based on prostaglandin activity in the rat brain and spleen (Flower

and Vane, 1972). In the early 1990s, a second COX form was cloned from mouse and chicken

fibroblast cell cultures and was named COX-2 (Kujubu et al., 1991; Xie etal., 1991; O'Banion et

al., 1992). This discovery would later prove to be of major importance in the field of

prostaglandin biology due to the development of drugs that could selectively inhibit the different

forms of cyclooxygenase.

Both forms catalyze the reactions that form prostaglandins from arachidonic acid, but

early studies suggested that COX-1 was constitutively expressed in many tissues, while COX-2

was an inducible isoform that was expressed in response to physiological stimuli (Funk, 2001).

Because basal expression of COX-2 was low in some cell types and COX-2 was found to be

expressed much more so than COX-1 in response to mitogens and cytokines, it was assumed that

COX-1 was constitutively expressed at basal levels and served a housekeeping function and that

COX-2 was inducibly expressed to serve in inflammatory functions (Kujubu et al., 1991; Xie et

al., 1991; O'Banion et al., 1992). However, this was later shown to be an oversimplification

(Funk, 2001). Notably, it was shown that COX-2 is constitutively expressed in the mammalian









kidney, where it regulates blood flow and ion transport in the Loop of Henle (Harris and Breyer,

2001) and plays a role in cell survival in medullary interstitial cells during dehydration (Yang et

al., 2002; Yang, 2003).

Despite their potential functional differences in different tissues, COX-1 and COX-2 are

biochemically very similar. Both are inhibited by nonsteroid anti-inflammatory drugs

(NSAIDs), such as aspirin, due to competition with arachidonic acid for the active sites of the

enzymes or irreversible acetylation (Vane, 1971). Both proteins in humans are -600 amino acids

and share about 63% sequence similarity with each other. However, the proteins originate from

different genes on different chromosomes and encode different mRNA transcripts. COX-1 comes

from a much larger gene (22 kilobases) than COX-2 (8 kilobases). The amino acid residues that

form the binding and catalytic sites (the functionally important sites) as well as the adjacent

amino acids are all identical in human COX-1 and COX-2, except for two substitutions. At

positions 434 and 523, valine is substituted for isoleucine in COX-2 when compared to COX-1.

These substitutions have been implicated as the most likely reason behind the biochemical

differences between COX-1 and COX-2. COX-1 appears to be more selective in terms of the

fatty acids it will accept as substrates than COX-2 (Otto and Smith, 1995). COX-2 selective

inhibitors have offered an insight into the biochemistry behind the different forms. These

inhibitors can differentiate between COX-1 and COX-2 with over 1000 fold specificity

(Griswold and Adams, 1996). The rationale behind these selective inhibitors is that the smaller

size of valine on position 523 in COX-2 allows the inhibitor to bind to a side pocket in COX-2,

but the larger isoleucine on position 523 of COX-1 appears to prevent this binding. When this

position is altered in both forms to the alternate amino acid, the selectivity for inhibitors is

reversed (Wong et al., 1997; Gierse et al., 1996; Guo et al., 1996), suggesting that this residue









plays a critical role in differing biochemical activity. COX-2 selective inhibitors were thought to

be a major medical breakthrough due to the ability to stop disease induced inflammation without

affecting normal prostaglandin levels produced by COX-1 and therefore causing minimal side

effects. However, due to the constitutive expression of COX-2 in certain tissues (especially the

kidney), COX-2 selective inhibitors may not be as affective as once thought. It is important to

remember that COX-1 and COX-2 are very structurally similar and that their main difference is

due to expression.

As outlined above, COX form and function have been extensively studied extensively in

mammals, especially humans. However, non-mammalian COX forms and their functions have

not been examined in great detail in other chordates. The goal of this research was to

characterize COX evolution in the chordates, with specific attention on the more evolutionarily

ancestral chordates. Describing ancestral COX forms is the first step in elucidating the evolution

of COX function, which may lead to a better understanding of overall COX function, and even

the development of new drugs. In this chapter, I give a brief overview of COX evolution and

function in the chordates, concluding with a brief summary of the research described in the

following chapters.

Cyclooxygenase Evolution

Like mammals, most vertebrates appear to have two homologous copies of

cyclooxygenase named COX-1 and COX-2. However, other COX forms exist throughout the

chordates, including variants of COX-1 and COX-2 as well as forms representing independent

origins of COX that are not closely related to COX-1 or COX-2. There are also COX forms in

corals and other invertebrates (Jarving et al., 2004), but the focus of this section is to review the

forms found in the chordates.









Mammals

COX-1 and COX-2 sequences have been either cloned or derived from genomic

sequences for most of the classic mammalian model species, including mouse (Accession#

NP_032995 and NP_035328), rat (Accession# NP_058739 and NP_058928), dog (Accession#

NP_001003023 and NP_001003354), cow (Accession# XP_869575 and NP_776870), sheep

(Accession# NP_001009476 and NP_001009432), and rabbit (Accession# NP_001076150 and

NP_001075857). COX sequences also exist for rhesus monkey, guinea pig, pig, horse, and

mink, but these are either partial sequences or are not available for both forms.

A third COX variant has been proposed to occur in mammals (Flower and Vane, 1972)

based on inhibitory studies (see below). This form is known as COX-3 or COX-lb because of its

close similarity to COX-1. Recently, a potential COX-3 (Accession# AY547265) was cloned

from mouse (Kis et al., 2006) and rat (Accession# AY523672, Snipes et al., 2005). However,

these forms likely do not participate in prostaglandin synthesis (Kis et al., 2006) and are not a

separate COX form. Furthermore, a similar study using an artificially created human COX-lb

found no inhibitory similarity to the proposed COX-3 enzyme (Censarek et al., 2006).

Therefore, while a third, genuinely different COX variant may exist in mammals, its sequence

remains elusive.

Analysis of the recently completed gray short-tailed opossum genome yielded the

predicted COX-1 and COX-2 forms (Accession# XP_001370514 and XP_001375945), as well

as two additional COX-1 forms, named COX-lb and COX-lb2 (Accession# XP_001370542 and

XP_001370595). These forms are complete and differ in amino acid composition and length.

These are the first COX-1 variants in a mammal that do not represent modified versions of

normal COX-1 and although is has not been suggested, it is possible that one of these forms









represents the elusive COX-3 variant. Functional studies to determine how these forms differ

from normal COX have not been published to date.

Fishes

Cyclooxygenase has been sequenced in several fishes, including the rainbow trout

(Oncorhynchus mykiss) (Zou et al., 1999), brook trout (Salvelinusfontinalis) (Roberts et al.,

2000), Atlantic croaker (Micropogonias undulates), and zebrafish (Danio rerio) (Grosser et al.,

2002). These fishes, along with genomic sequence data from the stickleback (Gasterosteus

aculeatus), green spotted puffer (Tetraodon nigroviridis), pufferfish (Takifugu rubripes), and

Japanese medaka (Oryzias latipes) suggest that fishes posses both COX-1 and COX-2 forms

(Jarving et al., 2004). Furthermore, COX-2 has recently been sequenced from European sea bass

(Dicentrarchus labrax) (Buonocore et al., 2005) and euryhaline killifish (Fundulus heteroclitus)

(Choe et al., 2006), supporting this prediction.

A cyclooxygenase has also been sequenced from the spiny dogfish shark (Squalus

acanthias) (Yang et al., 2002). However, this form was not designated as COX-1 or COX-2

because of its near equal identity to both forms (it is only slightly more similar to COX-1). This

form was named sCOX by the authors and may represent an evolutionarily distinct form of

cyclooxygenase found in the elasmobranchs.

Ishikawa and colleagues have recently further characterized COX evolution in the

teleosts by demonstrating that both the zebrafish (Ishikawa et al., 2007) and rainbow trout

(Ishikawa and Herschman, 2007) have two functional COX-2 genes (named COX-2a and COX-

2b). In addition, by analyzing sequence databases, they concluded that not all teleosts have two

COX-2 forms, but that the stickleback, green spotted puffer, pufferfish, and Japanese medaka

have two COX-1 forms instead (named COX-la and COX-lb) (Ishikawa et al., 2007). They

concluded that a genome duplication before the teleosts, and a subsequent loss of either one









COX-1 or COX-2 form characterizes the species mentioned (Ishikawa and Herschman 2007)

(Fig. 1-2). This is supported by the apparent genome duplication events in teleosts (Crollius and

Wessenbach, 2005).

Other Vertebrates

Although functional studies on prostaglandin roles and cyclooxygenase are not

uncommon in other vertebrates, cyclooxygenase sequences from non-mammalian and non-

teleost vertebrates are lacking. Cyclooxygenase forms have been predicted from the chicken

genome (Gallus gallus), suggesting that birds have both COX-1 and COX-2 forms (Urick and

Johnson, 2006). Both forms have also been found in the African clawed frog (Xenopus laevis)

and COX-2 has been found in the western clawed frog (Xenopus tropicalis), suggesting that both

forms occur in amphibians (Klein et al., 2002). Functional studies suggest COX exists in reptiles

as well (Seebacher and Franklin, 2003) and the recently completed Anolis carolinensis genome

contains both COX forms.

Taken together, the presence of COX-1 and COX-2 forms in the mammals, teleosts,

birds, and amphibians suggest that both forms originated in the early vertebrates, possibly from

the duplication of an ancestral COX gene. The presence of sCOX in the dogfish indicates that

COX forms in the early vertebrates may not have diverged (in sequence or possibly in function)

to the extent that allows them to be labeled as COX-1 or COX-2, although it is predicted that two

COX forms exist in all vertebrates, since sCOX groups with COX-1 and is not ancestral to the

COX-1/COX-2 split (Fig. 1-2, unpublished phylogeny generated using the online program

PHYML) (Guindon and Gascuel, 2003; Guindon et al., 2005).

Other Chordates

Although the subphylum Vertebrata includes the majority of species in the phylum

Chordata, two other subphyla are included in the chordates: the Urochordata (sea squirts or









tunicates) and the Cephalochordata amphioxuss or lancelets). These other subphyla are basal to

the vertebrates and represent evolutionarily distinct monophyletic groups (Fig. 1-3). There is

some debate as to whether the urochordates or cephalochordates represent the base of the

chordate phylogeny, although urochordates have been generally been considered basal (Pough et

al., 2005).

Interestingly, the genomes of sea squirts (Ciona intestinalis and Ciona savignyi) predict

COX genes that are not homologous to COX-1 and COX-2. These forms (named COXa and

COXb) are basal to vertebrate COX forms and indicate an evolutionarily distinct branch in COX

evolution (Jarving et al. 2004) (Fig. 1-4). This separate branch is situated between the COX of

corals and the vertebrate COX forms (Fig. 1-4). This suggests that the COX-1 and COX-2 forms

originated sometime after the origin of the sea squirts but before the origin of the elasmobranchs

(around 550-600 mya).

Cyclooxygenase Function in Mammals

Cyclooxygenase was first described from mammals, and due to its medical importance,

its function has been intensely examined in mammals, specifically humans. Since COX

stimulates the production of prostaglandins, COX function is intrinsically linked to the roles that

prostaglandins play in everything from water balance to reproduction. Here, I review the major

functions of COX-1 and COX-2 in various mammalian physiological systems (modeled after

Vane et al., 1998), but it should be noted that mammalian COX studies are ongoing and that

COX likely plays many roles not described here.

Gastrointestinal Tract

In the stomach, prostaglandins play a cytoprotective role, guarding the stomach lining

against digestive acids and enzymes. The mechanism behind this protection is an increased

mucosal blood flow caused by the vasodilating properties of prostaglandins (specifically









Prostaglandin 12). It has generally been accepted that these prostaglandins are synthesized by

COX-1, although COX-2 is present in small amounts (Kargman et al., 1996). This is supported

by studies showing that irradiated mice have further reduced numbers of crypt cells

(cytoprotective mucosal cells) when indomethacin (a non-specific COX inhibitor) was

administered, but not when a COX-2 selective inhibitor was administered (Cohn et al., 1997).

COX-2 expression in the esophagus of patients with gastroesophageal reflux disease was

upregulated in response to acid exposure, suggesting a protective function in the distal esophagus

(Lurje et al., 2007). This may indicate that throughout the gastrointestinal (GI) track, COX-2 is

inducibly expressed in response to inflammation responses.

Furthermore, throughout the GI track, inflammatory mediators such as COX may also

serve to repair damaged mucosa (Martin and Wallace, 2006). While it seems that COX-1 is

responsible for baseline prostaglandin expression in the GI tract, COX-2 and not COX-1 seems

to be more involved in response to GI tract cancers (Ristimaki et al., 1997). However, recent

studies on patients with gastric cancer from Dalian, China showed that COX-2 expression was

infrequent in gastric cancers due to hypermethylation and that a COX-2 focused strategy for

treatment would likely be ineffective (Huang et al., 2006).

The Kidney

Prostaglandins can modulate blood flow in the kidney, as in other organs (Vane and

Botting, 1994). In addition, COX-2 has been shown to play a major role in ion regulation and

water balance in the mammalian kidney (Harris et al., 1994). It has also been shown that the

COX-2 selective inhibitor SC-58236 and several non-selective COX-inhibiting NSAIDs

(including sulindac, ibuprofen, and indomethacin) cause medullary interstitial cell (MIC) death

in the kidney, but the COX-1 selective inhibitor SC-58560 was 100-fold less potent for inducing

MIC death (Hao et al., 1999). COX-2 knockout mice also develop terminal nephropathy









(abnormal kidney function) which results in death by the third month (Morham et al., 1995).

These results suggest that COX-2, and not COX-1, plays a critical role in kidney function via

MIC cell survival. This is contrary to the original description of COX-1 as the constitutively

expressed form responsible for maintaining homeostasis and COX-2 as the inducible form

responsible for inflammatory responses.

In the kidney, COX-2 is present in a subset of tubular epithelial cells located in the cortex

and outer medulla (Vio et al., 1997). These cells are part of the medullary thick ascending limb

(MTAL), the part of the nephron that regulates extracellular fluid volume by establishing the

osmotic gradient in the medulla that concentrates urine (Hebert et al., 1981; Hebert and Andrioli,

1984). In vitro MTAL preparations have demonstrated high levels of Prostaglandin E2 (PGE2)

(Ferreri et al., 1984; Hirano et al., 1986; Lear et al., 1990) which has been shown to inhibit

basolateral Na+/K -ATPase (NKA) and apical Na+/K+-2C1 cotransporter (NKCC) in the MTAL

(Kaji et al., 1996; Stokes and Kokko, 1976; Wald et al., 1990). These proteins are critical for

NaCl reabsorption in the MTAL. Furthermore, the COX-2 selective inhibitor NS-398 has been

shown to prevent tumor necrosis factor (TNF) mediated PGE2 production in cultured MTAL

cells (Ferreri et al., 1999). TNF is a cytokine expressed in the MTAL that may be an important

mediator of ion transport by inhibiting ion uptake via a prostanoid dependant mechanism

(Escalante et al., 1994; van-Lanschot et al., 1991). Taken as a whole these results show that

COX-2 may influence ion uptake via TNF mediated prostaglandin production in the MTAL.

Therefore, COX-2 is involved in cell survival and ion transport in the mammalian kidney.

This finding is critical for treatment protocols using COX inhibitors. NSAIDs are the

most common drugs used to treat pain and inflammation with over 30 million uses per day

worldwide (Singh and Triadafilopoulos, 1999). Although the negative side effects









(hypertension, renal ischemia, and GI toxicity) associated with chronic NSAID use have been

known for some time, the discovery of COX-2 selective inhibitors suggested a treatment for

inflammation without causing disruption of normal COX function (thought to be mediated by

COX-1). The role of COX-2 in the kidney has reduced the supposed power of these new drugs

and negative side effects associated with COX-2 selective inhibitors are now well documented

(see Harris and Breyer, 2006 for a review). Furthermore, in 2005 the Food and Drug

Administration (FDA) required additional warning labeling of celecoxib to highlight side effects

and required Pfizer to remove valdecoxib from the market, both of which are selective COX-2

inhibitors.

Platelets

Interestingly, COX-1 seems to be the only form expressed in mammalian platelets.

COX-2 was not detected in human platelets using western blotting techniques (Reiter et al.,

2001) or in canine platelets using northern blotting techniques (Kay-Mugford et al., 2000). In

the platelet, COX-1 is involved in platelet aggregation (clotting) through the production of

Thromboxane A2 (TXA2). When inhibited with aspirin, COX-1 mediated production of TXA2 is

irreversibly inhibited, causing reduced platelet aggregation (blood thinning). This effect occurs

about an hour after oral ingestion of even low doses of aspirin and can inhibit platelet function

for several days after a dose (Vane et al., 1998; Vane, 1971). Inhibition of COX-1 by resveratrol

and other components of red wine is responsible for the anti-platelet activity associated with the

cardiovascular benefits of red wine consumption (Szewczuk and Penning, 2004).

Reproduction

Cyclooxygenase and the prostaglandins it produces play critical roles in almost every

aspect of reproduction in mammals, although its role in pregnancy has been studied the most









intensely. Both isoforms are likely involved in reproductive processes, with COX-1 expression

constitutive and COX-2 expression inducible (Chakraborty et al., 1996).

In a recent study, COX-2 was shown to contribute to fertility in male mice (Balaji et al.,

2007). COX-2 expression was intense in the epithelial cells of mice vas deferens and the COX-2

specific inhibitor nimesulide was shown to decrease sperm motility six hours after administration

(Balaji et al., 2007). Interestingly, although prostaglandin levels initially dropped after

nimesulide administration, they tended to rise after sustained COX-2 inhibition (Balaji et al.,

2007). This suggests that COX-1 expression may recover COX function or at least contributes

heavily to prostaglandin production in the vas deferens. A similar situation exists with respect to

human female fertility, in which COX-1 expression can recover function in cases where COX-2

is lost or inhibited (Wang et al., 2004). It also seems that COX-2 is critical for follicle

development and ovulation in women and that COX inhibitors can have severe effects on female

fertility (Sirois et al., 2004).

Prostaglandins are also important during pregnancy and labor. Generally, COX-1 is

expressed more than COX-2 during normal stages of pregnancy, and COX-1 may play an active

role in maintaining a healthy pregnancy (Trautman et al., 1996). COX-1 is expressed at high

levels is the fetal brain, kidneys, heart, and lungs (Gibb and Sun, 1996). However, both COX-1

and COX-2 are expressed in the uterine epithelium during early pregnancy and may play roles in

implantation and placental formation (Chakraborty et al., 1996). This was recently confirmed

for COX-2 in rats (Diao et al., 2007). COX-2 may play a dominant role in inducing labor.

During labor, contractions are stimulated by prostaglandins and therefore any effect causing

increased COX activity will induce labor. Up-regulation of COX-2 during intrauterine infection

may increase prostaglandins to the point of causing premature labor (Spaziani et al., 1996).









Furthermore, a similar up-regulation in sheep may reduce progesterone levels to the point that

pregnancy cannot be maintained (McLaren et al., 1996). However, it was recently shown that

during the course of pregnancy there is a gradual change in COX expression, with COX-1

dominating during early pregnancy and COX-2 dominating in later stages (Johnson et al., 2006).

It was concluded that while COX-2 is expressed at high levels in late stages of pregnancy, it is

not up-regulated suddenly to induce labor (Johnson et al., 2006). COX inhibitors can halt

premature labor by inhibiting prostaglandin production (Sawdy et al., 1997). However, as in

treatment strategies that use COX inhibitors for other problems, there are negative side effects

associated with the kidneys: renal clearance and glomerular filtration were reduced during the

first weeks of life in babies that were exposed to COX inhibitors to stop premature birth

(Allegaert et al., 2006). As in other cases, COX-2 selective inhibitors were thought to eliminate

these negative side effects but have not proven more effective than non-specific NSAIDs (Olson

2005).

Nervous System

COX-1 and COX-2 are both expressed in the mammalian nervous system and may

participate in nerve transmission, sensory processing, thermoregulation, and pain effects. As in

other systems, the use of COX inhibitors to treat neural diseases (such as Alzheimer's) is

prevalent, but is accompanied by undesired side effects that are not prevented by using COX-2

selective inhibitors.

The brain is one of the few organs that constitutively expresses COX-2 and contains high

amounts of COX-2 in the cortex, hippocampus, hypothalamus, and spinal cord (Breder et al.,

1995), whereas COX-1 is abundantly expressed in the forebrain, where it may be involved in

sensory information processing (Yamagata et al., 1993). COX-2 is expressed in both neuronal









and non-neuronal cells in the brain and is up-regulated in response to abnormal nerve activity,

suggesting a role in nerve transmission (Breder et al., 1995; Breder and Saper, 1996).

COX-2 in the spinal cord has been shown to modulate pain responses (nociception) in

both humans and rats due to peripheral pain stimulation (Martin et al., 2007; Gardiner et al.,

1997; Willingale et al., 1997). These results are expected since prostaglandins are generated at

the ends of sensory neurons during inflammatory pain (Ferreira 1972; Woolf et al., 1997).

However, the up-regulation of COX-2 over COX-1 in the spinal cord during inflammatory pain

suggests a role during pain processing in the central as well as the peripheral nervous system.

This up-regulation has been detected via increases in COX-2 mRNA (Gardiner et al., 1997;

Beiche et al., 1996) in the spinal cord of rats during inflammatory stimuli. Furthermore, COX-2

selective inhibition also affected the withdrawal reflex of rats when stimulated using a non-

inflammatory, electrical stimulus (Willingale et al., 1997). This supports the general role of

COX-2 in nociception in the spinal cord.

COX-2 also plays a thermoregulatory role in the brain by increasing prostaglandin

production in the hypothalamus during periods of fever. Injection of bacterial

lippopolysaccarides (LPS) into mammals causes a monophasic fever characterized by a single

rise in core body temperature. It has long been known that PGE2 levels increase in the

hypothalamus during fever and likely mediate the response (Ivanov and Romanovsky, 2004).

Early studies suggested that an up-regulation of COX-2 was the cause of increased PGE2 levels

during fever (Cao et al., 1997). This initial hypothesis has been confirmed, recently in COX-2

deficient mice which do not respond to LPS injection, in contrast to COX-1 deficient mice or

control mice (Steiner et al., 2005).









Cyclooxygenase Function in Non-Mammals

Although COX has been extensively studied in mammals due to its role in inflammation

and the correlating drug market surrounding COX inhibition, studies in non-mammals are

lacking. Unfortunately, it seems that although non-mammals posses the mammalian COX-1 and

COX-2 forms, these forms may be substantially different (both in structure and function) from

their mammalian counterparts (Grosser et al., 2002) and may also represent independent

evolutionary events, such as duplications of an isoform (teleosts) or independent origins (sea

squirts). In this section, I review the major studies investigating COX function in non-

mammalian chordates.

Fishes

Cyclooxygenase has likely been studied in the fishes more than other non-mammals due

to their status as model organisms. As noted above, COX forms have been found in several

fishes and along with genomic sequence data there is a general consensus that fishes possess

both COX-1 and COX-2 forms that are homologous to those found in mammals. However, there

is recent evidence that teleosts also possess an additional form of COX-1 or COX-2 (Ishikawa et

al., 2007; Ishikawa and Herschman, 2007) and some functional data also exist for these forms.

Osmoregulation

In fishes, the gills are the dominant site of acid/base regulation, nitrogenous waste

secretion, gas exchange, and ion transport (Evans et al., 2005). Several studies have investigated

the osmoregulatory role of COX in the gills of the euryhaline killifish, Fundulus heteroclitus.

This teleost can withstand instant salinity transfer between full strength seawater and fresh water

without any apparent physiological stress (Wood and Laurent, 2003). One study showed that

short circuit current across the opercular epithelium (a tissue with known ion transport

capabilities) was reduced by using a non-specific COX inhibitor (Evans et al., 2004). This









suggests that COX-2 plays a role in ion transport in fishes that may be similar to its function in

the kidneys of mammals. Further studies supported this initial hypothesis by showing that COX-

2 is expressed most abundantly in the gill, opercular epithelium, and kidney of the killifish (Choe

et al., 2006), tissues that are known to be involved in ion transport in fishes. This is supported in

the zebrafish, which shows the highest levels of COX-2 in the gills (Grosser et al., 2002).

Furthermore, COX-2 was localized in mitochondrion-rich cells in the gills of killifish, the main

sites of ion uptake and secretion in the gills (Choe et al., 2006). Finally, COX-2 expression was

shown to significantly increase following either hypotonic or hypertonic salinity transfers,

suggesting COX-2 may play a role in maintaining cell homeostasis or promoting cell survival

during periods of osmotic shock (Choe et al., 2006). Taken together, these results strongly

suggest that COX-2 in the gills of teleosts acts in the same way as COX-2 in the kidneys of

mammals, including regulating ion transport and promoting cell survival.

Cyclooxygenase has also been shown to play an osmoregulatory role in the rectal gland

of the dogfish shark, Squalus acanthias. Sharks rely on the rectal gland rather than the gills for

regulation of salt secretion and a COX form (sCOX) has been cloned from the rectal gland (Yang

et al., 2002). This form was expressed most abundantly in the rectal gland of the shark, where

PGE2 production was also high (Yang et al., 2002). Finally, using a COX-2 specific inhibitor,

vasoactive intestinal peptide mediated chloride secretion decreased in the rectal gland, but then

recovered following removal of the inhibitor (Yang et al., 2002). Even though sCOX is slightly

more similar to COX-1 of mammals than COX-2, this result can be explained by the presence of

valine at position 523 instead of isoleucine (and thus conferring COX-2 inhibitory properties).

This result suggests that sCOX plays a role in ion transport in the rectal glands of sharks that

may be similar to the gills of teleosts or the kidneys of mammals. The osmoregulatory role of









COX in fishes needs to be examined in greater detail using other species and diverse techniques.

This may be highly feasible because COX sequences exist for a wide range of teleosts.

Reproduction

As in mammals, COX has been shown to play a role in reproduction in fishes. By using

the non-specific COX inhibitor indomethacin, it was shown in the Atlantic croaker

(Micropogonias undulatus) that COX pathways may play a role in the maturation of ovarian

follicles and ovulation through prostaglandin formation, although other proteins may play a more

dominant role (Patifio et al., 2003). Results from the European sea bass also indicate a similar

role for prostaglandins in ovulation, with indomethacin inhibiting follicle maturation (Sorbera et

al., 2001). However, it has been shown in the brook trout that indomethacin does not block

ovulation (Goetz et al., 1989), although it does in other fish species (Goetz et al., 1991). This

apparent loss of function in the brook trout may be explained by changes in the levels of COX-1

and COX-2 during ovulation. It was shown that COX-1 levels remained constant and high

during ovulation but that COX-2 levels did not increase prior to ovulation as they do in mammals

(Roberts et al., 2000). This is supported by data from the zebrafish which show high levels of

COX-1 but not COX-2 in the ovaries (Grosser et al., 2002). This suggests that COX-2 function

in the reproduction of the brook trout (and perhaps other fishes) is different than in other

vertebrates. In the Japanese medaka, it was shown that low chronic levels (perhaps comparable

to those found in waste water) (Metcalfe et al., 2003) of the non-specific COX inhibitor

ibuprofen causes altered reproduction by decreasing the number of spawning events but

increasing the number of eggs per spawning event (Flippin et al., 2007). This conforms to

delayed pregnancies associated with NSAID use in mammals. These results indicate that COX

likely plays a role in fish reproduction, although it may vary among species.









Differential functions of novel forms

Recently, the discovery of novel COX-2 forms in the zebrafish (Ishikawa et al., 2007)

and rainbow trout (Ishikawa and Herschman, 2007) and analyses of genomic sequence data

indicates that all teleosts may posses at least three COX forms: either COX-la, COX-lb, and

COX-2 or COX-1, COX-2a, and COX-2b. Preliminary data on the described COX-2 forms

suggests that they are functionally different from each other. In the zebrafish, COX-2a and

COX-2b are expressed at different levels in the gill, kidney, and other tissues and respond

differently to 12-O-tetradecanoylphorbol-13-acetate (TPA, which is known to induce COX-2

expression in mouse) (Ishikawa et al., 2007). A similar result was found in the rainbow trout,

with differential expression of the two forms due to TPA (Ishikawa and Herschman, 2007).

Interestingly, COX-2a and not COX-2b was up-regulated in response to lippopolysaccarides

(Ishikawa and Herschman, 2007). Clearly, the discovery of these new forms indicates a new

chapter in studying COX function and further studies are needed to determine the functional

differences among the teleosts' forms of COX.

Other functions

Cyclooxygenase has also been implicated in determining body plan development during

embryonic stages in the zebrafish (Grosser et al., 2002; Cha et al., 2005; Cha et al., 2006a; Cha

et al., 2006b; Yeh and Wang, 2006). Briefly, COX may function to signal cell motility during

gastrulation (Cha et al., 2006b), and COX-1 function has been shown to participate in vascular

tube formation during development (Cha et al., 2005). COX-2 expression has also been shown

to increase in response to lippopolysaccarides in the rainbow trout (Brubacher et al., 2000;

Holland et al., 2002), suggesting an immunoregulatory role for COX-2 in fish as in mammals

(e.g., fever induction). Clearly, the function of COX in fishes is just beginning to be explored,









and further studies will likely indicate more similarities and differences with COX functions in

mammals.

Amphibians

As in other vertebrates, there is evidence that COX is involved in ovulation and

reproduction in amphibians. Indomethacin was shown to inhibit oocyte ovulation in the frog

(Rana temporaria), while Prostaglandin F2u was shown to promote ovulation (Skoblina et al.,

1997). This suggests another mediator may be influencing PGF2, production in the frog, but this

contrast between seemingly related proteins has not been confirmed in other species. COX may

also function in testosterone synthesis via PGE2 in the crested newt, Triturus carnifex (Gobbetti

and Zerani, 2002). Ion and water transport have also been studied and non-specific COX

inhibitors reduce short circuit current and voltage potential across the frog skin, bladder, and

corneal epithelium (Shakhmatova et al., 1997; Carrasquer and Li, 2002). As in reptiles, fever

and thermoregulatory changes are behaviorally induced in amphibians. Injection of

lippopolysaccarides (LPS) normally causes a behaviorally induced fever in toads. However,

when indomethacin is administered, the fever induced by LPS is completely blocked in the toad

(Bufoparacnemis) (Bicego et al., 2002). This indicates that the COX pathway plays a role in the

behaviorally induced fever caused by LPS and that this response has an ancient origin that may

be conserved in all tetrapods.

Reptiles

Cyclooxygenase has not been studied extensively in the lizards, crocodilians, snakes, and

turtles although some data suggest prominent roles in thermoregulation. Reptiles undergo

behavioral thermoregulation to adjust their body temperatures, and this process is amplified by

changes in heart rate. Specifically, an increase in heart rate (heart-rate hysteresis) accompanies

an increase in temperature. Prostaglandins and cyclooxygenase were shown to play a role in this









process in the lizard Pogona vitticeps. Inhibition of COX-1 and COX-2 resulted in no increased

heart rate during heating (Seebacher and Franklin, 2003). Furthermore, administration of

Prostaglandin F2a and Prostaglandin 12, but not Thromboxane B2, caused an increased heart rate

(Seebacher and Franklin, 2003). However, this effect was not seen in crocodiles (Crocodylus

porosus) (Seebacher and Franklin, 2004) or the lizard Phrynocephalusprzewalskii (Liu et al.,

2006), suggesting that thermoregulatory functions of COX via changes in heart rates may vary

between closely related species.

Interestingly, COX was also implicated in tail regeneration in the house lizard. Twenty

days after tail removal (the time period associated with tissue differentiation), an increase in

cyclooxygenase activity occurred along with the appearance of endogenous Prostaglandin E2,

which may signal a cascade resulting in tissue differentiation (Jayadeep et al., 1995). Finally,

indomethacin was used in the turtle bladder to show that prostaglandins likely do not play a role

in the inhibition ofNa+ or H+ transport due to high intracellular calcium (Arruda, 1982). COX

function in reptiles obviously needs further study as many functions associated with COX in

mammals have yet to be investigated in reptiles and there have been no COX targeted

sequencing efforts.

Birds

Cyclooxygenase and prostaglandin function have been studied somewhat in model avian

species, specifically chickens, where there is support that COX is involved in similar processes

as in mammals, (e.g., reproduction). COX-2 (but not COX-1) and prostaglandin activity was

shown to increase with administration of transforming growth factor in granulosa cells of white

leghorn hen follicles, suggesting that COX-2 and not COX-1 plays a role in granulosa cell

proliferation during follicular development (Li et al., 1996). It was also shown that

indomethacin reduced the proliferation of granulosa cells (Jin et al., 2006). When using COX-1









and COX-2 specific inhibitors, it was found that both types reduced granulosa cell proliferation,

but that COX-2 specific inhibitor showed a stronger effect, supporting original studies that COX-

2 is the dominant form in granulosa cell proliferation (Jin et al., 2007). However, COX-1 was

found to be expressed highly in the brain and seminal vesicle of the chicken (Reed et al., 1996).

COX-1 (but not COX-2) is also up-regulated in ovarian cancers occurring in hens, suggesting a

target for treatment (Urick and Johnson, 2006). COX inhibitors, both specific and non-specific,

were shown to decrease sperm motility in the domestic turkey, demonstrating a further role for

COX in bird reproduction (Kennedy et al., 2003).

As in mammals, COX-2 has been implicated with the detection and persistence of

peripheral inflammatory pain in chickens. The number of COX-2 containing neurons increased

significantly in laminae under inflammatory conditions, but not under control conditions, 12-24

hours after injection of Freund's adjunvant (Yamada et al., 2006). This suggests that the numbers

of COX-2 containing neurons are related to inflammatory pain as in mammals. It appears that

COX may not play a thermoregulatory role in birds as it does in mammals because indomethacin

did not cause any change in rectal temperature during heat stress in chickens (Furlan et al.,

1998). However, COX and its prostaglandins may play a role in memory retention in birds,

because inhibitors produce amnesic effects (Holscher, 1995). Finally, COX-2 is expressed in the

kidneys during development in chickens (Mathonnet et al., 2001) and at high levels in the

kidneys of adults (Reed et al., 1996).

Study Overview

As outlined above, the family of cyclooxygenase genes has an interesting and

functionally important history in the vertebrates. Drugs derived from cyclooxygenase function

are among the most important and widely used treatment methods in medicine. However, COX

function and evolution is not fully understood in the ancestral chordates, specifically the teleosts,









sharks, hagfish, lampreys, cephalochordates, and urochordates. Indeed, the origin of the

mammalian forms of COX is unclear since the sea squirt possesses evolutionarily distinct COX

forms. Resolving the evolution of COX in the chordates could prove invaluable in determining

new targets for drug development (Searls, 2003).

In Chapter 2, I describe the cloning and characterization of 9 new COX genes in the

chordates. These include COX-1 and COX-2 forms from teleosts as well as novel forms from

the hagfish, lamprey, and amphioxus. In phylogenetic analyses, these forms indicate that several

origins of COX occurred during chordate evolution, with the mammalian COX-1 and COX-2

forms likely originating with the hagfish craniatess). Data from the teleosts supports the findings

of Ishikawa and colleges that teleosts in the Acanthopterygii posses two COX-1 forms and one

COX-2 form, whereas earlier teleosts such as the zebrafish and rainbow trout posses two COX-2

forms and one COX-1 form. Furthermore, analyses of protein alignments from novel sequences

indicate conserved and derived functional residues and areas between novel COX forms and the

COX-1 and COX-2 forms of mammals.

In Chapter 3, I summarize the findings of this research and suggest future avenues of

research based on the results presented here. I also present preliminary data investigating the

possible function of cyclooxygenase in these ancestral chordates.














Arachidonic Acid


COX-1
or
COX-2


0
0






Prostaglandin 12



HO C ,-OOH


OH OH
6-keto-Prostaglandin F1

OH


o OH
Prostaglandin D2


ODH
Prostaglandin G2

COX-1
or
COX-2

: %-Q--^G^^OOH
0 .... V
OH
Prostaglandin H2








H iH
-- oOH


OH
Thromboxane A2




HO C -H
OH
Thromboxane B2

v\ COOH

OH OH
Prostaglandin E2


Prostaglandin F2a



Figure 1-1. Cascade of reactions depicting the conversion of arachidonic acid to the primary
prostaglandins via catalyzation with either cyclooxygenase (COX) form. Arachidonic
acid is obtained from the conversion of linoletic acid found in plants or directly from
other animals. It is then oxidized by COX to produce Prostaglandin G2 which is
subsequently reduced by COX to form Prostaglandin H2. Prostaglandin H2 can then
be converted into the other prostaglandins via other enzymes (Vane et al., 1998).











COX-2a Danio


I COX-2a Oncorhynchus
COX-2b Danio
COX-2b Oncorhynchus
COX-2 Acanthopterygians


sCOX Squalus
COX-la Acanthopterygians
COX-1 b Acanthopterygians
COX-1 Danio

COX-1 Oncorhynchus
Figure 1-2 General phylogeny representing the different COX forms in the fishes. Based on
recent studies, it appears that teleosts have multiple forms of COX-1 or COX-2
(Ishikawa et al., 2007; Ishikawa and Herschman, 2007). It is hypothesized that with
the origin of the teleosts, COX-1 and COX-2 underwent duplication, but that in one
lineage (represented by the zebrafish and rainbow trout) one COX-1 form was lost
and in another lineage (represented by the Acanthopterygians) one COX-2 form was
lost. Also included is the form cloned from the spiny dogfish (Yang et al., 2002)
which loosely groups with COX-1 forms, although it shares near equal identity with
COX-1 and COX-2. This phylogeny was simplified from an unpublished phylogeny
generated using the online program PHYML (Guindon and Gascuel, 2003; Guindon
et al., 2005). Abbreviations: Danio = Danio rerio (zebrafish), Oncorhynchus =
Oncorhynchus mykiss (rainbow trout), Squalus = Squalus acanthias (spiny dogfish).
The Acanthopterygians clade is represented by the stickleback (Gasterosteus
aculeatus), green spotted puffer (Tetraodon nigroviridis), pufferfish (Takifugu
rubripes), and Japanese medaka (Oryzias latipes).













Precambrian Paleozoic Mesozoic Cenozoic
Cambrian Ordovician Silurian Devonian Carboniferous Permian Triassic Jurassic Cretaceous Tertiary
600 mya 543 490 443 417 354 290 251 206 144 65

To
tetrapods



-Teleostei


Petromyzontoidea

Myxinoidea

Cephalochordata

*Urochordata



Figure 1-3 Accepted evolution of ancestral chordates (Pough, et. al, 2005). Black lines show
relationships only; they do not indicate times of divergence nor the unrecorded
presence of taxa in the fossil record. Bars shaded red indicate ranges of time when
the taxon is known to be present. The subphylum Vertebrata includes the
Myxinoidea hagfishess), Petromyzontoidea (lampreys), Teleostei (teleosts), and
tetrapods. There is some controversy over whether the Urochordates (sea squirts) or
the Cephalochordates (lancelets) represent the most basal chordate lineage. Also, it
has historically been suggested that the hagfish and lamprey constitute a
monophyletic group (the Agnathans or Cyclostomes).










COX-1 Vertebrates




COX-2 Vertebrates


COXb Urochordates

COXa Urochordates

Invertebrate COX forms


Figure 1-4 Evolution of COX forms in the chordates, based on Jarving et al. (2004). The
Urochordates (represented by Ciona intestinalis and Ciona savignyi) have COX
forms that represent an evolutionary distinct branch ancestral to the COX-1 and
COX-2 forms of the vertebrates. In this tree, the vertebrates are represented by the
teleosts, mammals, Xenopus, and Gallus









CHAPTER 2
CYCLOOXYGENASES IN THE ANCESTRAL CHORDATES: SEQUENCE AND
PHYLOGENETIC ANALYSES

Introduction

Cyclooxygenase (COX) is the enzyme that catalyzes the oxidation and subsequent

reduction of arachidonic acid to form Prostaglandin G2 and Prostaglandin H2 (PGH2). PGH2 can

then undergo additional reactions to produce the primary prostaglandins, which act as vascular

tone regulators in the vertebrates. Prostaglandins and COX participate in a variety of

physiological functions in the vertebrates, including inducing fever, maintaining pregnancy, and

regulating ion transport in the kidneys (Steiner et al., 2005; McLaren et al., 1996; Harris et al.,

1994). These functions have been extensively studied in mammals (Vane et al., 1998), but

comparatively little functional data exist for other animals. However, COX has been sequenced

in several evolutionarily more ancestral chordates, particularly the teleosts (Zou et al., 1999;

Roberts et al., 2000; Choe et al., 2006). Based on functional studies of COX in the teleosts, it

seems that some functions are conserved (Choe et al., 2006; Sorbera et al., 2001; Brubacher et

al., 2000; Holland et al., 2003) while others may be altered in some species (Goetz et al., 1989)

or novel (Cha et al., 2006b).

In mammals there are two main forms of cyclooxygenase. The first form was isolated

from sheep seminal vesicles in 1988 and later named COX-1 (DeWitt and Smith, 1988; Merlie et

al., 1988; Yokoyama et al., 1988). A second form was isolated from mouse and chicken

fibroblast cell cultures in the early 1990s and named COX-2 (Kujubu et al., 1991; Xie etal.,

1991; O'Banion et al., 1992). Originally, COX-1 was considered to be a constitutive form that

maintained normal cell functions and COX-2 was considered to be an inducible form that was

up-regulated in inflammatory responses (Funk, 2001). However, studies have shown that this is

an oversimplification and COX-2 is expressed constitutively in the brain (Breder et al., 1995)









and kidneys (Harris and Breyer, 2001) of mammals. This has led to the abandonment of COX-2

selective inhibitors (e.g., celecoxib and valdecoxib) which were thought to treat inflammatory

pain without the negative side effects associated with non-selective COX inhibition. Although

structurally and biochemically similar, COX-1 and COX-2 vary in expression in the vertebrates

and participate in different functions. For example, COX-2 but not COX-1 plays a role in

granulosa cell proliferation in chicken follicles (Li et al., 1996; Jin et al., 2007). Although the

amino acid sequences of COX-1 and COX-2 share about 63% similarity, the presence of valine

in COX-2 at position 523 instead of isoleucine is thought to be responsible for their differences

in substrate selectivity and sensitivity to specific inhibitors (Otto and Smith, 1995). Inhibitory

studies in mammals have suggested a COX-3 form also exists, but it has not been characterized

(Kis et al., 2006; Censarek et al., 2006).

Analyses of genomic sequence and targeted cloning efforts have demonstrated that, like

mammals, other vertebrates have COX-1 and COX-2 forms (Jarving et al., 2004). However,

some variation exists in the COX-1 and COX-2 dichotomy, notably in the more evolutionarily

ancestral chordates. Ishikawa and colleagues (Ishikawa et al., 2007; Ishikawa and Herschman,

2007) have recently shown that teleosts posses three forms of COX. The zebrafish (Danio rerio)

and the rainbow trout (Onchorhynchus mykiss) both posses two COX-2 forms (named COX-2a

and COX-2b) and one COX-1 form, whereas the stickleback (Gasterosteus aculeatus), green

spotted puffer (Tetraodon nigroviridis), pufferfish (Takifugu rubripes), and Japanese medaka

(Oryzias latipes) posses two COX-1 forms (named COX-la and COX-lb) and one COX-2 form.

They also showed that COX-2a and COX-2b are functionally different in the zebrafish and

rainbow trout. Also, a COX form (named sCOX) has been cloned from the spiny dogfish

(Squalus acanthias) and groups with COX-1 in phylogenetic analyses, but shares strong









sequence identity to COX-2 as well (Yang et al., 2002). Furthermore, sea squirts subphylumm

Urochordata) possess two forms of COX (named COXa and COXb) that do not correspond to

the COX-1 or COX-2 of vertebrates and represent an ancestral origin of COX in the chordates

(Jarving et al., 2004).

The current view of the evolution of cyclooxygenase in the chordates represents an

interesting but incomplete account of this gene family, both from a phylogenetic and functional

standpoint. Therefore, the first goal of this study was to pinpoint the origin of COX-1 and COX-

2 in the chordates by searching for COX forms in the sea lamprey (Petromyzon marinus),

Atlantic hagfish (Myxine glutinosa), and amphioxus (Branchiostoma lancelolatum). A second

goal was to confirm the findings of Ishikawa and colleagues by searching for the 3 forms of

COX in the euryhaline killifish (Fundulus heteroclitus) and longhorn sculpin (Myoxocephalus

octodecemspinosus). It was hypothesized that both teleosts would posses COX-la, COX-lb, and

COX-2 since both are in the Acanthopterygii clade of Teleostei and all other acanthopterygiians

posses COX-la, COX-lb, and COX-2. Here, I report the cloning of 9 novel COX sequences

from the species mentioned above. Sequence and phylogenetic analyses suggest that ancestral

COX forms may have had similar functions as COX-1 and COX-2 in mammals, which likely

originated with the origin of the vertebrates, although the hagfish and lamprey may have COX-1

and COX-2 forms that represent novel lineages.

Materials and Methods

Animals and Holding Conditions

All procedures were approved prior to beginning the experiment by the University of

Florida Institutional Animal Care and Use Committee (IACUC). Euryhaline killifish (Fundulus

heteroclitus) were captured from Northeast Creek near the Mount Desert Island Biological

Laboratory (MDIBL), Salisbury Cove, Maine using minnow traps. They were transported to the









MDIBL where they were kept in fiberglass tanks containing 100%, flowing seawater from the

Gulf of Maine. The tanks were exposed to natural conditions. Longhorn sculpin

(Myoxocephalus octodecemspinosus) were purchased from fishermen and were housed in a

similar way at the MDIBL. Atlantic hagfish (Myxine glutinosa) were also purchased from

fishermen and were housed at the MDIBL in 100% seawater and maintained on a 12 h light/dark

cycle. Female, non-migratory lampreys (Petromyzon marinus) were a generous gift from the

USGS Great Lakes Science Center at the Hammond Bay Biological Station in Millersburg,

Michigan and were dissected there. Lancelets (Branchiostoma lancelolatum) were purchased

from Gulf Marine Specimens (Panacea, FL) and were kept in the bags they were shipped in for a

few days before they were sacrificed. All housed animals were fed to satiation regularly.

Some killifish were shipped to the University of Florida (Gainesville, FL) for further

experiments. These killifish were housed as described previously (Choe et al., 2006). Some

hagfish were also transported to the University of Florida (Gainesville, FL) for further

experiments. These hagfish were housed for a few days in a large Rubbermaid tank in 100%

seawater at 4 C before they were killed.

Tissue Collection

After initial anaesthetization with MS-222 (-600 mg L-1), killifish, sculpin, and lampreys

were pithed and/or decapitated. The gill arches (1st and 2nd arches for lampreys, 2nd and 3rd

arches for teleosts) were then removed using sterile, RNAse free dissecting tools. Hagfish were

decapitated and all gill baskets were then removed using sterile, RNAse free tools. Lancelets

were cut in half with sterile, RNAse free tools. After removal, tissues were immediately placed

in liquid nitrogen and stored at -80 OC for 2 weeks to 4 months before further processing.









Reverse Transcription, Primer Design, PCR, Cloning, and Sequencing

Reverse transcription, PCR, cloning, and sequencing were performed as described

previously (Choe et al., 2006) with modifications. Total RNA was isolated from the gills of

killifish, lampreys, sculpin, and hagfish as well as the anterior half of lancelets using TRI-reagent

(Sigma, St. Louis, MO), and reverse transcribed with a SuperscriptTM II or SuperscriptTM III

reverse transcriptase kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocol and

using oligo-dT as a primer. The resulting cDNA was stored at -20 OC until used for PCR.

Degenerate primers (those labeled as CH COX, Table 2-1) were first designed to amplify

non-specific cyclooxygenases in the hagfish, lamprey, and lancelet based on conserved amino

acid sequences between COX-1 and COX-2 of Mus musculus, COX-2 of Fundulus heteroclitus,

sCOX of Squalus acanthias (GenBank accession numbers: NP_032995, NP_035328, and

AAS21313), and predicted COXa and COXb of Ciona intestinalis based on genomic data.

These primers were also later used to amplify COX-2 in sculpin. Another set of degenerate

primers (those labeled as CH COX-1 in Table 2-1) were designed to amplify COX-1 forms

specifically over COX-2 forms in teleosts based on conserved amino acid sequences between

COX-1 in Oncorhynchus mykiss, Danio rerio, Salvelinusfontanalis (GenBank accession

numbers: CAC10360, NP_705942, and AAF14529), and a predicted COX-1 of Tetraodon

nigroviridis based on genomic data. These primers first amplified a COX-la form in sculpin and

a COX-lb form in killifish (see below). A further set of degenerate primers (those labeled as CH

COX-la in Table 2-1) were designed to amplify COX-la forms specifically over COX-lb in

teleosts based on conserved amino acid sequences between predicted COX-la forms in

Gasterosteus aculeatus, Oryzias latipes, and Tetraodon nigroviridis based on genomic data and a

COX-la found in sculpin earlier in this study (see below). A final set of degenerate primers

(those labeled as CH COX-lb in Table 2-1) were designed to amplify COX-lb forms specifically









over COX-lb in teleosts based on conserved amino acid sequences between predicted COX-lb

forms in Gasterosteus aculeatus, Oryzias latipes, and Tetraodon nigroviridis based on genomic

data and a COX-lb found in killifish earlier in this study (see below). All degenerate primers

were designed using the online program Consensus-Degenerate Hybrid Oligonucleotide Primers

(CODEHOP) (Rose et al., 1998).

Initial PCR reactions were performed on 1/20th of a reverse transcriptase reaction with a

TaKaRa Ex TaqTM Hot Start DNA Polymerase Kit (Takara Bio Inc., Japan) in a PCR Express

thermocycler (ThermoHybaid, Franklin, MA) with the following parameters: initial denaturing at

95 C for 5 minutes, then 35 cycles of 30 seconds at 95 OC, 30 seconds at 60 OC, and 1.5 minutes

at 72 C. A final elongation step of 7 minutes at 72 C was performed for each PCR and then the

reaction was held at 4 OC. Initial PCR products were visualized by ethidium bromide staining on

1-2% agarose gels and then ligated into PCR4-TOPO vectors and transformed into TOP 10

chemically competent cells using a TOPO TA Cloning Kit (Invitrogen) following the

manufacturer's protocol. Plasmids were then isolated using a High Pure Plasmid Isolation Kit

(Roche, Germany) following the manufacturer's protocol and plasmid DNA was sequenced in

both directions at the Marine DNA Sequencing Center at the MDIBL.

After initial fragments were sequenced, Primer Express software (Applied Biosystems,

Foster City, CA) was used to design specific primers based on the initial fragments (Tables 2-2,

2-3, 2-4, 2-5, and 2-6). These specific primers, along with the original degenerate primers, were

used to extend COX sequences. PCR was completed as above with slight modifications to

annealing temperature and elongation time for each reaction, as well as nested PCR reactions in

some cases to increase specificity. Products were visualized, cloned, and sequenced as above.









5' and 3' Rapid Amplification of cDNA Ends (RACE)

After initial sequences were extended with specific primers, the 5' and 3' ends were

sequenced using 5' and 3' RACE. Following the manufacturer's protocols, a GeneracerTM Kit

(Invitrogen) was used to make 5' and 3' cDNA for hagfish, killifish, and sculpin. Initial

sequence data was used to make 5' and 3' RACE specific primers for each species (those labeled

with 5' or 3' in Tables 2-2, 2-3, 2-4, 2-5, and 2-6). These primers, along with kit primers that

anneal to the ends of the 5' and 3' cDNA, were used in touchdown PCR reactions with the

following parameters: 5 cycles of 30 seconds at 94 OC and 1.5 minutes at 72 C; 5 cycles of 30

seconds at 94 C and 1.5 minutes at 70 OC; and 25 cycles of 30 seconds at 94 OC, 30 seconds at

60 C, and 1.5 minutes at 72 C. As above, a final elongation step of 7 minutes at 72 C

terminated the reaction, which was held at 4 OC. For some reactions, annealing temperatures and

elongation times were slightly altered and nested PCR reactions were performed for some

reactions using specific primers (Tables 2-2, 2-3, 2-4, 2-5, and 2-6) to increase specificity.

Products were visualized, cloned, and sequenced as above.

Sequence Analysis

Initial sequences from degenerate primers, sequence extensions, and 5' and 3' RACE

were assembled using GeneTools software (BioTools Inc., Edmonton, Alberta) and the resulting

sequences were searched for open reading frames. The predicted amino acids were aligned with

other COX proteins using PepTools software (BioTools Inc.) for phylogenetic analysis. Multiple

alignments were also generated using ClustalW to search for conserved protein domains across

COX sequences. Alignments were manually adjusted and the N and C-terminal areas were

realigned using Microsoft Word based on previous COX alignments (Ishikawa et al., 2007; Yang

et al., 2002; Kulmacz et al., 2003). For the novel sequences, COX forms were aligned with









other relevant COX proteins and annotated using functional amino acids and domains

highlighted from Ishikawa et al. (2007), Yang et al. (2002), and Kulmacz et al. (2003).

Phylogenetic Analysis

To investigate the evolutionary relationships of the different forms of COX in the

chordates, phylogenies were generated using the novel amino acid sequences generated in this

study and several other COX protein sequences from GenBank and genome databases. All novel

protein sequences were included in the data set. COX sequences from teleosts were included in

the analysis only if all 3 forms of COX were available. These included the zebrafish, rainbow

trout, green spotted puffer, stickleback, euryhaline killifish, and longhorn sculpin. COX

sequences from non-teleosts were included in the analysis if at least two forms of COX were

available and both sequences were designated as Reference Sequences (RefSeqs) in GenBank.

These included the chicken, frog, mouse, rat, human, rabbit, dog, cow, sheep, and opossum.

COX sequences were also included if they represented an evolutionarily interesting group.

These included the dogfish shark and the sea squirt. In total, 50 protein sequences were included

in the analyses (Table 2-7).

Models of evolution to be used in phylogenetic analyses were evaluated to account for

amino acid substitutions, among-site variation, and invariable sites. Using likelihood ratio tests,

a model using the WAG rate matrix (Whelan and Goldman, 2001) and the gamma (F)

distribution for among-site variation was chosen (Table 2-8).

A distance phylogeny was first generated using Molecular Evolutionary Genetics

Analysis 4 (MEGA) software (Tamura et al., 2007) with a minimum evolution (ME) analysis

and 1000 bootstrap replicates. In this analysis the a parameter was fixed to the value estimated

from maximum likelihood (ML) analysis (a = 0.737, see below). Additionally, a JTT rate matrix

(Jones et al., 1992) was used instead of a WAG matrix because a WAG matrix is not available









for MEGA 4. A complete deletion option was also selected. After comparing results between

ML, ME, and Bayesian Phylogenetics (BP), lamprey and hagfish sequences were identified as

possible rouges and another distance analysis with identical parameters (but with a = 0.742) was

performed excluding these two sequences.

A maximum likelihood phylogeny (ML) was generated using the program PhyML

(Guindon and Gascuel, 2003). An initial phylogeny was generated using WAG analysis, 1000

bootstrap replicates, 8 substitution rate categories, and a gamma shape parameter with an

estimated a. Again, the analysis was repeated with the rouge lamprey and hagfish sequences

removed. To evaluate support for monophyletic groups, an approximate likelihood ratio test

(aLRT, Anisimova and Gascuel, 2006) was performed both including and excluding the lamprey

and hagfish sequences. This test used the minimum of Chi-Squared and Shimodaira-Hasegawa

(SH) support values.

Finally, A Bayesian phylogenetics (BP) analysis was also performed using an initial run

with 2 million generations and a final run with 10 million generations using the program

MrBayes 3.1.2. Once again, analyses were performed with and without hagfish and lamprey

sequences. Each run consisted of one cold and three heated chains (T= 0.2). The WAG + F

model was used with an estimated a. After discarding the first 10% of each run as burning,

posterior probabilities were calculated based on trees sampled every 500 generations.

Due to inconsistencies in tree topologies between the three methods used above,

alternative topologies were generated in which the positions of the hagfish and lamprey

sequences were forced to be included in different clades (Figure 2-15). These alternative

topologies were generated and viewed using TreeView 1.6.6. Alternative topologies were









evaluated using Ln L scores generated by maximum likelihood criterion with online execution in

PhyML (Guindon et al., 2005).

Results

Molecular Identification of Cyclooxygenases

Branchiostoma

Initial PCR reactions using the degenerate primers CH COX F3 and CH COX R2 (Table

2-1) amplified two 532 bp products from the lancelet (Branchiostoma lancelolatum). These two

products (referred to hereafter as COXc and COXd) were found to share the most identity with

COXa and COXb from the tunicate. COXc was 50.9% identical to COXa from the tunicate and

COXd was 58.8% identical to COXb from the tunicate. All attempts to extend these sequences

using specific primers designed against these sequences (Table 2-2) were unsuccessful. After

searching for open reading frames, two putative 177 amino acid proteins were predicted from the

lancelet.

Myxine

Initial PCR reactions using the degenerate primers CH COX Fl, CH COX F2, CH COX

R1, and CH COX R2 (Table 2-1) were used to amplify a single 1454 bp product from the hagfish

(Myxine glutinosa). This product (hereafter referred to as hCOX) was found to be 63.8%

identical to COX-1 of the zebrafish (Danio rerio). RACE primers (5' and 3', Table 2-3) were

designed against this initial product and used with kit primers to completely sequence the 2416

bp product. Searching for open reading frames yielded a putative 610 amino acid protein.

Petromyzon

Initial PCR reactions using degenerate primers CH COX F3 and CH COX R1 (Table 2-1)

amplified a single 541 bp product from gill tissue of the lamprey (Petromyzon marinus). This

product (hereafter referred to as 1COX) was later extended using degenerate primers (Table 2-1)









and specific primers designed against the initial 541 bp product (Table 2-4). Despite all attempts

to amplify the complete 1COX product (including RACE) the sequence was only extended to 860

bp. A 286 amino acid open reading frame was predicted from this product.

Fundulus

Because the COX-2 sequence for the euryhaline killifish (Fundulus heteroclitus) was

previously completed (Choe et al., 2006), the degenerate primer CH COX-1 F2 (Table 2-1)

designed to amplify COX-1 forms over COX-2 forms was used with the degenerate primer CH

COX R1 (Table 2-1) to initially amplify a non-COX-2, 1335 bp product (hereafter referred to as

COX-lb Fundulus) from killifish gill tissue. This product was then extended and completed

using specific primers and RACE primers (Table 2-5). The completed 2226 bp sequence

contains a 598 amino acid open reading frame that was nested inside the teleost COX-lb clade in

all phylogenetic analyses (see below), prompting the design of degenerate primers (Table 2-1)

that were used to amplify COX-la forms in teleosts over COX-lb forms. Using the degenerate

primers CH COX-la Fl and CH COX-la R1 (Table 2-1), a 759 bp product was initially

amplified in PCR reactions. Using RACE primers designed to complete this sequence (Table 2-

5), the 3' end of this product was completed, resulting in a 1655 bp product (hereafter referred to

as COX-la Fundulus) that contains a 452 amino acid reading frame. However, the 5' end of this

product was not sequenced despite multiple attempts. This putative protein grouped with the

COX-la sequences of other teleosts in all phylogenetic analyses (see below), leading to the

conclusion that the COX-la, COX-lb, and COX-2 sequences included in the analyses all

represent different COX products expressed in the gill tissue of the euryhaline killifish.

Myoxocephalus

Using the degenerate primers CH COX Fl and CH COX R2 (Table 2-1), a 977 bp

product was initially amplified in PCR reactions using cDNA from the gills of the longhorn









sculpin (Myoxocephalus octodecemspinosus). This product (hereafter referred to as COX-2

Myoxocephalus) was then extended and completed using RACE and specific primers (Table 2-

6), resulting in a 2618 bp product that contains a 605 amino acid open reading frame. This

sequence grouped with COX-2 forms from other teleosts in all phylogenetic analyses, inciting

the use of COX-1 degenerate primers (Table 2-1) CH COX-1 F2 and CH COX-1 R2 in PCR

reactions to amplify a 1000 bp product (hereafter referred to as COX- la Myoxocephalus). This

product was then extended and completed using RACE primers (Table 2-6), resulting in a 2633

bp product encoding a 622 amino acid open reading frame. This product grouped with COX-la

forms from other teleosts in all phylogenetic analyses, prompting the design of degenerate

primers used to amplify COX-lb forms in teleosts over COX-la forms (Table 2-1). Using the

degenerate primers CH COX-lb Fl and CH COX-lb RO (Table 2-1), an initial 895 bp product

(hereafter referred to as COX-lb Myoxocephalus) was amplified in PCR reactions. This product

was extended and completed using specific RACE primers (Table 2-6), resulting in a 2332 bp

product containing a 600 amino acid open reading frame. This product grouped with other

teleost COX-lb forms in all phylogenetic analyses, suggesting that the COX-2, COX-la, and

COX-lb forms sequenced represent 3 different COX forms expressed in the gills of the longhorn

sculpin.

Sequence Analyses

Teleosts COX-la, COX-lb, and COX-2

Three different COX forms are now available from the teleosts studied here: the COX-2

sequenced from the euryhaline killifish (Choe et al., 2006), the COX-2 sequenced here from the

longhorn sculpin, and two the COX-1 forms sequenced here from both the killifish and sculpin,

named as COX-la and COX-lb (following Ishikawa and Herschman, 2007). COX-la and COX-

lb predicted proteins from the killifish were more similar to Mus COX-1 than COX-2 (70%









versus 64% for COX-la and 68% versus 59% for COX-lb, respectively). Similarly, COX-2

from the sculpin was more similar to Mus COX-2 than Mus COX-1 (70% versus 60%) and

COX-la and COX-lb were more similar toMus COX-1 than Mus COX-2 (53% versus 47% for

COX-la and 67% versus 57% for COX-lb, respectively).

An alignment of COX forms from the killifish (Figure 2-1) shows that all the important

amino acid residues for cyclooxygenase function are conserved in all forms. However, because

COX-la is incomplete it is unknown whether the N-terminal characteristics are conserved in this

form. These include an active site tyrosine (Tyr-385, using ovine COX-1 numbering), haem-

binding histidines (His-207 and His-338), and the aspirin acetylation site (Ser-530) (Ishikawa et

al., 2007; Ishikawa and Herschman, 2007; Yang et al., 2002; Kulmacz et al., 2003). In addition

the COX-2 form contains a C-terminal amino acid insertion and the COX-1 forms contain an N-

terminal amino acid insertion. This is consistent with the COX-1 and COX-2 forms of other

vertebrates (Herschman et al., 2003; Kulmacz et al., 2003), although killifish COX-2 seems to

have a shorter than average insertion. In mammals, the amino acid residues thought to give

COX-1 and COX-2 their differing affinities for substrates are positions 513 (His in COX-1 and

Arg in Cox-2) and 523 (Ile in COX-1 and Val in COX-2) (Guo et al., 1996). However, as

previously noted in other teleosts (Grosser et al., 2002; Choe et al., 2006; Ishikawa et al., 2007)

these differences appear to be absent in teleosts, with Arg and Val present in positions 513 and

523, respectively, in both teleost COX-1 and COX-2. This is also the case in the killifish with

Arg in position 513 and Val in position 523 in all COX forms reported here.

As in the killifish, alignment of the three COX forms sequenced from the sculpin (Figure

2-2) shows conserved amino acid residues characteristic of COX function in all three forms. The

characteristic terminal amino acid insertions found in mammalian COX forms are also present in









their respective sculpin counterparts. However, COX-lb from the sculpin seems to have both N

and C-terminal insertions. Disulfide bonds near the n-terminus of sculpin COX-la also seem to

be altered due to the substitution of other amino acids for cysteine. It also appears that some

functional amino acid residues near the C-terminus of sculpin COX-la are not conserved with

the other forms. These include the substitution of Leu for Arg at position 513, Leu for Val at

position 523, and Phe for Ser at position 530.

Lamprey, hagfish, and amphioxus COX forms

The one incomplete putative COX protein obtained from the gills of the lamprey shares

near equal identity (63%) with Mus COX-1 and COX-2. The gene, referred to here as lamprey

COX (1COX) (Yang et al., 2002) shares the most identity (70%) with a form from the lancelet

(COXc), but shares notable identity (64%) with the form from the hagfish as well. Based on an

alignment of COX forms sequenced from select non-mammalian and non-teleost chordates

(Figure 2-3), the partial COX form sequenced from the gills of the lamprey has the predicted

histidine site (His-207) critical for peroxidase activity as well as conserved haem-binding

domains. However, the membrane binding regions near the Arg-277 loop that differ between

COX-1 and COX-2 forms appear to be different from both COX-1 and COX-2 in the lamprey

form. This is seen when the lamprey form is aligned with COX-1 and COX-2 forms from

vertebrates (Figure 2-4).

The putative COX protein from the gills of the hagfish shares slightly more identity with

Mus COX-2 than Mus COX-1 (63% versus 60%). The gene, referred to here as hCOX, shares

the most identity (> 60%) with vertebrate COX-2 forms, but also shares notable identity with

1COX (64%) and forms from amphioxus (> 60%). An alignment of hCOX with other non-

mammalian, non-teleost COX proteins (Figure 2-3) shows that all important amino acid residues

and binding sites critical to cyclooxygenase activity are conserved in hCOX. An alignment of









hCOX with COX-1 and COX-2 proteins from various vertebrates (Figure 2-5) shows that hCOX

has an insertion of amino acids near the C-terminal; characteristic of COX-2 proteins in

vertebrates. However, there are several regions where COX-1 differs from COX-2 but hCOX

shows no identity to COX-1 or COX-2 amino acids (indicated as sites with a + over them in

Figure 2-5).

Two putative incomplete COX sequences were obtained from amphioxus. These

sequences, referred to here as COXc and COXd, are most identical (81%) to each other, but also

share notable identity with hCOX (66%), 1COX (67%), vertebrate COX-1 (> 60%), and

vertebrate COX-2 (> 60%). They are the least identical to Ciona COXa (48%) and COXb

(55%). Based on an alignment of non-mammalian, non-teleost COX proteins (Figure 2-3), it

appears that amino acids critical to COX function are conserved in COXc and COXd. However,

the first haem-binding domain seems to be very different in the amphioxus forms. This is also

the case for Ciona COX forms and COXa, COXb, COXc, and COXd all share notable identity in

this region as shown by alignment (Figure 2-6).

Phylogenetic analyses

A phylogeny (Figure 2-7) generated under a distance optimality criterion with 1000

bootstrap replicates using VMEGA4 shows predicted COX clades. COX-la and COX-lb forms

from teleosts form monophyletic sister clades. The novel COX-1 forms from the killifish and

sculpin group together with other teleosts in these clades as expected based on their designation

here. Eutherian COX-1 forms group together in a monophyletic clade sister to the teleost COX-1

clade. The COX-1 proteins from Gallus, Monodelphis, and Xenopus are located at the base of

this monophyletic vertebrate COX-1 clade, with sCOX being the most basal. Vertebrate COX-2

forms group together in another monophyletic clade that is sister to the vertebrate COX-1 clade.

There are two monophyletic clades within the COX-2 group: a teleost clade and a tetrapod clade.









The forms from lamprey and hagfish (1COX and hCOX) are basal to the vertebrate COX clade,

while the forms from amphioxus (COXc and COXd) form a monophyletic group basal to these

forms. The forms from Ciona (COXa and COXb) form another monophyletic group that is basal

to all other COX forms. Bootstrap values give robust support for vertebrate clades, the

amphioxus clade, and the Ciona clade, but give weak support for placement of hCOX, 1COX,

and sCOX. Based on these results and other generated phylogenies (see below), hCOX and

1COX were identified as possible rogue sequences due to their inconsistent positions depending

on the type of analysis used and another distance analysis was completed without including these

sequences (Figure 2-8). The topology of this phylogeny is very similar to the complete COX

phylogeny. However, sCOX is basal to the vertebrate COX clade and not included with the

other vertebrate COX-1 forms. Additionally, bootstrap values for most clades are considerably

more robust than when hagfish and lamprey sequences are included.

To further test relationships between COX proteins, a phylogeny (Figure 2-9) was

generated using the maximum likelihood criterion, 1000 bootstrap replicates, and the program

PhyML (Guindon and Gascuel, 2003). The topology of this tree is very similar to the one

generated using distance criterion. Briefly, it contains well supported, monophyletic clades for

COX-1 teleostt COX-la, teleost COX-lb, teleost COX-1, mammalian COX-1, and vertebrate

COX-1 with sCOX as the most basal sequence) and COX-2 teleostt COX-2, mammalian COX-2,

and vertebrate COX-2). The lamprey (1COX) is also basal to the vertebrate COX clade, the

amphioxus COXc and COXd form a basal monophyletic clade, and the most basal clade in the

tree is a monophyletic COXa and COXb from the sea squirt. However, in the ML analysis, the

hagfish (hCOX) sequence groups at the base of the vertebrate COX-2 clade instead of being

basal to the vertebrate COX clade. Again, the analysis was repeated without hCOX and 1COX









(Figure 2-10), causing bootstrap values to increase dramatically. Also, the estimated gamma

shape parameter increased slightly from 0.737 to 0.742.

To further evaluate support for COX clades, an aLRT analysis (Anisimova and Gascuel,

2006) was performed. This type of analysis provides values of support similar to Felsenstein's

bootstrap support values but is much faster because bootstrap sampling requires many (1000 in

the previous analyses) runs while aLRT is run only once. The fundamental difference between

the two is that bootstrap support values are based on repeatability whereas the aLRT is a measure

of the likelihood gain of including a branch versus collapsing it (Anisimova and Gascuel, 2006).

The resulting aLRT statistics can be interpreted using parametric Chi-Squared distributions or a

non-parametric Shimodaira-Hasegawa-like (SH) procedure (Anisimova and Gascuel, 2006). The

former seems to be more liberal (giving values similar to Bayesian posterior probabilities) and

the later more conservative (Anisimova and Gascuel, 2006). Here, the most conservative

approach was used and values represent the minimum of Chi-Squared and Shimodaira-Hasegawa

(SH) support values. When all species are included (Figure 2-11) clades of interest have robust

support (> 0.90) except for the vertebrate COX-2 clade (containing hCOX, 0.683) and the clade

containing 1COX (0.87). When hCOX and 1COX are excluded from the analysis (Figure 2-12),

support values generally increase, with a notable increase in the vertebrate COX-2 clade (0.883).

A Basesian phylogenetics (BP) analysis was also performed (Figure 2-13) using the

program MrBayes 3.1.2, with one initial 2 million generation run, and one final 10 million

generation run. The topology of the consensus tree (with burnin = 10%) is similar to those

described above, with well-supported aforementioned vertebrate COX clades. However, in BP

analysis 1COX groups at the base of the amphioxus COXc/COXd clade instead of being the basal

member of the vertebrate COX clade. This grouping is well-supported by posterior probability









scores (0.93). As in ML analysis, hCOX groups with vertebrate COX-2 proteins at the base of

the clade. Also similar to previous analyses, when 1COX and hCOX are excluded (Figure 2-14)

scores improve and all clades have robust support.

Due to the contrasting topologies generated using distance, ML, and BP methods, 21

different tree topologies (Figure 2-15) were generated by using the most likely tree from ML

analysis (Figure 2-9) and relocating hCOX and/or 1COX sequences to other branches of the tree.

Positions of all other sequences were not altered from the most likely tree. A Ln likelihood score

(Ln L) was generated for each topology using online execution of the program PhyML (Guindon

et al., 2005). Likelihood scores were compared to the most likely tree. These tests indicate that

1COX and hCOX can be moved somewhat freely around the base of the tree without resulting in

a noticeably different Ln L than the most likely topology (Table 2-9). It appears that moving

1COX to the base of the COX-1 clade causes the smallest change in Ln L, whereas moving

hCOX or 1COX deeply into COX-1 or COX-2 clades causes the largest changes in Ln L.

Discussion

Here, I present the first forms of the enzyme cyclooxygenase (COX) sequenced from the

hagfish, lamprey, and amphioxus. I also show that the killifish and sculpin conform to the

current hypothesis that teleosts posses additional COX-1 and COX-2 forms. The phylogenetic

relationships among the various COX forms are still not definite, although the results presented

here allow some conclusions to be drawn and predictions to be made. Specifically, it appears

that COX sequences in the chordates form at least 3 well supported phylogenetic groups: the

urochordata, cephalochordata, and vertebrata. Hagfish and lamprey positions are variable

depending upon the analysis used, but likelihood scores seems to favor placement within the

vertebrata clade.









Novel COX Forms

Although the two COX sequences from amphioxus were not able to be extended, they

aligned readily with other COX sequences. They show a first haem-binding domain that shares

little conservation with vertebrate forms (20%), as in the urochordates. However, the second

haem-binding domain is completely conserved in these forms, as is histidine-207 which is crucial

for peroxidase activity. The arginine-277 loop region does not resemble COX-1 or COX-2

forms. Due to the truncated nature of these proteins, it is difficult to determine if COX in the

cephalochordates plays similar roles as in the vertebrates, but some functions are likely

conserved. Following the nomenclature used by Jarving et al. (2004) these forms are named

COXc and COXd. The COX sequence found in the gills of the hagfish was extended to

completion and readily aligned with other COX forms. It has conserved amino acids, binding

domains, and bonds critical for COX function. Therefore, it likely plays a similar role in the

hagfish. The hagfish COX has an N-terminus deletion and valine-523 characteristic of COX-2

forms, but there are multiple sites where it shares equal or no homology with COX-1 and COX-2

of vertebrates. Therefore, following the nomenclature of Yang et al. (2002) this form is named

hCOX. Although incomplete, the one COX sequence cloned from the gills of the lamprey

readily aligns with other COX sequences and contains amino acids and regions critical to COX

function. However, like the hagfish sequence it shares no biased homology with COX-1 or

COX-2 forms. This form is therefore named 1COX. As predicted based on phylogenetic

position, COX-la, COX-lb, and COX-2 were found in the gills of the euryhaline killifish and the

longhorn sculpin. With the exception of COX-la in the sculpin, all COX forms from these

teleosts contain conserved regions critical to COX function. As has been reported in other

teleosts (Ishikawa et al., 2007), these forms all contain valine at position 523, unlike in

mammals. COX-la from the sculpin does not have several amino acids critical for COX









function located near the C and N terminus of the sequence. These include disruption of

disulfide bonds near the N terminus due to cystine replacements, little conserved homology in

the membrane binding domain, and alternate residues for arginie-513 leucinee), valine-523

leucinee), and serine-530 phenylalaninee). This is not the case in the other acanthopterygiians,

where all COX forms have these conserved regions. This may indicate that in the sculpin COX-

la has partially lost function, and COX-lb or COX-2 may therefore have additional

responsibilities.

Phylogenetic Analyses

As has been seen when tracing the evolutionary histories of other gene families in

evolutionary deep time (Abbasi and Grzeschik, 2007; Goudet et al., 2007), the pedigree of the

COX family is littered with duplications, losses, and uncertainty. Despite utilizing a multi-

faceted approach to depict COX evolution in the chordates, the results indicate that COX has a

complicated and under-examined history of evolution in the chordates. This is not surprising,

considering the complex functions COX has been shown to have in the chordates. Because of

these complex functions, duplications, partitioning of roles, differential expression, and multiple

forms with multiple functions are to be expected.

In all phylogenetic analyses, the more recent history of COX evolution is more clear and

consistent than the relationships of the "ancestral" taxa. The teleosts consistently form

monophyletic COX clades nested within the gnathostomes, as expected. The gnathostomes

consistently form two COX sister clades in phylogenetic analyses: COX-1 and COX-2. This is

expected based on previous phylogenetic analyses in the vertebrates (Gu, 2001; Gu, 2006). The

COX-1 clade is consistently rooted with the sequence from the dogfish (sCOX), although

support values are not robust for this relationship when hagfish and lamprey sequences are

included. In distance analysis excluding hagfish and lamprey sequences, sCOX groups outside









the COX-1 and COX-2 clades, representing another COX gnathostome lineage. These groupings

are as expected, based on Yang et al.'s (2002) original description of sCOX as neither a COX-1

nor COX-2. These are the first phylogenetic analyses of COX to include sCOX and it is

concluded that sCOX is most likely a basal member of the COX-1 clade.

In all analyses, the COX-1 clade contains robustly supported, monophyletic teleost and

eutherian mammal clades. In the teleost clade, COX-lb and COX-la form well-supported,

monophyletic clades. Although this is expected based on initial studies (Ishikawa et al., 2007;

Ishikawa and Herachmann, 2007), this is the first study to use sophisticated tree-building

techniques to show the monophyly and legitimacy of the COX-1 forms in teleosts. It is also

shown in each analysis that the non-duplicate COX-1 forms from the zebrafish and trout are

more related to COX-lb forms from the acanthopterygiians, grouping with them at the base of a

monophyletic COX-1/COX-lb clade.

The mammalian COX-1 group is not as robustly supported using all types of analyses.

The expected relationships (based on assumed evolutionary relationships in the tetrapods) are

shown and supported with some confidence in the maximum likelihood analysis (ML) when the

hagfish and lamprey sequences are excluded. In this anlaysis, the tetrapods form a monophyletic

group with the frog and chicken at the base of the clade, followed by the opossum sequences,

and then the eutherians. Even in this analysis, the frog and chicken sequences group together,

contrary to what is biologically expected (the frog should be more basal, representing an

evolutionary ancient non-amniote lineage, and the chicken should be a sister to the mammals).

However, this grouping is not well-supported in any analysis. When hagfish and lamprey are

included in ML analysis, the chicken/frog clade occurs at the base of the mammalian/teleost

clade, contrary to biological predictions. However, this relationship is poorly supported. These









low support values are confirmed in the aLRT analysis. In Bayesian and distance analyses, the

frog is basal to the chicken, but these sequences are again at the base of a biologically irrelevant

teleost/mammal clade. However, unlike distance and ML methods, Bayesian posterior

probabilities strongly support this relationship. Bayesian posterior probabilities traditionally

overestimate support values for a variety of reasons (Cummings et al., 2003: Lewis, pers.

comm..) and can give incorrect clades strong posterior probabilities. This may explain why this

biologically unexpected relationship is strongly supported only in the Bayesian trees. Therefore,

ML analyses (supported by bootstrap and aLRT scores) show the most probable scenario for

COX-1 evolution in the gnathostomes: a basal sCOX, followed by a teleost COX-1 clade (as

described above) sister to a tetrapod COX-1 clade.

The COX-2 groups within the gnathostomes mirrors the same general trends as the COX-

1 clade. Teleosts consistently form a well-supported, monophyletic group. The COX-2a

sequences from zebrafish and trout group together to form the most basal members of this clade.

The COX-2b sequences group with the non-duplicate COX-2 sequences from the

acanthopterygiians to form the other teleost COX-2 clade. Sister to the teleosts, the tetrapods

form another COX-2 clade in the gnathostomes. In this tetrapod COX-2 clade, frog and chicken

sequences form the most basal (although not well-supported) clade, with the mammals as a sister

clade. This biologically relevant relationship between COX-2 sequences is predicted in all

analyses.

Basal to the COX-1 and COX-2 clades in the gnathostomes are two clades that are

consistently and robustly supported in all phylogenetic analyses: a urochordate COX clade and a

cephalochordate COX clade. The cephalochordate clade consists of the two COX sequences

obtained in this study (COXc and COXd) and the urochordate clade consists of the two









sequences from Ciona (COXa and COXb). The urochordate COX forms always forms the base

of the entire tree and the cephalochordate COX forms always group as a sister clade to the

gnathostome COX clade. This study is the first to show this biologically relevant relationship

between COX forms in these "ancestral" chordates.

The hagfish (hCOX) and lamprey (1COX) sequences have alternate phylogenetic

positions depending on the technique used to reconstruct their evolutionary histories. Also,

when these sequences are removed from analysis, bootstrap, aLRT, and posterior probability

scores supporting COX clades increase. Because of their uncertain placement in COX evolution,

these sequences are designated as "rogues" and analyses excluding them likely show a more

accurate representation of COX evolution. In distance analysis, both hCOX and 1COX are

placed outside the gnathostome COX clade and form two separate COX lineages between the

cephalochordate and gnathostome clades. In ML analysis, 1COX remains basal but hCOX is

placed at the base of the gnathostome COX-2 clade. In BP analysis, hCOX remains in the COX-

2 clade but 1COX is placed within the cephalochordate clade. Support values for hCOX and

1COX placement are weak in all but BP analyses. Clearly, the placement of hCOX and 1COX in

COX evolution is not clear and without further evidence, placing them with any degree of

certainty becomes difficult. In an attempt to find the most likely placement of these sequences,

21 different phylogenies were generated based on the most likely tree with hCOX and 1COX

placed in variable positions. The trees were then evaluated in a maximum likelihood framework.

It is important to note that although Ln L scores were generated for each topology, other

biological factors may influence which topology or type of topology is actually the most likely.

For example, placing hCOX in the COX-2 clade and 1COX at the base of the tree (technically the

most likely tree) implies that another COX lineage exists for all vertebrates (the lineage









represented by 1COX) because the hagfish is biologically ancestral to the lamprey. This would

mean that dozens of novel COX forms have yet to be discovered in well-examined groups such

as the mammals and teleosts. With genomes of several of these groups available, it is unlikely

that such a vast amount of sequence data have yet to be found. Therefore, the most likely

scenarios for COX evolution would imply that either hagfish is basal and lamprey is derived,

both are basal, or both are derived. However, placing 1COX, hCOX, or sCOX in the COX-1 or

COX-2 clade implies that another COX form has yet to be found in these species.

It appears that moving 1COX into the base of the COX-1 clade causes less of a change in

Ln L scores than moving hCOX outside the gnathostome COX clade. However, it is apparent

that either sequence can be moved somewhat freely throughout the tree without resulting in

noticeably worse (-10 Ln L) tree scores. This leads to the conclusion that although hCOX and

1COX may represent independent, basal lineages of COX, they are most likely basal members of

the COX-2 and COX-1 clades (respectively) in the gnathostomes. If this is the case then at least

one other form of COX should exist in the hagfish and lamprey, as well as in the dogfish shark.

Summary

Presented here are the first COX sequences from amphioxus, the hagfish, and the

lamprey. All predicted COX sequences from the killifish and sculpin are also described. These

sequences all likely represent functional forms of COX found in the chordates based on

conserved amino acids and domains critical to COX function. For the first time, a sophisticated

phylogenetic analysis of COX forms from the majority of representative chordate lineages was

attempted. Although still not definitive, a general hypothesis for the evolutionary history of

COX in the chordates can be made (Figure 2-16). In this scenario, there are three main COX

groups corresponding to the three subphyla found in the phylum Chordata: the urochordata,

cephalochordata, and vertebrata. Each contains two main COX groups, with the well-









documented COX-1 and COX-2 forms only found in the vertebrata clade. These forms contain

monophyletic groups for the teleosts and the tetrapods, with multiple forms in the teleosts

confirming previous hypotheses (Ishikawa et al., 2007; Ishikawa and Herschman, 2007).

Hagfish and lamprey placement is uncertain, but most likely these sequences represent basal

members of the vertebrata clade. This does not support the timing of genomic duplications in the

chordates according to the most recent view of the 2R Hypothesis (Kasahara, 2007), indicating

two rounds of genome duplication after the origin of the urochordates but prior to the radiation of

the jawed vertebrates. However, several developmentally important proteins have been shown to

not support this timing and indicate an earlier duplication (Hughes, 1999). Multiple COX-1 and

COX-2 genes in the teleosts do support the hypothesis that the teleosts are characterized by

another round of genome duplication after the origin of the vertebrates (Li et al., 2007). Clearly,

this uncertain period representing some 600 million years of evolution is characterized by many

losses and duplications and remains to be resolved definitively.










Table 2-1. Degenerate primers used in PCR


Name
CH COX Fl
CH COX F2
CH COX F2.25
CH COX F2.5
CH COX F3
CH COX F5
CH COX R1
CH COX R2
CH COX R3
CH COX-1 F4
CH COX-1 F5
CH COX-1 R2
CH COX-la Fl
CH COX-la F2
CH COX-la F3
CH COX-la F4
CHCOX-laF5
CHCOX-la R1
CH COX-la R2
CH COX-la R3
CH COX-lb Fl
CH COX-lb RO
CH COX-lb R3


Orientation
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Antisense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Antisense
Antisense


Nucleotide sequence (5' to 3')
ATG GAC GAC TAC CAG TGY GAY TGY AC
GAT GTT TGC ATT TTT CGC TCA RCA YTT YAC
TGG CGT GGA CCT AGG TCA NRT NTA YGG
CGA GCT GCG GTT CCA TAA ARA YGG NAA RYT
CCA CTA TAT GGC TGC GGG ARC AYA AYM G
CAT GTG GAA TTC CAT CAC CTG TAY CAY TGG CA
CCC CGA AGG TGG ATG GYT TCC AVY A
GCA TCA GCG GGT GCC ART GRT A
TCC TCT TTT AGT ATG TCG CAG ACT CKR TTR TGY TC
TGC CAG ACA GCA TCC ACA THG AYG GNG A
CAG CAG ACA ATG CGC AGG NCA RAT HGG
TGG GTG TAT GTT GTG TCC TCC NCC DAT YTG
CCC CAC CAA CCT ACA ATA CCA ART AYG GNT A
AAA GTG CTG ACT GAR MGN TTY TT
GGA CTA ATC TGA TGT TCG CGT TYA TGG CNC A
AGC GGA AGG CGG CTT YAC NAA NGC
CAA ATA GTA AAT GGG GAR AYN TAY CC
ATA GGG AAT GTC GTC NCC RTC DAT
GGG GCA CGT TTT CCG GRT ANA YCA T
ACC TCA GTG ACA GTG GGA GGR TAN RTY TC
CGG ATG GGT GTG GGC TTY ACN AAR GC
GGA GAA GGG AGC ACC CAT YTC NAC CAT
CCA GCG TCC GCG CDA TYT CYT C










Table 2-2. Amphioxus COX specific primers used in PCR


Name
COXa Fl
COXaF2
COXa F2.5
COXa F3
COXaF4
COXaF5
COXa R1
COXa R2
COXa R3
COXa 3' Fl
COXa 3' F2
COXa 5' R1
COXa 5' R2
COXa 5' R3
COXbFO
COXb Fl
COXb F1.5
COXbF2
COXb F3
COXb R1
COXb R2
COXb R3
COXb 3' Fl
COXb 3' F2
COXb 3' F3
COXb 5' R1
COXb 5' R2
COXb 5' R3


Orientation
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Antisense
Antisense
Antisense


Nucleotide sequence (5' to 3')
TAC TCC ACC GTG TGG CTG C
TGG GAC GAC GAG AGG CTC
ACC GGT GAG ACC ATC AAC ATC AT
CAC CTG AGC GGC TAC AAC TTT
GAC CTG TTC TGG GAC CCT GAG
CCA GTA CCA GAA CCG CAT CTT C
CGT ACG GCG GGT AGA TCA TG
GCT GCC GCT CCA CGG
CCC GTA GAT ATG GCT CAG GTC A
GAG GCT CTT CCA GAC AGC CA
CAC CTG AGC GGC TAC AAC TTT
TGG CTG TCT GGA AGA GCC TC
TGG AGT AGA CGA ACA GGC CC
CCC CGT AGA TAT GGC TCA GGT
CAG ACA GCT AGA CTC ATT CTT ATC AGT GA
TAT CAA CAT CGT CAT TGG AGA GTA TG
GGC TGG CAA AAA CTT CCA ACT
TGC AGT ACC AGA ACA GCA TAT TTG
AGC ATA TTT GTG GAG TTT AAC CAC TTG
CGT CCG GAG GGT AAA TCA TGT
ACT TCA GTT TCC CGT CCT GGA
CGT AAA CAT GGC TCA TGT CCA CT
GTC CCC GAG AAG AAG CGA TT
GAA CGG CTC TAC CAG ACA GCT AG
TGT TCT GGG ACC CTG AGC TG
TGG TAG AGC CGT TCG TCG TC
GGA GGG TAA ATC ATG TGT ACG GA
ACA TGG CTC ATG TCC ACT GC











specific primers used in PCR


Name
hCOX 3' Fl
hCOX 3' F2
hCOX 3' F3
hCOX 3' F0.8
hCOX 3' F0.6
hCOX 3' F0.4
hCOX 3' F0.2
hCOX 3' F0.1
hCOX 3' FO
hCOX mid Fl
hCOX mid F2
hCOX mid R1
hCOX mid R2
hCOX 5'R1
hCOX 5' R2
hCOX 5' R3
hCOX 5' R4
hCOX 5' R5
hCOX 5'R6


Orientation
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Antisense
Antisense
Antisense
Antisense
Antisense


Nucleotide sequence (5' to 3')
TTA ACC ACC TGT ACC ACT GGC AC
CTC AAC CAT GGA GTA CGA GGT CT
CAA TGC TAT GGA GTT CTA CCT GGG
CAG TAC AGC AAC CGC ATC TCA
AAT GGG ACG ACG AGA GAA TCT TT
GGT TTG CTG TTG GTC ACG AAG
AGT CCA CCC ACA TAC AAT GCC
CTC ACG CAC TTT GCT CCC TT
GAA CTG CAC CTA CCC CGA GAC
GTT TCG AGT CCA CCC ACA TAC AA
CAT ACT CCA ACC TCA GCT ACT TCA CT
CAC ATC GAG TAC GTC ACA CAC G
AAG GGT CCC GTA GAG CAT GA
TCT CCG AAT AAA GCT CCC TGT C
TGC TGC CAT TTC CGT TTC TC
CAT GTT TGA TGG ATG CTG TTG C
AGG CGA GTG AAG TAG CTG AGG TT
CCT CCC AGG ACT TGT ACC GTA AT
GAG GAA GTA ATG CAC GGT ATC TGG


Table 2-3. Hagfish COX











Table 2-4. Lamprey COX specific primers used in PCR


Name
1COX Fl
1COX R1
1COX R2
1COX R3
1COX 3' Fl
1COX 3' F2
1COX 3' F3
1COX 5' R1
1COX 5' R2


Orientation
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Antisense
Antisense


Nucleotide sequence (5' to 3')
GAG GAA CGC TTG AGA GAC AAC A
TCT TGT TGA TGT TTC GAC CGC
CCA CCG CCA ATC TGG C
AAT CAG GAA GCT CTC AGG CAT C
GAG TTC CTC TTC AAC CCC GG
TCG AAA CAT CAA CAA GAA CCT CC
TTC ACC TCC TTC CTG GAG CTC
TGC TGC ACG TAC TCC TCG ATC
GCA GCT GGT GTT GTC TCT CAA











Table 2-5. Killifish COX specific primers used in PCR


Name
Fh COX-lb 3' Fl
Fh COX-lb 3' F2
Fh COX-lb 3' F3
Fh COX-lb 5' R1
Fh COX-lb 5' R2
Fh COX-lb 5' R3
Fh COX-la 3' Fl
Fh COX-la 3' F2
Fh COX-la 3' F3
Fh COX-la 5' R1
Fh COX-la 5' R2
Fh COX-la 5' R3


Orientation
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Antisense
Antisense
Antisense


Nucleotide sequence (5' to 3')
CTT CAC CGA TAG CGA GGA GAT AG
AGC TCT ACG GTG ACA TCG ACA CT
GGG TAA CCC CAT ATG TTC TCC AC
CCC ATG TGA GAG AGC CTT AGT GA
CAG CCT CTC AAA CAA CAC CTG AG
GTA GTA GGA TTC CCA GCT GAG GTA GT
GGT GTA CCC CGA AGG TTT CC
GAG TAC GTG CAG CAC CTG AGC
AGT ACA CCA ATC GCA TCG CC
GAT GAT AAG TCT GGC GGT CTG G
GAT GGT GGC ATA CAC GGT GA
TTT ATG AAG CCG AAG CTG GTG











Table 2-6. Sculpin COX specific primers used in PCR


Name
Mo COX-2 3' Fl
Mo COX-2 3' F2
Mo COX-2 3' F3
Mo COX-2 3' F4
Mo COX-2 3' F5
Mo COX-2 3' F6
Mo COX-2 mid Fl
Mo COX-2 mid F2
Mo COX-2 mid R1
Mo COX-2 mid R2
Mo COX-2 5' R1
Mo COX-2 5' R2
Mo COX-2 5' R3
Mo COX-la 3' Fl
Mo COX-la 3' F2
Mo COX-la 3' F3
Mo COX-la 3' F4
Mo COX-la 3' F5
Mo COX-la 3' F6
Mo COX-la 5' R1
Mo COX-la 5' R2
Mo COX-la 5' R3
Mo COX-lb 3' Fl
Mo COX-lb 3' F2
Mo COX-lb 3' F3
Mo COX-lb 5' R1
Mo COX-lb 5' R2
Mo COX-lb 5' R3


Orientation
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Antisense
Antisense
Antisense


Nucleotide sequence (5' to 3')
CCC CGG TCT GAT GAT GTA CG
ACC GAG TGT GTG ACG TGT TGA
TCG AGG ACT ACG TGC AGC AC
CAG GTC TAA CGC AAT CTT TGG G
CCT CAA GGG CTT AAT GGG AAA C
AAC ATC GTC AAC ACC GCC TC
ACA GTT CAT TCC GGA TCC ACA
CAC CAG CCT GAT GTT TGC ATT
CGA AGC GGT GAG ATT CAG GA
CTC CAT CCA GGA CCT GAT ATT TAA G
TCC GGA ATG AAC TGT CTT CTC A
AGG TCG GAG GAC TAT CAA TCA AGT
GAA GGA GAT GGA GTT AAT GAT GTT CC
CAT CAC CTC CGG CTT CAT AAA
GAG GGA GCA TAA CAG ACT CTG TGA
CTG GAG TTC TGC CAC CTC TAC C
GCA GCC CTT CAA TGA ATA CAG G
CAT GCT GGA GAA GAC CCT TCC
GAG TAT GTT GGA GAT GGG TGC TC
GCC GAA ATG TCT TCC TCC TAA A
CAT CGG TAA AGG GCA GTC CTC
TGA CCG TCA GCA CTA ATC TCA TG
TCA CAC ACT ACG GCA TCG AGA
TTG GCG GTG GCT TTA ACA TC
CAG CCC TTC AAC GAG TAC AGG
GAG CGA TAC GGT TCC CAT ACT G
GTT GTG CTC CCT GAG CCA GA
AAT AGC CAT CTG AGC CTC AGG A









Table 2-7. Sequences included in phylogenetic analyses


Name
COX1a Fundulus
COXlb Fundulus
COX2 Fundulus
COX1 a Myoxocephalus
COXlb Myoxocephalus
COX2 Myoxocephalus
COXa Ciona
COXb Ciona
COXc Branchiostoma
COXd Branchiostoma
hCOX Myxine
1COX Petromyzon
COXla Oryzias
COXlb Oryzias
COX2 Oryzias
COXIa Gasterosteus
COXlb Gasterosteus
COX2 Gasterosteus
COX1a Tetraodon
COXlb Tetraodon
COX2 Tetraodon
COX1 Oncorhynchus
COX2a Oncorhynchus
COX2b Oncorhynchus
COX1 Danio
COX2a Danio
COX2b Danio
COX1 Monodelphis
COXlb Monodelphis
COXlb2 Monodelphis
COX2 Monodelphis
COX1 Gallus
COX2 Gallus
COX1 Bos
COX2 Bos
COX1 Xenopus 1
COX2 Xenopus 1
COX1 Canis
COX2 Canis
COX1 Homo
COX2 Homo
COX1 Rattus
COX2 Rattus
COX1 Mus


Organism
Fundulus heteroclitus killifishh)
Fundulus heteroclitus killifishh)
Fundulus heteroclitus killifishh)
Myoxocephalus octodecimspinosus (lh. Sculpin)
Myoxocephalus octodecimspinosus (lh. Sculpin)
Myoxocephalus octodecimspinosus (lh. Sculpin)
Ciona intestinalis (sea squirt)
Ciona intestinalis (sea squirt)
Branchiostoma lancelolatum amphioxuss)
Branchiostoma lancelolatum amphioxuss)
Myxine glutinosa (Atlantic Hagfish)
Petromyzon marinus (Sea Lamprey)
Oryzias latipes (Japanese medaka)
Oryzias latipes (Japanese medaka)
Oryzias latipes (Japanese medaka)
Gasterosteus aculeatus (stickleback)
Gasterosteus aculeatus (stickleback)
Gasterosteus aculeatus (stickleback)
Tetraodon nigroviridis (Green Spotted Puffer)
Tetraodon nigroviridis (Green Spotted Puffer)
Tetraodon nigroviridis (Green Spotted Puffer)
Oncorhynchus mykiss (rainbow trout)
Oncorhynchus mykiss (rainbow trout)
Oncorhynchus mykiss (rainbow trout)
Danio rerio (zebrafish)
Danio rerio (zebrafish)
Danio rerio (zebrafish)
Monodelphis domestic (gr. S.t. opossum)
Monodelphis domestic (gr. S.t. opossum)
Monodelphis domestic (gr. S.t. opossum)
Monodelphis domestic (gr. S.t. opossum)
Gallus gallus (rd. jungle fowl)
Gallus gallus (rd. jungle fowl)
Bos taurus (cattle)
Bos taurus (cattle)
Xenopus laevis (Afr. Claw. Frog)
Xenopus laevis (Afr. Claw. Frog)
Canis lupusfamiliaris (dog)
Canis lupusfamiliaris (dog)
Homo sapiens (human)
Homo sapiens (human)
Rattus norvegicus (norway rat)
Rattus norvegicus (norway rat)
Mus musculus (house mouse)


Size (AA)
452
598
610
622
600
605
653
600
177
177
610
286
605
606
609
598
598
620
1023
589
456
624
607
609
597
601
606
625
627
729
608
649
603
600
604
587
604
633
604
599
604
602
604
602










Table 2-7. Continued.
# Name
45 COX2 Mus
46 COX1 Oryctolagus
47 COX2 Oryctolagus
48 COX1 Ovis
49 COX2 Ovis
50 sCOX Squalus


Organism
Mus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
Oryctolagus cuniculus (rabbit)
Ovis aries (sheep)
Ovis aries (sheep)
Squalus acanthias (spiny dogfish)


Size (AA)
604
606
604
600
603
593









Table 2-8. LRTs between amino acid based models of COX evolution


Evolutionary Model*
WAG
WAG + I
WAG + F
WAG + I + F


Ln likelihood (Ln L) Score (Ln Li-LnL2) 2 A d.f.@
-26632
-26315 317 1
-25294 1021 1
-25294 0 2


*Evolutionary models are arranged from the most simple to the most complex. Only models
using the WAG matrix were evaluated. I represents an invariable sites parameter and F
represents an among site variation parameter
A d.f. indicates the difference in free parameters between the complex and simple models
#P values represent the comparison of simple to complex models and were obtained using a chi-
squared distribution test


< 0.01
< 0.01
> 0.05









Table 2-9. LRTs between tree topologies with altered positions of hCOX and 1COX
Tree Topology Name* Ln likelihood (Ln L) Score Estimated ag (Ln L1-LnL2)-2T
COX2 Hag (most likely) -25294.22 0.737
COX1Lamp/COX2Hag -25295.57 0.737 2.70
COX2 Agnatha -25299.09 0.737 9.74
Lamp/Amp -25299.27 0.737 10.1
COX2 Vert -25299.61 0.736 10.7
COX1 Hag -25300.92 0.735 13.4
COX1 Vert -25301.22 0.735 14.0
Ancestral Hag -25301.56 0.735 14.6
COX1 Agnatha -25301.78 0.735 15.1
COX1Hag/COX2Lamp -25302.19 0.735 15.9
Ancestral agnatha -25302.55 0.735 16.7
COX1 Lamp -25303.25 0.735 18.1
COX2 Lamp -25303.85 0.735 19.3
Agnatha/Amp -25308.82 0.735 29.2
Hag/Amp -25308.90 0.735 29.4
Hag Internal -25321.15 0.737 53.9
Lamp Internal -25322.77 0.736 57.1
Lamp Mammal -25341.41 0.732 94.4
Lamp Teleost -25344.68 0.735 101
Hag Teleost -25350.30 0.734 112
Hag mammal -25408.58 0.731 229

*Each tree has the same topology as the most likely tree (Figure 2-9) but with alternative
placements of hagfish (hCOX) and lamprey (1COX) sequences as noted in Figure 2-15.
@a represents the shape value under the F model
Where Lnl is the most likely tree score and Ln2 is the tree score of the topology in question










COX-lb MRASVLGSVCALLVLLREPGCQGDEVTTSTVNPCCYLPCKHWSVCVRYGEDKYECDC 57

COX-2 MNRITFAVFLLALC--------FSFHKEVLGNACCSEPCQNRGVCTAMGSDSYECDC 49
-N 68 .........................................................................................
COX-lb THTGYYGENCSIPELWTRVRQFLKPSPDVVHYILTHFHWLWDIINN-TFLRNVLMRL 113
COX-2 TRTGYRGQNCTTPEFLTWIKISLKPTPNTVHYILTHFKGFWNIINNISFLRDAIMKY 106
*R120 *N144 -
COX-la I" NLSYYTRLLPPVPKDCPLPMGTKGKPVLP 29
COX-lb TARSNLIPSPPTFNSKYNYLSWESYYNLSYYTRILPPVPEDCPTPLGVKGRNGLP 170
COX-2 LLTSRSHMIDSPPTYNADYGYKSWEAYSNLSYYTRALPPVPEDCPTPMGVVGKKELP 163
Q203/H207
COX-la DIKEISERYFKRTEFRPDPQGTNLMFAFMAQHFTHQFFKTSHKVDAGFT LGHVD 86
COX-lb DPQVLFERLLKRRTFRPDPQGSNIMFAFFAQHFTHQFFKTYNRMGLGFT[ALSH[VD 227
COX-2 DVKVLAEKLLVRRRFIPDPQGTSLMFAFFAQHFTHQFFKSDMKNGPAFTAKGHVD 220
------- -----R277----
COX-la ASNIYGEELERQHQLRLHKDGKLKYQLINGEMYPPTVSEVPVHMVYPEGFPAEQRLA 143
COX-lb AGHIYGDSLERQHLLRLFRDGKLKYQLIDGEVYPPSVTDAPVRMSYPPGIPVEKQMA 284
COX-2 LGHIYGENLEKQHKLRLFKDGKLKYTMVNGEVYPPLVKDVGVEMHYPPHVPDSQRFA 277

COX-la IGQEVFGLVPGLTVYATIFLREHNRVCDELKGEHPTWDDEQLFQTARLIIIGEIINI 200
COX-lb IGQEVFGLLPGLSLYATLLREHNRVCD LKAEHPTWDDEQLFQTTRLIIIGETIRI 341
COX-2 VGHEAFGLVPGLLMYATIILREHNRVCD LKGVHPDWDDERLFQTTRLILIGETIKI 334
Y385/H388
COX-la IIEEYVQHLSGYYLKLKYDPSLLFGVRFQYTNRIALEFCYLYHWHPLMPDSFLIDGD 257
COX-lb VIEEYVQHLSGYLLQLKFDPTLLFNSNFQYGNRIALEFSQLYHWHPLMPDSFHISGD 398
COX-2 VIEDYVQHLSGYHFKLKFDPELLFNQRFQYQNRIASEFNTLYHWHPLMPDSFHIEEK 391
*N410
COX-la ELPYSQFLYNTSILMHYGVEKLVDAFSRQPAGQIGGGRNIHQAVLRVAEMVIRDSRA 314
COX-lb ELSYSQFLFNTSVLTHYGVEKLVDAFSRQAAGQIGGGHNINAVITKVIVGTIEESRQ 455
COX-2 DYSYKEFVFNTSVVTEHGINNLVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRK 448
*R513
COX-la ARLQPFNQYRKRFNLKPYSSFYELTGDEEMARGLEELYGDIDALEFYPGLLLEKTRP 371
COX-lb LRIQPFNEYRKRFNLEPYTSFRDFTDSEEIASTLEELYGDIDTLEFYPGLLLEKTRP 512
COX-2 MRYQSLNAYRKRFSMKPYTSFEDLTGEKEMAAILEELYGDVDAVELYPGLLVEKPRT 505
*V523 *S530
COX-la SSIFGESMVEMGAPFSLKGLLGNPINSPEYWKPSTFGGETGFNIIKTSTLKKLVCLN 428
COX-lb GAIFGESMVEMGAPFSLKGLLGNPICSPQYWKPSTFGGKVGFDIVNSASLKKLVCLN 569
COX-2 NAIFGETMVEMGAPFSLKGLMGNPICSPEYWKPSTFGGTAGFDIVNTASLQRLVCNN 562

Figure 2-1. Amino acid alignment of the forms of COX sequenced from the gills of the
euryhaline killifish (Fundulus heteroclitus). The COX-2 sequence is from GenBank
(Accession #AAS21313) and the others were obtained in this study. Amino acids that
share at least 60% identity across sequences are highlighted. *Indicates amino acids
(numbered based on ovine COX-1) involved in COX function: active site of COX
(tyrosine-385, histidine-388, and serine-530), substrate binding site (arginine-120), N-
glycosylation sites (asparagine-68, 144, and 410), sites for peroxidase activity
(glutamine-203 and histidine-207), and the two sites which define conformational
differences in channels between COX-1 and COX-2 in mammals (arginine-513 and
valine-523) (Ishikawa et al., 2007). The black boxes indicate the two domains that
define the haem-binding sites and the dashed box indicates the membrane binding
domain (Kulmacz et al., 2003; Ishikawa et al., 2007). The solid lines represent
disulfide bonds, with the beginning and end of the longest bond represented by
arrows (Kulmacz et al., 2003). The dashed line represents the arginine-277 loop
region, which has been shown to differ between COX-1 and COX-2 forms in
mammals (Yang et al., 2003). The numbers to the right of the sequences indicate the
amino acid number of each sequence.











COX-la TK-WCPYVDFHVPRNEEERKPS---------------------TEL 452
COX-lb SR-TCPYVAFSVPAEEEPGRNDGKERS--------------SEL 598
COX-2 VRGPCPVASFYVPDVKETGSMTINSSTSHSRDSNINPTVILKERTSEL 610

Figure 2-1. Continued.













COX-la MKRANLSNYFLETNHCEQNISDNYHFITLITIL-SRES--KSLASFPSGLTFCCWGA 54
COX-lb MRSPVLGPVCALLLL-LREPAC------------------RGDEVTSDTVNPCCY-- 36
COX-2 MYRFTFA-VF---LLALGVLVC--------------------- EGGNPCC--- 25
N6 8 : ................ ..............
COX-la HVLLTQVLIPVVITLVRTQECVCDSVQIT-TNVTAL LASMETTALSRSSGPEFVRS 110
COX-lb --FPCQHWGVCVRYGEDKYECDCTRTGYTGGNCTVPEFWSRVHQFL-KPS-PEVLHY 9
COX-2 -SEPCQNRGVCTALGTDNYECDCTRTGYHGHNCTTPEFLTWVKISL-KPS-PNTVHY ,79
...................................................... .......... RY.U ...................................
COX-la RTRRSSSSSPTSIGCWHLVNN-SFLRGTVMRLVETVRSDLIPSPPTYNTKYGYLNWE 166
COX-lb LTHFN-------WLWDIINH-TFLRDVLMRMVLTVRSNLIPSPPTYNSKYDYLSWE 138
COX-2 .LTHF.K....-......-.GFWNLNSSF.F.RDAIMR TSRSHLIDSPPTFNADYGYKSWE 129
*N144
COX-la SYYNISYYTRLLPPVPEDCPLPMGTKGRPDLPDPKVLTERFFRRKTFRPDPQGANLM 223
COX-lb SYYNLSYYTRILPPVPKDCPTPLGVKGKAGLPDPELLVERLLKRRTFRPDPQGSNLM 195
COX-2 AYSNLSYYTRTLPPVPEDCPTPMGVVGKKELPDAKLLAEKLFMRRQFIPDPQGTSLM 186
*Q203/H207
COX-la FAFMAQHFTHQFFKTDHELQGGFT LGVDAGNIYGDNLAKQHHLRLHKDGKLKY 280
COX-lb FAFFAQHFTHQFFKTYNRMGVGFT ALAH VDAGHVYGDNLQRQLKLRLHKDGKLKY 252
COX-2 FAFFAQHFTHQFFKSDMKKGPAFT ATG VDLNHVYGGSMERQHKLRLRQDGKLKY 243
--------------*R277----
COX-la QIVNGETYPPTTSEAPVHMMYPEDVPPEKRLAIGQEVFGLLPGLTMYATI LREHNR 337
COX-lb QLVDGQIYPPSVVDAPVKMSYPPGVPPEAQMAICQEVYGLLPGLGMFATL LREHNR 309
COX-2 QVLDGEVYPPTVKEVGADMHYPPHVPESHRFAVGHEAFGLVPGLMMYATILREHNR 300

COX-la LCDILKAEHPTWDDEQLFQTTRLIVIGEIINIIIEEYVQQLSGYQLKLKFDPTLLFN 394
COX-lb VCDELKAEHPTWDDEQLFQTARFVIIGETIKIVIEEYVQQLSGYLLQLKFDPALLFN 366
COX-2 VCDJLKEVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYNFKLKFDPELLFN 357
S* *Y385/H388 *N410
COX-la ERFQYSNRIALEFCHLYHWHSLMPDSFLIDGDDIPYSQFFYNTSILMHYGVEKLVDA 451
COX-lb SNFQYGNRIALEFSQLYHWHPLMPESFLINGDELPYKRFLFNNTVLTHYGIENLVTA 423
COX-2 QRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHGIGNLVES 414

Figure 2-2. Amino acid alignment of the three forms of COX sequenced from the gills of the
longhorn sculpin (Myoxocephalus octodecemspinosus): COX-la, COX-lb, and COX-
2. All sequences were obtained during this study. Amino acids that share at least
60% identity across sequences are highlighted. *Indicates amino acids (numbered
based on ovine COX-1) involved in COX function: active site of COX (tyrosine-385,
histidine-388, and serine-530), substrate binding site (arginine-120), N-glycosylation
sites (asparagine-68, 144, and 410), sites for peroxidase activity (glutamine-203 and
histidine-207), and the two sites which define conformational differences in channels
between COX-1 and COX-2 in mammals (arginine-513 and valine-523) (Yang et al.,
2002; Ishikawa et al., 2007). The black boxes indicate the two domains that define
the haem-binding sites and the dashed box indicates the membrane binding domain
(Kulmacz et al., 2003; Ishikawa et al., 2007). The solid lines represent disulfide
bonds, with the beginning and end of the longest bond represented by arrows
(Kulmacz et al., 2003). The dashed line represents the arginine-277 loop region,
which has been shown to differ between COX-1 and COX-2 forms in mammals
(Yang et al., 2003; Kulmacz et al., 2003). The numbers to the right of the sequences
indicate the amino acid number of each sequence.











COX-la FSHQPAGQIGGGHNSHAVVLKVAEMVIRESRETRVQPFNEYRKKFNLQPYTSFYDLT 508
COX-lb FSRQVAGQIGGGFNINAAVTKVSVLTIKESRKLRMQPFNEYRKRFNLKPYTSFREFT 480
COX-2 FTNQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFSMKPYSSFEDMT 471
*R513 *V523 *S530
COX-la GDIEMAKGLEELYGDIDAVEFYPGLMLEKTLPTRIFGESMLEMGAPLFPERPVGKPH 565
COX-lb DNEEIARELEEFYGDVDALEFYPGLLLERTREGSIFGESMVEMGAPFSLKGLLGNPI 537
COX-2 GEKEMAAILEEFYGHVDAVELYPGLLVEKPRSNAIFGETMVEMGAPFSLKGLMGNPI 528

COX-la LLPV-LEAQHLWGRDGFQYRNFHSEETGVPQHQVVSIRGL-PCSPKGRRNQTNKSI- 620
COX-Ib CSPVYWKPSTFGGKVGF---DIVNSAT---LKKLVCLNTR-TCSYVAFRVPTEEQLK 587
COX-2 CSPEYWKPSTFGGSEGF---NIVNTAS---LQRLVCNNVQGPCPVASFSGPDVKDSG 579

COX-la -----------------------YTL 622
COX-lb TGNDDSKTRT------------ DEL 600
COX-2 SMIINSSTSNSDINPTVILKERTTEL 605


Figure 2-2. Continued.











sCOX MEAARIILLLLPLCFLKMADTTAINPCCYYPCQNKGICVNVGKEGYECDCTRT 53
hCOX MTSEVFVVLCVAVVFAGAA--AADDPCCGSPCENKGVCVSVGFEDYECDCTRT 51
t *N68 .................................................................................
sCOX GYYGVNCTFPFSWSRVHKFLKPSPSSMHHVLTHYKWLWYIINNISFFSDTLMR 106
hCOX GYYGSNCTYPTWTWIVNMLKPLPDTVHYFLTHFAPFWSLVNRAAFLRDRVMR 104
*R120 N1 -""
sCOX L.LTVRANIIPSPPTYNSDYTYVSWEGYSNISYLTRLLPPVPKDCPTPTGTQG 159
hCOX -.LMSRAHMVSSPPTYNADYGYKSWEAYSNLSYFTRLLPPVPQGCPTPMGRTG 157
*Q203/H207
sCOX YKKLPDSEQLAEEFLLRRKFIPDPQGSNLMFAFFAQHFTHQFFRTDLDRGPGF 212
1COX HQFFNTDPVRGPAF 14
hCOX KKELPDAQKLAERFLLRRTFIPDPQGSNLLFAFFAQHFTHQFFKTDFKRGPGF 210
COXc HQFFKTDFKKGAGR 14
COXd HQFFKTDFKKGPGR 14

sCOX TCALGH3VDLTHIYGDSLERQHHLRLFKDGKLKYQVVNGEVFPPSVKEAPIQM 265
ICOX TPALGH[VDLNHIYGGTLERQHQLRLFKDGKLKFQMIDGEAYPPVVRDAPVHM 67
hCOX TCALGH3VDLSHIYGDTLDKQHKLRLHNNGKLKFQMIDGEVFPPLVSEAPVDM 263
COXc TY-GDH3VDLSHIYGETVERQHQLRSFTDGKLKFQRVEGEVYPPSLADAPVHM 66
COXd TV-SDHVDMSHVYGETVERQRQLRSFQDGKLKYQLVDGEAFPPSLQDASVHM 66
--*R277----
sCOX KYPSTLPEEKRLAIGHDTFGLIPGLMMYATI LREHNRVCDILKEEHPVWSDE 318
1COX VYPEHVPASLRFAVGHEVYGLLPGLLVYATVILREHNRVCDJLHARHPRWDDE 120
hCOX IYPPHVPEAARFAVGHEAFGLVPGLMLYGTL'LREHNRVCDJLDVDHPEWDDE 316
COXc IYPPYVPEGKRFAIGHEFFGLLPGLFVYSTVILREHNRVCDJMKELHPDWDDE 119
COXd IYPPDVPEKKRFALGHEFFGLLPGLFVWATV LREHNRVCDJMKDLHPDWDDE 119

sCOX QLFQTTRLILIGETIKIVIEDYVQHLSGYNFRMMFNPELLFTEHFQYSNRIAV 371
1COX RLFQTARLILTGETMKIVIEEYVQHLSGYNFHLKFDPTLLFGVNFQYSNRMSL 173
hCOX RIFQTARLILIGETIKIVIEDYVQHLSGYHFKLKFDPKLLFGQPFQYSNRISV 369
COXc RLFQTARLILTGETINIIINEYVQHLSGYNFDLFWDPELLFSDQFQYQNRIFV 172
COXd RLYQTARLILISETINIVIGEYVQHLAGKNFQLFWDPELLFEEQXQYQNSIFV 172

Figure 2-3. Amino acid alignment of the forms of COX sequenced from the gills of the lamprey
(Petromyzon marinus), hagfish (Myxine glutinosa), and amphioxus (Branchiostoma
lancelolatum), along with sCOX from the dogfish (Squalus acanthias, Accession
#AAL37727). Amino acids that share at least 60% identity across sequences are
highlighted. *Indicates amino acids (numbered based on ovine COX-1) involved in
COX function: active site of COX (tyrosine-385, histidine-388, and serine-530),
substrate binding site (arginine-120), N-glycosylation sites (asparagine-68, 144, and
410), sites for peroxidase activity (glutamine-203 and histidine-207), and the two
sites which define conformational differences in channels between COX-1 and COX-
2 in mammals (arginine-513 and valine-523) (Yang et al., 2002; Ishikawa et al.,
2007). The black boxes indicate the two domains that define the haem-binding sites
and the dashed box indicates the membrane binding domain (Kulmacz et al., 2003;
Ishikawa et al., 2007). The solid lines represent disulfide bonds, with the beginning
and end of the longest bond represented by arrows (Kulmacz et al., 2003). The
dashed line represents the arginine-277 loop region, which has been shown to differ
between COX-1 and COX-2 forms in mammals (Yang et al., 2003; Kulmacz et al.,
2003). The numbers to the right of the sequences indicate the amino acid number of
each sequence.














*Y385/H388 *N410
sCOX EFDHLYHWHPLMPDSFIVKGQDFSYKDFLFNTDILLNLGVDALVESFSKQIAG 424
1COX EFNHLYHWHPLMPDSLLIDGRNYSYDEFLFNPGLLADKKLMPLVRSFMRQRAG 226
hCOX EFNHLYHWHGLNPDAFRVGTQEYQYSQFLFNNTILLNHGVRGLLEAFNVQQAG 422
COXc EFNHL 177
COXd EFNHL 177

sCOX RIGGGRNIHQSLLHIAIATIEHGRLLRFQPYNEYRKKLGLTPYKSFQELTGER 477
1COX TVSGGRNINKNLLHVATSIIEHGRTLRLQSLNQYRHRFNMRPFTSFLELTGDE 279
hCOX RIGGGQNIHGALLHVATASIKHGRKMRFQSLNQYRKQFGLQPYQSFEQLTGET 475
*V523 *S530
sCOX EVAARLEKLYGHIDAMEFYPALLLEAPNKNSIFGESMVEMGAPFSLKGLMGNP 530
1COX AMAAEME 286
hCOX EMAADLAELYSDINAMEFYLGLMVEKPRQGALFGETMVEAGAPFSLKGLMGNA 528

sCOX ICSPDYWKPSTFGGKTGFDIVNTATFEKLICLNVK-KCPYVGFHVPY------ 582
hCOX ICSPEYWKPSTFGGNRGFEIVNSASLRRLVCLNLQGPCPDVAFHVPRDNQQDV 581

sCOX NVDNDYEREKGKP----------- STEL 593
hCOX VVNVTGSQGGSDGVTTTPHYVADQQSREL 610

Figure 2-3. Continued.














COX1 Mus
COX1 G.gal
COX1 Danio
1COX
COX2 Mus
COX2 G.gal
COX2 F.het

COX1 Mus
COX1 G.gal
COX1 Danio
1COX
COX2 Mus
COX2 G.gal
COX2 F.het

COX1 Mus
COX1 G.gal
COX1 Danio
1COX
COX2 Mus
COX2 G.gal
COX2 F.het

COX1 Mus
COX1 G.gal
COX1 Danio
1COX
COX2 Mus
COX2 G.gal
COX2 F.het

COX1 Mus
COX1 G.gal
COX1 Danio
1COX
COX2 Mus
COX2 G.gal
COX2 F.het


*H207
HQFFKTSGKMGPGFT ALGH VDLGHIYGDNLERQYHLRLFKDGKLKYQVLDG
HQFFKTSGKMGRGFT ALGH VDLGHLYGDNLQRQHQLRLFQDGKLKFQVVNG
HQFFKTHNRVGLGFT GLGH VDAGHIYGDSLDRQLELRLHKDGKLKYQVLNG
HQFFNTDPVRGPAFT ALGH VDLNHIYGGTLERQHQLRLFKDGKLKFQMIDG
HQFFKTDHKRGPGFT GLGH VDLNHIYGETLDRQHKLRLFKDGKLKYQVIGG
HQFFKTDHKKGPGFTCAYGH VDLNHIYGETLERQLKLRLRKDGKLKYQMIDG
HQFFKSDMKNGPAFT AKGH VDLGHIYGENLEKQHKLRLFKDGKLKYTMVNG
-------- --*R 77----
EVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTIILREHNRV
EVYPPSVTEVPVHMVYPPAIPKEKQLAMGQEVFGLLPGLCMYATLLREHNRV
DIYPPTVLHAQVKMSYPPSVPPEQQLAIGQEVFGLLPGLGMYATLLREHNRV
EAYPPVVRDAPVHMVYPEHVPASLRFAVGHEVYGLLPGLLVYATVILREHNRV
EVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIILREHNRV
EMYPPTVKDTQAEMIYPPHVPEHLQFSVGQEVFGLVPGLMMYATI LREHNRV
EVYPPLVKDVGVEMHYPPHVPDSQRFAVGHEAFGLVPGLLMYATI LREHNRV

7D jLKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLSGYFLQLKFDPEL
:DILKQEHPTWGDEQLFQTARLILIGETIKIVIEDYVQHLSGYFLNLKFDPEL
:EILKQEHPTWGDEQLFQTARLIIIGETIRIVIEEYVQHLSGYRLKLHFDPTL
:DJLHARHPRWDDERLFQTARLILTGETMKIVIEEYVQHLSGYNFHLKFDPTL
:DILKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL
:DJLKQEHPEWDDEQLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL
LDLKGVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL
*Y385/H388 *N410
LFRAQFQYRNRIAMEFNHLYHWHPLMPNSFQVGSQEYSYEQFLFNTSMLVDYG
LFEQQFQYRNRIAVEFNQLYHWHALMPDSFTIQGQEYSYEQFLYNTSMLMDYG
LFNSQFQYQNRISVEFNQLYHWHPLMPDSFYIDGDHIQYSKFIFNTSILTHYG
LFGVNFQYSNRMSLEFNHLYHWHPLMPDSLLIDGRNYSYDEFLFNPGLLADKK
LFNQQFQYQNRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHG
LFNQRFQYQNRIAAEFNTLYHWHPLLPDTFQIHNQEYTFQQFLYNNSIMLEHG
LFNQRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHG

VEALVDAFSRQRAGRIGGGRNFDYHVLHVAVDVIKESREMRLQPFNEYRKRFG
VEALAESFSMQTAGRIGGGQNINANVLGVAVGVIEESRQLRLQPFNEYRKRFG
LEKLVEAFSIQPAGQIGGGHNIHPVVSGVAERVIVESRELRLQPFNEYRKRFN
LMPLVRSFMRQRAGTVSGGRNINKNLLHVATSIIEHGRTLRLQSLNQYRHRFN
LTQFVESFTRQIAGRVAGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFS
LSHMVKSFSKQSAGRVAGGKNVPAAVQKVAKASIDQSRQMRYQSLNEYRKRFM
INNLVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFS


Figure 2-4. Amino acid alignment ICOX sequenced from the gills of the lamprey (Petromyzon
marinus), with COX-1 and COX-2 forms from vertebrates (Accession # NP 032995,
NP 035328, XP425326, XP422297, AAS21313, and NP_705942). Amino acids
that share at least 60% identity across sequences are highlighted. *Indicates amino
acids (numbered based on ovine COX-1) involved in COX function: active site of
COX (tyrosine-385 and histidine-388), N-glycosylation site (asparagine-410), and
sites for peroxidase activity (glutamine-203 and histidine-207) (Yang et al., 2002;
Ishikawa et al., 2007). The black boxes indicate the two domains that define the
haem-binding sites (Ishikawa et al., 2007). The dashed line represents the arginine-
277 loop region, which has been shown to differ between COX-1 and COX-2 forms
in mammals (Yang et al., 2003; Kulmacz et al., 2003). The numbers to the right of
the sequences indicate the amino acid number of each sequence.















COX1 Mus LKPYTSFQELTGEKEMAAEL 493
COX1 G.gal LKPYTSFQELTGEEDKAAEL 536
COX1 Danio LKPYTSFAELTGEQEMSKEL 491
1COX MRPFTSFLELTGDEAMAAEM 285
COX2 Mus LKPYTSFEELTGEKEMAAEL 477
COX2 G.gal LKPFKSFEELTGEKEMAAEL 477
COX2 F.het MKPYTSFEDLTGEKEMAAIL 482

Figure 2-4. Continued.














COX1 Mus
COX1 Danio
COX1 Ovis
hCOX
COX2 Mus
COX2 F.het
COX2 Ovis

COX1 Mus
COX1 Danio
COX1 Ovis
hCOX
COX2 Mus
COX2 F.het
COX2 Ovis

COX1 Mus
COX1 Danio
COX1 Ovis
hCOX
COX2 Mus
COX2 F.het
COX2 Ovis


++ +
MSRRSLSLWFPPLLLLLLPPTPSVLLADPGVPSPVNPCCYYPCQNQGVCVRFG
MRECNFLLKWTVI--LLLSVSFCAGEESPTSSNTANPCCYYPCQNQGICVRYG
MSRQSISLRFPLL--LLLLSPSPVFSADPGAPAPVNPCCYYPCQHQGICVRFG
MTSEVFVVLCVAVVFA-GAAAAD------------DPCCGSPCENKGVCVSVG
MLFRAVLLCAALGLSQAA-----------------NPCCSNPCQNRGECMSTG
MNRITFAVFLLALCFSFHKEVLG------------NACCSEPCQNRGVCTAMG
MLARALLLCAAV-VCGAA-----------------NPCCSHPCQNRGVCMSVG
---- +*N68 ,...+...........+..... ..................
LDNYQCDCTRTGYSGPNCTIPEIWTWLRNSLRPSPSFTHFLLTHGYWLWEFVN:
LERYECDCTRTGYYGENCTIPLWTRVYRLLKPSPNVVHYILTHFDWLWDLINi
LDRYQCDCTRTGYSGPNCTIPEIWTWLRTTLRPSPSFIHFLLTHGRWLWDFVN:
FEDYECDCTRTGYYGSNCTYPETWTWIVNMLKPLPDTVHYFLTHFAPFWSLVN:
FDQYKCDCTRTGFYGENCTTP FLTRIKLLLKPTPNTVHYILTHFKGVWNIVNi
SDSYECDCTRTGYRGQNCTTPEFLTWIKISLKPTPNTVHYILTHFKGFWNIIN:
FDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPDTVHYI.THFKGVWNIVN
+ ++ + *R120+ *N144 +
.A'Tn'REV'LM~A7LfLTVRSNLIPSPPTYNSAHDYISWESFSNVSYYTRILPSVP
R-SFLRDWLMRK LTVRANLIPSPPTYNSRYDYLNWEAYSNITYYTRILPPVP
-ATFIRDTLMRL LTVRSNLIPSPPTYNIAHDYISWESFSNVSYYTRILPSVP
kAAFLRDRVMRHVLMSRAHMVSSPPTYNADYGYKSWEAYSNLSYFTRLLPPVP
NIPFLRSLIMKY LTSRSYLIDSPPTYNVHYGYKSWEAFSNLSYYTRALPPVA
SISFLRDAIMKY LTSRSHMIDSPPTYNADYGYKSWEAYSNLSYYTRALPPVP
ISF.LRNMIMR.LTSRSHLIESPPTYNVHYSYKSWEAFSNLSYYTRALPPVP


Figure 2-5. Amino acid alignment of ICOX sequenced from the gills of the lamprey
(Petromyzon marinus), with COX-1 and COX-2 forms from vertebrates (Accession #
NP 032995, NP035328, XP_425326, XP_422297, AAS21313, and NP705942).
Amino acids that share at least 60% identity across sequences are highlighted.
*Indicates amino acids (numbered based on ovine COX-1) involved in COX
function: active site of COX (tyrosine-385, histidine-388, and serine-530), substrate
binding site (arginine-120), N-glycosylation sites (asparagine-68, 144, and 410), sites
for peroxidase activity (glutamine-203 and histidine-207), and the two sites which
define conformational differences in channels between COX-1 and COX-2 in
mammals (arginine-513 and valine-523) (Yang et al., 2002; Ishikawa et al., 2007).
+Indicates amino acids unique to hCOX. The black boxes indicate the two domains
that define the haem-binding sites and the dashed box indicates the membrane
binding domain (Kulmacz et al., 2003; Ishikawa et al., 2007). The solid lines
represent disulfide bonds, with the beginning and end of the longest bond represented
by arrows (Kulmacz et al., 2003). The dashed line represents the arginine-277 loop
region, which has been shown to differ between COX-1 and COX-2 forms in
mammals (Yang et al., 2003; Kulmacz et al., 2003). The numbers to the right of the
sequences indicate the amino acid number of each sequence.











S+ + + *Q203/H207
COX1 Mus KDCPTPMGTKGKKQLPDVQLLAQQLLLRREFIPAPQGTNILFAFFAQHFTHQF 211
COX1 Danio NDCPTPMGTKGKIKLPDPKLLVEKFMLRRNFRLDPQGTNLMFAFFAQHFTHQF 209
COX1 Ovis RDCPTPMDTKGKKQLPDAEFLSRRFLLRRKFIPDPQSTNLMFAFFAQHFTHQF 209
hCOX QGCPTPMGRTGKKELPDAQKLAERFLLRRTFIPDPQGSNLLFAFFAQHFTHQF 199
COX2 Mus DDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMMFAFFAQHFTHQF 195
COX2 F.het EDCPTPMGVVGKKELPDVKVLAEKLLVRRRFIPDPQGTSLMFAFFAQHFTHQF 200
COX2 Ovis DDCPTPMGVKGRKELPDSKEVVKKVLLRRKFIPDPQGTNLMFAFFAQHFTHQF 194
+
COX1 Mus FKTSGKMGPGFT( ALGH3VDLGHIYGDNLERQYHLRLFKDGKLKYQVLDGEVY 264
COX1 Danio FKTHNRVGLGFTKGLGH3VDAGHIYGDSLDRQLELRLHKDGKLKYQVLNGDIY 262
COX1 Ovis FKTSGKMGPGFTCALGH3VDLGHIYGDNLERQYQLRLFKDGKLKYQMLNGEVY 262
hCOX FKTDFKRGPGFTCALGH3VDLSHIYGDTLDKQHKLRLHNNGKLKFQMIDGEVF 252
COX2 Mus FKTDHKRGPGFTrGLGH3VDLNHIYGETLDRQHKLRLFKDGKLKYQVIGGEVY 248
COX2 F.het FKSDMKNGPAFT[AKGH3VDLGHIYGENLEKQHKLRLFKDGKLKYTMVNGEVY 253
COX2 Ovis FKTDIERGPAFTk KM3VDLSHVYGESLERQHNRRLFKDGKMKYQMINGEMY 247
--+-----+---- *R277 +--
COX1 Mus PPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTILREHNRVCD-j 317
COX1 Danio PPTVLHAQVKMSYPPSVPPEQQLAIGQEVFGLLPGLGMYATLILREHNRVCEr 315
COX1 Ovis PPSVEEAPVLMHYPRGIPPQSQMAVGQEVFGLLPGLMLYATIILREHNRVCD- 315
hCOX PPLVSEAPVDMIYPPHVPEAARFAVGHEAFGLVPGLMLYGTLLREHNRVCDV 305
COX2 Mus PPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATI4LREHNRVCDj 301
COX2 F.het PPLVKDVGVEMHYPPHVPDSQRFAVGHEAFGLVPGLLMYATIrLREHNRVCD 306
COX2 Ovis PPTVKDTQVEMIYPPHIPEHLKFAVGQEVFGLVPGLMMYATILREHNRVCD 300
+
COX1 Mus LKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLSGYFLQLKFDPELLFR 370
COX1 Danio LKQEHPTWGDEQLFQTARLIIIGETIRIVIEEYVQHLSGYRLKLHFDPTLLFN 368
COX1 Ovis LKAEHPTWGDEQLFQTARLILIGETIKIVIEEYVQQLSGYFLQLKFDPELLFG 368
hCOX LDVDHPEWDDERIFQTARLILIGETIKIVIEDYVQHLSGYHFKLKFDPKLLFG 358
COX2 Mus LKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 354
COX2 F.het LKGVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 359
COX2 Ovis LKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 353
+ *Y385/H388 + *N410 + ++
COX1 Mus AQFQYRNRIAMEFNHLYHWHPLMPNSFQVGSQEYSYEQFLFNTSMLVDYGVEA 423
COX1 Danio SQFQYQNRISVEFNQLYHWHPLMPDSFYIDGDHIQYSKFIFNTSILTHYGLEK 421
COX1 Ovis AQFQYRNRIAMEFNQLYHWHPLMPDSFRVGPQDYSYEQFLFNTSMLVDYGVEA 421
hCOX QPFQYSNRISVEFNHLYHWHGLNPDAFRVGTQEYQYSQFLFNNTILLNHGVRG 411
COX2 Mus QQFQYQNRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQ 407
COX2 F.het QRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHGINN 412
COX2 Ovis QQFQYQNRIAAEFNTLYHWHPLLPDVFQIDGQEYNYQQFIYNNSVLLEHGVTQ 406
++ + + + ++ + +
COX1 Mus LVDAFSRQRAGRIGGGRNFDYHVLHVAVDVIKESREMRLQPFNEYRKRFGLKP 476
COX1 Danio LVEAFSIQPAGQIGGGHNIHPVVSGVAERVIVESRELRLQPFNEYRKRFNLKP 474
COX1 Ovis LVDAFSRQPAGRIGGGRNIDHHILHVAVDVIKESRVLRLQPFNEYRKRFGMKP 474
hCOX LLEAFNVQQAGRIGGGQNIHGALLHVATASIKHGRKMRFQSLNQYRKQFGLQP 464
COX2 Mus FVESFTRQIAGRVAGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKP 460
COX2 F.het LVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFSMKP 465
COX2 Ovis FVESFTRQIAGRVAGRRNLPAAVEKVSKASLDQSREMKYQSFNEYRKRFLLKP 459
*V523
COX1 Mus YTSFQELTGEKEMAAELEELYGDIDALEFYPGLLLEKCQPNSIFGESMIEMGA 529
COX1 Danio YTSFAELTGEQEMSKELEELYGHIDAMEFYPALLLEKTRPGAVFGESMVEMGA 527
COX1 Ovis YTSFQELTGEKEMAAELEELYGDIDALEFYPGLLLEKCHPNSIFGESMIEMGA 527
hCOX YQSFEQLTGETEMAADLAELYSDINAMEFYLGLMVEKPRQGALFGETMVEAGA 517
COX2 Mus YTSFEELTGEKEMAAELKALYSDIDVMELYPALLVEKPRPDAIFGETMVELGA 513
COX2 F.het YTSFEDLTGEKEMAAILEELYGDVDAVELYPGLLVEKPRTNAIFGETMVEMGA 518
COX2 Ovis YESFEELTGEKEMAAELEALYGDIDAMELYPALLVEKPAPDAIFGETMVEAGA 512

Figure 2-5. Continued













COX1 Mus
COX1 Danio
COX1 Ovis
hCOX
COX2 Mus
COX2 F.het
COX2 Ovis

COX1 Mus
COX1 Danio
COX1 Ovis
hCOX
COX2 Mus
COX2 F.het
COX2 Ovis


*S530 + + + + +
PFSLKGLLGNPICSPEYWKPSTFGGDVGFNLVNTASLKKLVCLNTK-TCPYVS
PFSLKGLMGNPICSPDYWKPSTFGGKTGFDIVNSATLKKLVCLNTK-WCPYVS
PFSLKGLLGNPICSPEYWKASTFGGEVGFNLVKTATLKKLVCLNTK-TCPYVS
PFSLKGLMGNAICSPEYWKPSTFGGNRGFEIVNSASLRRLVCLNLQGPCPDVA
PFSLKGLMGNPICSPQYWKPSTFGGEVGFKIINTASIQSLICNNVKG-CPFTS
PFSLKGLMGNPICSPEYWKPSTFGGTAGFDIVNTASLQRLVCNNVRGPCPVAS
PFSLKGLMGNPICSPEYWKPSTFGGEVGFKIINTASIQSLICSNVKG-CPFTS
+ + + ++
FR-VPDYPGDDGSVLVRR-------------------STEL
FHTPPSDYKPQRTS-----------------------HGEL
FH-VPDPRQEDRPGVERP-------------------PTEL
FH-VPRDNQQDVVVNVTGSQGGSDGVTTTPHYVADQQSREL
FN-V-QDPQPTKTATINASASHSRLDDINPTVLIKRRSTEL
FY-VP-DVKETGSMTINSSTSHSRDSNINPTVILKERTSEL
FS-V-QDAHLTKTVTINASSSHSGLDDINPTVLLKERSTEL


Figure 2-5. Continued













COXa Ciona
COXb Ciona
COXc
COXd

COXa Ciona
COXb Ciona
COXc
COXd


*H207
HMFFKTDPMKGMPYQ-GDQ-VDLSQIYGHGEKRQHELRSHVNGKLKVSLVDGH
HQFFKTNTIKGMPFQIGEH3VDLSHVYGHTIQRQHELRSHIDGKLKVFETNGE
HQFFKTDFKKGAGRTIGDH3VDLSHIYGETVERQHQLRSFTDGKLKFQRVEGE
HQFFKTDFKKGPGRTISDH4VDMSHVYGETVERQRQLRSFQDGKLKYQLVDGE
------------* 77----
EFPPLSNQTTANMSNINLLPQEYQFVFGHQGFSLMPTFLIWSTI LREHNRIC
VFPPLTESANVTMSGEKLM-RGRKFAIGHPGFGAFPSFFVIATLLREHNRVC
VYPPSLADAPVHMIYPPYVPEGKRFAIGHEFFGLLPGLFVYSTVILREHNRVC
AFPPSLQDASVHMIYPPDVPEKKRFALGHEFFGLLPGLFVWATVILREHNRVC


COXa Cional D[IKEENPAWDDERIFQTARLVLTGETIKVVIEDYVQHLSGFHYKLLYDPELV 385
COXb Cional DLKDLHPDWDDERLFQTARLILTGETLKIIVEDYVQHVSGFHFQLSYDPEIL 332
COXcI DMKELHPDWDDERLFQTARLILTGETINIIINEYVQHLSGYNFDLFWDPELL 159
COXd DJMKDLHPDWDDERLYQTARLILISETINIVIGEYVQHLAGKNFQLFWDPELL 159

COXa Ciona QGGSHSFHNQIHVEFQL 402
COXb Ciona HKSTFSYNNQIHAEFHI 349
COXc FSDQFQYQNRIFVEFNH 176
COXd FEEQXQYQNSIFVEFNH 176

Figure 2-6. Amino acid alignment of COXc and COXd sequences from the lancelet
(Branchiostoma lancelolatum) with COXa and COXb sequences from the sea squirt
(Ciona intestinalis). Amino acids that share at least 60% identity across sequences
are highlighted. *Indicates the amino acid (numbered based on ovine COX-1)
involved in peroxidase activity (histidine-207) (Yang et al., 2002; Ishikawa et al.,
2007). The black boxes indicate the two domains that define the haem-binding sites
(Ishikawa et al., 2007). The dashed line represents the arginine-277 loop region,
which has been shown to differ between COX-1 and COX-2 forms in mammals
(Yang et al., 2003; Kulmacz et al., 2003). The numbers to the right of the sequences
indicate the amino acid number of each sequence.














94 COX1b Gasterosteus
44 0ICOX1b Myoxocephalus
60 COX1b Oryzias

50 ICOX1b Fundulus
3 COX1 b Tetraodon
72 0COX1 Oncorhynchus

54 COX1 Danio
SCOX1 a Tetraodon

70 COX1a Fundulus
92 0COX1a Oryzias

22 79 COX1a Gasterosteus
55 0cOX1a Myoxocephalus
Sr COX1 Mus
COX1 Rattus

30 COX1 Oryctolagus
2 COX1 Homo
87 COX1 Canis

45 0COX1 Bos
86 COX1 Ovis
COX1 Gallus
81 0 COX1 Monodelphis

3 COX1b Monodelphis
56 84 COX1 b2 Monodelphis
COX1 Xenopus
sCOX Squalus
83 COX2 Gasterosteus
44 ICO2 Myoxocephalus
538 COX2b Oncorhynchus
53
40 COX2 Oryzlas
3 COX2 Fundulus
34 COX2 Tetraodon
74 COX2b Danio
COX2a Danio
27 COX2a Oncorhynchus

77 -COX2 Xenopus
94 COX2 Bos
26
23 COX2 Ovis
80 COX2 Gallus
4 COX2 Homo
434 COX2 Canis

57 COX2 Oryctolagus
38 COX2 Monodelphis
2 COX2 Mus
32- COX2 Rattus
1COX Petromyzon I
|hCOX Myxine
[COXc Branchiostoma
99 0-COXd Branchiostoma
COXa Clona
100 COXb Ciona


0.05









Figure 2-7. Evolutionary history of COX sequences inferred using the minimum evolution
method with a distance optimality criterion. The optimal tree with the sum of branch
length = 6.63322594 is shown. The percentage of replicate trees in which the
associated taxa clustered together in the bootstrap test (1000 replicates) is shown next
to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the phylogenetic
tree. The evolutionary distances were computed using the JTT matrix-based method
(Jones et al., 1992) and are in the units of the number of amino acid substitutions per
site (scale bar = 0.05 amino acid substitutions per site). The rate variation among sites
was modeled with a gamma distribution (shape parameter = 0.737). The ME tree was
searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of
1. The Neighbor-Joining algorithm was used to generate the initial tree. All positions
containing gaps and missing data were eliminated from the dataset (Complete
deletion option). There were a total of 147 positions in the final dataset. Phylogenetic
analyses were conducted in MEGA4 (Tamura et al., 2007). Sequences enclosed in a
box are novel and reported here for the first time.














94 COX1 b Gasterosteus
44 I= COX1b Myoxocephalus
61 COXlb Oryzias
51 COX1b Fundulus

37 COX1b Tetraodon
1 COX1 Oncorhynchus

60 COX1 Danio
I- COX1a Tetraodon

71 COX1a Fundulus
92 COX1a Oryzias

26 80 -[ COX1a Gasterosteus
57 COX1a Myoxocephalus

99 COX1 Mus
COX1 Rattus

32 8 86 COX1 Bos
98
COX1 Ovis

87 COX1 Oryctolagus

35 34 COX1 Homo
62 COX1 Canis
COX1 Gallus

91 COX1 Monodelphis

99 COX1b Monodelphis
85 COXlb2 Monodelphis
COX1 Xenopus

82 COX2 Gasterosteus
43'1 COX2 Myoxocephalus

52 3 COX2b Oncorhynchus
41 COX2 Oryzias

40 COX2 Fundulus
34= COX2 Tetraodon
0 -- COX2b Danio

COX2a Danio
26 CO)X2a Oncorhynchus

77 COX2 Xenopus

S94 COX2 Bos
0 COX2 Ovis
83 CO)X2 Gallus

3 COX2 Homo

383 COX2 Mus
COX2 Rattus

38 COX2 Monodelphis

13 COX2 Canis
41 COX2 Oryctolagus
sCOX Squalus
COXc Branchiostoma
99 COXd Branchiostoma
COXa Ciona
100 COXb Ciona


01









Figure 2-8. Evolutionary history of COX sequences excluding hagfish (hCOX) and lamprey
(1COX) sequences inferred using the minimum evolution method with a distance
optimality criterion. The optimal tree with the sum of branch length = 6.18990857 is
shown. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is shown next to the branches
(Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units
as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the JTT matrix-based method (Jones et
al., 1992) and are in the units of the number of amino acid substitutions per site (scale
bar = 0.1 amino acid substitutions per site). The rate variation among sites was
modeled with a gamma distribution (shape parameter = 0.742). The ME tree was
searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of
1. The Neighbor-Joining algorithm was used to generate the initial tree. All positions
containing gaps and missing data were eliminated from the dataset (Complete
deletion option). There were a total of 147 positions in the final dataset. Phylogenetic
analyses were conducted in MEGA4 (Tamura et al., 2007).












COXb Clona
COXa Clona


Canis


COX2 Xenopus
COX2a Oncorhynchus
COX2aDanio


COX2 Tetraodon


sCOX Squalus
CCX1 Monodelphis
995 60 COXlb2 Monrdelphis
100 COX b Monodelphis
978 r-l I Rattus

10Q X1 Oryatolagus
1 66*i1X1 Homo
34 -83Eril Canis





ICOXla Fundulus7
COXb a Tetraodon

ICOX II Ca taratmsus
I COXI a1 :aaphals
COXI aryziaa
614 COXIa Fundulus






0-7
SCOXI Cncorhynchus
CCXI Xenopus
L .57 COXI Gallus
4 CXd BranchiOs tora-4

0.1



Figure 2-9. Evolutionary history of COX sequences inferred using a maximum likelihood
optimality criterion. The most likely tree with an Ln L score = -25294.222410 is
shown. The number of replicate trees in which the associated taxa clustered together
in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein,
1985). The tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary distances
were computed using the WAG matrix-based method (Whelan and Goldman, 2001)
and are in the units of the number of amino acid substitutions per site (scale bar = 0.1
amino acid substitutions per site). The rate variation among sites was modeled with a
gamma distribution (shape parameter estimated = 0.737). Phylogenetic analyses were
conducted using PhyML (Guindon and Gascuel, 2003). Sequences enclosed in a
rectangle are novel and reported here for the first time.











COXb Ciona
COXa Ciona


COX2 Monod
QOX2 Canis


COX2 Xenop
Gallu
COX2a Onco
COX2a Dani
IX2b Dani
OX2b Onco
COX2 Tetra
,SY2 Myoxo


COX a Tetr


4 /Ud COX1a Myox
691 COX a Oryz
L 9 COX1a Fund
997
8 COX1b Tetr
COX b Myox
1000
I OXl b Gast
954
1 dCOX1b Fund
681 675
65 COXlb Oryz
946
1000 2 COX1 Danio
1000 724
COX1 Oncor
COX1 Xenop
COX1 Gallu
COX1 Monod
6G9 COXlb2 Mon
1000
COX1b Mono
981 COX1 Rattu
Mus
Oox1 Oryct
693DX1 Homo
80COX1 Canis
7Q 1 Ovis
Cu(1 Bos
COXd Branc
979
COXc Branc
0.1









Figure 2-10. Evolutionary history of COX sequences excluding sequences from the hagfish
(hCOX) and lamprey (1COX) inferred using a maximum likelihood optimality
criterion. The most likely tree with an Ln L score = -24242.30059 is shown. The
number of replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The
tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary distances
were computed using the WAG matrix-based method (Whelan and Goldman, 2001)
and are in the units of the number of amino acid substitutions per site (scale bar = 0.1
amino acid substitutions per site). The rate variation among sites was modeled with a
gamma distribution (shape parameter estimated = 0.742). Phylogenetic analyses were
conducted using PhyML (Guindon and Gascuel, 2003). Sequence names are
abbreviated from Figure 2-9.














COX Petromyzon
COX Myxine
COX2 Monodelphis

,&W canis
- COX2 Oryctolagus
0 Rattus
_QR6g2 Mus
000 COX2 Homo


SCOY2 Bos
COX2 Gallus
)0000
- COX2 Xenopus


Danio
b Oncorhynchus


! Oryzias


Monodelphis


COX1a Myoxoi


1 a Oryzias
I a Fundulus


I ... COXlaTetraodon

0.999 COX1 b Tetraodon
1 b Myoxocephalus
C- OX1 b Gasterosteus
0.939000
.74 0 0.59901 b Fundulus
COX1b Oryzias
COX1 Danio
0.776000
COX1 Oncorhynchus
COXd Branchiostoma
-- C c Branchiostoma
0.1


COXb Ciona
COXa Ciona


COM1 Bos









Figure 2-11. Alternate likelihood ratio test (aLRT) scores for branch support of COX sequences.
The most likely tree is shown and the topology, branch lengths, and model of
evolution are the same as in Figure 2-9. Scores represent the gain in likelihood when
collapsing a particular branch and are independent of bootstrap scores (Felsenstein,
1985). Scores were interpreted using a conservative estimate of the minimum of
parametric chi-squared and non-parametric Shimodaira-Hasegawa-like (SH) support
value interpretations (Anisimova and Gascuel, 2006). Analysis was performed using
PhyML (Guindon and Gascuel, 2003). Support values of interest to the present study
are enclosed in boxes.












COXb Ciona
SCOXa Ciona


COX2 Monod
CQX2 Canis


X2b Onco
COX2 Tetra


COX1a Myox


COXa Tetr


COX b Oryz


COX b Mono


O"uWX1 Canis

0.O99' 51)
L- CI1 BDos


COXd Branc
Branc


0.1









Figure 2-12. Alternate likelihood ratio test (aLRT) scores for branch support of COX sequences
excluding hagfish (hCOX) and lamprey (1COX) sequences. The most likely tree is
shown and the topology, branch lengths, and model of evolution are the same as in
Figure 2-11. Scores represent the gain in likelihood when collapsing a particular
branch and are independent of bootstrap scores (Felsenstein, 1985). Scores were
interpreted using a conservative estimate of the minimum of parametric chi-squared
and non-parametric Shimodaira-Hasegawa-like (SH) support value interpretations
(Anisimova and Gascuel, 2006). Analysis was performed using PhyML (Guindon
and Gascuel, 2003). Support values of interest to the present study are enclosed in
boxes.












COXb Ciona
- COXa Ciona


sCOX Squalus
- COX1 Xenopus
COXI Gallus
COXi Oryctolag a
1Fl.98COX1 Homo
00COX1 Canis
J 49?X1 Bos


COX1b Monodelphis


1 1. COXlaMyoxocephalusl
COXla Gasterosteus
COX1a Tetraodon
COX Myxine
COX2 Monodelphis
COX2 Oryctolagus
aOX2 Canis
53COX2 Homo
0.94 1 L po i 2 Bos
1.01 1 L COX2 Ovi
1C9X2 Mus
COX2 Rattus
COX2 Gallus
0.91
COX2 Xenopus
0.9 COX2b Danio
0COX2b Oncorhynchus
1.00 COX2 Tetraodon
COX2 Fundulus
S 1.0 COX2 Oryzias
1. OX2 Gasterosteus
-COX2 Myoxocephalus
SCOX2a Danio
SCOX2a Oncorhynchus
[COX Petromyzon
0.93 COXe Branchiostoma
0 COXd Branchiostoma
0.1



Figure 2-13. Evolutionary history of COX sequences inferred using a Bayesian optimality
criterion. The consensus tree is shown. The posterior probabilities (10 million
generations) are shown next to the branches. Trees were sampled every 500
generations with a burnin of 10% (401 trees). The tree is drawn to scale, with branch
lengths in the same units as those of the evolutionary distances used to infer the

phylogenetic tree. The evolutionary distances were computed using the WAG matrix-
based method (Whelan and Goldman, 2001) and are in the units of the number of
amino acid substitutions per site (scale bar = 0.1 amino acid substitutions per site).
The rate variation among sites was modeled with a gamma distribution (shape
parameter estimated = 0.672275). Phylogenetic analyses were conducted using
MrBayes 3.1.2. Sequences enclosed in a rectangle are novel and reported here for the
first time.












COXa Ciona
COXb Ciona
sCOX Squalus
COX1 Xenopus
1.) COX1 Gallus
COX1 Oryctolagus

1.00 98COX1 Homo
OOCOX1 Canis
1 9 OX1 Bos
1.00
SCOX1 Ovis
0.92 COX1 Mus
1. 1.00
COX1 Rattus
COX1 Monodelphis
S 1.00 d1 COX b Monodelphis

COXlb2 Monodelphis


0.97 COX1 Oncorhynchus
COX1 Danio
0 6 COX1b Tetraodon
COX1b Fundulus
9 COXlb Oryzias
0.99
1.00 r .0COX b Gasterosteus
1.00 1.00
COX1b Myoxocephalus
COX1a Fundulus
0.98
COXla Oryzias
1.i0 COX1a Myoxocephalus
COX1a Gasterosteus
C0.88
COXI a Tetraodon


S COX2 Monodelphis
COX2 Oryctolagus
8a0X2 Canis
75COX2Homo

1 00OX2 Bos


1 'XK
I "TTM 1.00


L COX2 Rattus
COX2 Gallus
0.96
COX2Xenopus
10 COX2b Danio

COX2b Oncorhynchus

1.00 COX2 Tetraodon
COX2 Fundulus

S 1.0 COX2 Oryzias
COX2 Gasterosteus
1.00
0COX2 Myoxocephalus
COX2a Danio
CO1.00
COX2a Oncorhynchus


COXc Branchiostorna
1.0 Branchistma
COXd Branchiostoma


- 1.00


I 1.0(


0.1









Figure 2-14. The evolutionary history of COX sequences excluding hagfish (hCOX) and
lamprey (1COX) sequences inferred using a Bayesian optimality criterion. The
consensus tree is shown. The posterior probabilities (10 million generations) are
shown next to the branches. Trees were sampled every 500 generations with a bumin
of 10% (401 trees). The tree is drawn to scale, with branch lengths in the same units
as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the WAG matrix-based method (Whelan
and Goldman, 2001) and are in the units of the number of amino acid substitutions
per site (scale bar = 0.1 amino acid substitutions per site). The rate variation among
sites was modeled with a gamma distribution (shape parameter estimated =
0.667978). Phylogenetic analyses were conducted using MrBayes 3.1.2.








a) b) .















c) d)
















Figure 2-15. The 21 alternate tree topologies generated to test the effects of relocating the
hagfish (hCOX) and lamprey (ICOX) sequences on the likelihood score (Ln L). Each
topology is simplified, but all relationships remain the same as in the most likely
topology (Figure 2-9) with the exception of hCOX and ICOX. Each topology was
generated using TreeViewl.6.6 based on the most likely topology. Ln L was
calculated for each topology online using PhyML (Guindon et al., 2005), branch
lengths and model parameters were estimated. The topologies are named in Table 2-9
as follows (from most to least likely): a) COX2 Hag (most likely) b) COX1
Lamp/COX2 Hag c) COX2 Agnatha d) Lamp/Amp e) COX2 Vert f) COX1 Hag g)
COX1 Vert h) Ancestral Hag i) COX1 Agnathaj) COX1 Hag/COX2 Lamp k)
Ancestral Agnatha 1) COX1 Lamp m) COX2 Lamp n) Agntha/Amp o) Hag/Amp p)
Hag Internal q) Lamp Internal r) Lamp Mammal s) Lamp Teleost t) Hag Teleost u)
Hag Mammal.





100






e) f)








g) h) .



L mi m



i)j)..
-,.







Figure 2-15. Continued.


101









k) 1)



--- ______________ ^ ^-- --- -- -'llli














m) n)


















o) p)













Figure 2-15. Continued




Figure 2-15. Continued.











q) c := r) c


u~tot

















s) t)}
_____ ______ Cm~ltianu m^







hillashwites







u)l
|- ---- .. ----- L-2--
L*"raxlhumar
























Figur 2-5.
^. -I103



)) t> ,nvs
ua :*Brram
SQ ~n ic, _co! l?
















coooor*au






Figure~ 2-15.Contnued
103 .r- =
I r ^ ':,*.'*
__________________ ~ BLJ| O.K'S. BE~tfifct`





L ^ f "- _F,.,.
J ^ f
l nh __ ^ ^
___________~r ""**_____
I-f C ^ ^ ^ ^ ^ ^ ^ f
^ -------------------- ^ 1

Ui "2 ^NL RI-,~I

I- ------- ^^^^ = ^ ^h.^.
-- 0 X L .----------

1~ ~ 1 TeE -tt-s --
---^----^ |i I&'l-iWU~i ^^^^^^^^^^^^ ^



^^|},-.K1








Figur I--lj- C01CS'Oida












103













CQOXd BrarLosboina
COxa Crona


COX2 GaRlus-
COxq Xerpus
CGO2 Ovi
COX2 Manodel*his
COX2 Cuiis


COX2 Pru
COX, MiVS

cox2 aa oi
cCX20 riasw
COX2-a OrLshynd s
COX2 Ga$terWisV
COX2tb Dan
COX2b OQnXWhyrthvS
COx2 Tw"raod
COX Oryzias
CoX2 Fridukis
G0X2 WOiaOMMlu
1COX Petronmyrmn
SCOX Squalus j


COX1 Bos
COX1 M~nidelpis

COXl Monodflophis
COX1 laindel s
coxi Ws
COX1 Rautus
COX1 Orycolagus
COX HTna'
Coxi Cams
COXi Crdas

COXIa Tetaodrn
COX 1a Galserosiem
COX 1 a Myoocephaks
COXa Funrdulus
COX1 Danio
COX Oncorwhhtus
COX1b Funailus
COX b Tetrankn
COXlb Gasierole-us
COXib Myonxccihakus
.U*i it .jr,:iia


Cephalochordata
Urochordata


2a


2b/2


















la


1



lb


Vertebrata-2






















Vertebrata-1


Figure 2-16. Unrooted phylogeny showing the most likely scenario for COX evolution in the
chordates based on several other analyses. Clades of interest are highlighted on the
right and sequences that represent unclear placements are highlighted in red boxes.









CHAPTER 3
SUMMARY AND FUTURE DIRECTIONS

Summary

This thesis is the first attempt to characterize the evolution of the enzyme cyclooxygenase

in the phylum chordata. Traditional molecular protocols such as cloning and polymerase chain

reactions were used to search for cyclooxygenase sequences in a wide range of evolutionarily

key chordates. Using these novel sequences and pre-existing sequences from GenBank and

genomic databases, phylogenetic analyses were performed to reveal the identity of the novel

sequences and create the most likely scenario for the evolutionary history of cyclooxygenase in

the chordates.

Novel COX Sequences

Chapter 2 describes the process of obtaining and characterizing novel cyclooxygenase

(COX) sequences in amphioxus (Branchiostoma lancelolatum), the Atlantic hagfish (Myxine

glutinosa), the sea lamprey (Petromyzon marinus), the euryhaline killifish (Fundulus

heteroclitus), and the longhorn sculpin (Myoxocephalus octodecemspinosus). Total RNA was

isolated and reverse transcribed from gill tissue (or whole animals in amphioxus) and the

resulting cDNA was exposed to PCR in the presence of degenerate and/or specific primers. The

products were then visualized and cloned. The resulting plasmids were sequenced at the Marine

DNA Sequencing Center at the MDIBL. Complete amino acid sequences of putative COX

proteins from the hagfish, killifish, and sculpin and incomplete amino acid sequences from

amphioxus and the lamprey were predicted based on the sequences found. These putative amino

acid sequences were aligned with known COX protein sequences and conserved domains,

regions, and residues critical to COX function were described. Based on the novel sequences'

identity to known COX forms and phylogenetic analyses (see below), it was concluded that









COX-la, COX-lb, and COX-2 are found in the sculpin; COX-la and COX-lb are found in the

killifish; a new COX form named 1COX is found in the lamprey; a new COX form named hCOX

is found in the hagfish; and two ancestral forms named COXc and COXd are found in

amphioxus.

Phylogenetic Analyses

Chapter 2 also describes the phylogenetic analyses used to reconstruct the evolutionary

history of cyclooxygenase in the chordates. The nine novel COX protein sequences described

here, as well as 41 additional COX protein sequences from GenBank and genomic databases,

were subjected to phylogenetic analyses using three optimality criteria: minimum

evolution/distance, maximum likelihood, and Bayesian phylogenetics. In each analysis, two

clades were well-supported in the gnathostomes: a COX-1 clade and a COX-2 clade. Each of

these clades contained a well supported teleost and tetrapod clade. In the teleost clades, clades

for COX-la, COX-lb, COX-2a, and COX-2b were well-supported, confirming the findings of

Ishikawa et al. (2007) and Ishikawa and Herschman (2007). The placement of the more

evolutionarily ancestral chordates was ultimately uncertain, although it appears that the

urochordates, cephalochordates, and vertebrates (containing COX-1 and COX-2) each represent

an independent origin of COX proteins. The hagfish and lamprey sequences most likely

represent basal members of the COX-2 and COX-1 clades of vertebrates, respectively; however,

these forms may belong closer to the root of the tree.

Future Directions

Although here I report 9 novel cyclooxygenase (COX) sequences from a range of

chordates and present a likely scenario for the evolution of this gene family in the chordates,

many questions remain regarding COX evolution and function in the chordates. These include

determining the true history of COX evolution in the earliest chordates, pinpointing the origin of









COX-1 and COX-2, and investigating the history of COX duplication and loss in the teleosts.

Finally, the physiological function of COX in the earliest chordates has only been preliminarily

investigated.

Completion of novel sequences

Several of the novel sequences documented here could not be completed despite multiple

efforts using many primer sets and PCR techniques. Notably, only 532 bp were obtained of the

sequences from amphioxus (COXc and COXd). Compared to the complete open reading frame

of COX transcripts in other chordates (ranging from about 1700-2500 bp), this indicates that

about 75% of these sequences remain unknown. Completing the rest of these sequences or

thoroughly searching the recently complete genome of Branchiostomafloridae may yield

putative proteins that provide novel groupings in phylogenetic analyses. Based on the available

sequence data, however, COXc and COXd are well-supported as being near the base of the

chordate COX lineage, sister to the vertebrate COX-1 and COX-2 clade. The 1COX sequence

obtained from the lamprey is also only about 40% complete. Completing the open-reading frame

of this putative protein or searching genomic databases could possibly give a more definite view

of its identity as this sequence groups with different clades depending on the phylogenetic

analysis used. In actuality, the short size of 1COX compared to the other COX sequences used in

the phylogenetic analyses may have contributed to its tendency to appear in different clades in

different analyses. Finally, the COX-la sequence obtained from the sculpin may warrant further

investigation and reaffirmation via additional sequencing since many residues critical to COX

function are missing.

Additional Phylogenetic Analysis

Unfortunately, the evolutionary relationship of the COX proteins in the chordates was not

able to be unequivocally resolved, despite the use of three different phylogenetic approaches.









This may be due to the incompleteness of some sequences and a lack of sufficient sampling

across taxa or may represent a history that is irresolvable under the current circumstances.

However, the phylogenetic techniques used here may also be improved upon in future analyses.

Phylogenetically uninformative areas of the sequences (such as near the C and N terminus,

which show little homology across sequences) could be removed. Here, protein sequences were

used to gain insight into the evolution of COX functionality, but insights could also be gained by

performing similar analyses using DNA sequence data instead of using their inferred protein

amino acid sequences. This would allow for greater complexity of models to estimate

substitution rates and could take additional factors into account such as codon bias. Using

nucleotide versus amino acid would also enable additional search strategies and phylogenetic

programs to be used. Choosing the optimum search strategy and computer program may be

critical in finding the optimum evolutionary reconstruction. For example, maximum likelihood

analyses PhyML (the program used in this study) may find suboptimal likelihood scores when

the data-set contains little phylogenetic information (Morrison, 2007). However, programs such

as RAxML (Stamatakis, 2006), GARLI (Brauer et al., 2002), and PAUP* (Rogers and Swafford,

1998) may perform better under the same circumstances (Morrison, 2007). Also, evaluating

alternative tree topologies using frameworks other than maximum likelihood may yield

alternative results.

Additional Sequences

Based on the most likely scenario of COX evolution in the chordates, there are several

COX sequences in the chordates examined in this study that remain to be characterized. If

hCOX in actuality represents a basal form of COX-2, then there should also be a COX-1 form in

the hagfish. Similarly, if ICOX and sCOX represent basal forms of COX-1 in the lamprey and

dogfish, then they should also contain versions of COX-2. These additional forms of COX may









be harder to clone and sequence due to accumulated mutations over evolutionary time, which

would render the sequences unidentifiable and possibly non-functional. However, this is not the

case in the other vertebrates, as COX-1 and COX-2 seem to each have critical functionality.

Using Southern blotting techniques (Southern, 1975) to identify COX DNA sequences in the

DNA of hagfish, lamprey, and dogfish could immediately reveal the presence of two forms of

COX in these animals. If two forms are present, it could be concluded that they likely represent

COX-1 and COX-2 forms, and thus the origin of these forms would coincide with the vertebrate

lineage (as predicted in the current phylogenetic analysis). However, if only one form of COX

was found the results would suggest a more recent origin of COX-1 and COX-2. This technique

could be applied to a wide diversity of chordates to determine how many COX variants they

contain.

Additional Taxa

In this study, a comparative evolutionary approach was taken by sampling a wide range

of chordates, most of which had no previous record of COX genes. However, there are still

many evolutionarily interesting chordates that have not been examined for COX genes. While

COX genes are known from multiple mammals and teleosts, other chordate groups have only

been minimally sampled. Most notably is the lack of any reported COX genes from any reptile.

COX has also only been documented from a single avian species (Gallus gallus), a single

elasmobranch species (Squalus acanthias), and a single marsupial (Monodelphis domestica.

These groups represent major lineages within the vertebrates and may contain novel forms of

COX. The results of this and other recent studies also warrant further investigation within

certain chordate groups. For example, evolutionarily ancestral fishes such as the sturgeon,

paddlefish, and bowfin could likely contain a single COX-1 and COX-2 gene, while the teleosts

studied here have multiple COX-1 or COX-2 genes. Ishikawa and Herschman (2007)









hypothesize that a genome duplication in the teleost lineage and subsequent loss of either a

COX-1 (in the zebrafish/trout lineage) or COX-2 gene (in the acanthopterygian lineage) is

responsible for this observation. If this is the case, then examining taxa near the base of the

teleost lineage may reveal fish with all four variants of COX (COX-la, COX-lb, COX-2a, and

COX-2b). The COX variants from the dogfish shark, lamprey, and hagfish all group weakly

with either COX-1 or COX-2. Obtaining COX sequences from other species of hagfish,

lampreys, and elasmobranchs may confirm this initial relationship or reveal novel groupings.

Finally, although support values for the urochordata and cephalochordata clades are strong

regardless of the phylogenetic technique used, only one species from each subphylum was

included in phylogenetic analyses. Including more species could give a more complete view of

COX evolution in the evolutionarily ancestral chordates. COX sequences from these additional

taxa may be readily available in the near future due to the growing regularity of genome

sequencing across many taxa. For example, the recently completed Anolis carolinensis genome

contains COX-1 and COX-2 genes. However, due to the length of time required for whole

genome projects, the limited number of genomes available, and the difficulty of searching

recently completed, non-annotated genomes, studies that specifically target sequencing COX

genes in these taxa are needed.

Function of Cyclooxygenases

As reviewed in Chapter 1, cyclooxygenase plays several roles in chordates because of its

role in prostaglandin production. These functions have been examined in some detail in the

mammals and to a lesser degree in the fishes. However, no studies have examined the functions

of the novel COX forms presented here or any non-vertebrate COX forms in the chordates.

Therefore, I have begun preliminary studies investigating the function of these COX forms.









One preliminary method used to infer possible functions for a gene is to determine where

its mRNA transcript or protein product is expressed in the organism. For example, Choe et al.

(2006) concluded that COX-2 may play a role in osmoregulation in the killifish partly due to its

high level of expression in the gills and its presence within mitochondrion-rich cells. Following

similar procedures, I have found that COX-lb mRNA is also expressed highly in the gills of the

killifish (Fig. 3-la). Similar results were also found for COX-la and COX-2 in the sculpin (Fig.

3-2a, Fig. 3-2b), hCOX in the hagfish (Fig. 3-3a), and 1COX in the lamprey (Fig. 3-4). To

investigate which cells express COX mRNA transcripts, in situ hybridization (ISH) was

performed using probes designed against the novel COX sequences described here using

previous protocols (Choe et al., 2006). However, no staining was observed for COX transcripts

in the gills of the killifish (Fig. 3-1b) or sculpin (Fig. 3-2b). This is likely due to errors in probe

design or hybridization protocol, since COX mRNA was abundant in the gills of both the

killifish and sculpin. In the hagfish, probes designed to show hCOX mRNA expression did

produce staining patterns in a population of epithelial cells lining gill filaments and lamellae

(Fig. 3-3b). This gill structure is similar to those ofteleosts but may be functionally different

(Evans et al., 2005) and since ion transport mechanisms in hagfish may not be directly related to

those in teleosts (Choe et al., 1999), it is difficult to assign an osmoregulatory function of hCOX

based on this staining pattern. Finally, since COX-2 mRNA expression was shown to increase

following salinity transfers using qPCR (Choe et al., 2006), a similar procedure was used to

investigate COX expression after salinity acclimation in the gills of the killifish (Fig. 3-1c) and

sculpin (Fig. 3-2c). The only significant result from this experiment was that COX-lb was

expressed more abundantly in killifish chronically acclimated to fresh water than seawater.

However, the same general trend shown for COX-2 in the killifish (Choe et al., 2006) was also









shown for COX-lb in the killifish: an increase in expression 3 hours after salinity transfer

(although P = 0.06 in this case) and a return to pre-transfer expression levels by 8 hours after

transfer. Taken as a whole, these preliminary results suggest that the different COX forms may

be playing various roles in osmoregulation in the killifish, sculpin, lamprey, and hagfish.

Clearly, COX function in the non-mammalian chordates has just begun to be investigated

and many questions still remain. Future studies may investigate COX roles in these chordates by

examining protein expression patterns using immunohistochemistry, blocking COX function via

commercially available COX inhibitors, or examining COX expression during reproductive

events. Of particular interest are the differential roles of the various COX forms in the teleosts

and the possible partitioning of functionality between COX-la and COX-lb or COX-2a and

COX-2b.









A)


COX-lb

18S


GOB HS K

1111111


Tkm(h)


Figure 3-1. Euryhaline killifish (Fundulus heteroclitus) COX-lb mRNA expression following
procedures outlined in Choe et al. (2006). A) COX-lb mRNA expression in various
tissues in the killifish. Expression was constant across tissues for the ribosomal
protein 18S. COX-lb expression was most abundant in the gills (labeled as G),
followed by the heart (H), kidney (K), opercular epithelium (0), stomach (S),
intestine (I), and brain (B). B) In situ hybridization of COX-lb in the gills of the
killifish. C) Expression of COX-lb mRNA in the gills of the killifish following
salinity transfers. Expression is normalized to the ribosomal gene L8 and is shown
relative to seawater (SW) (FW = fresh water).










GOBH S IK


iUUU11


A)







COX-la
18S


B)


40
35
30
25
20
15


05
00
24 hrs


72 hrs


GO BH SI K


[ [ [


iOX-2
18S-


COX-la 16 COX-2
o Seaw ater
14
20% Seaw ater

S10
10

W 06
04
02
00


24 hrs


72 hrs


Time
Time
Figure 3-2. Longhorn sculpin (Myoxocephalus octodecemspinosus) COX-la and COX-2 mRNA
expression following procedures outlined in Choe et al. (2006). A) COX-la and
COX-2 mRNA expression in various tissues in the sculpin. Expression was constant
across tissues for the ribosomal protein 18S. COX-la was most abundant in the
opercular epithelium (labeled as O), followed by the gills (G), kidney (K), heart (H),
brain (B), intestine (I), and stomach (S). COX-2 was most abundant in the gills,
followed by the opercular epithelium, and kidney. B) In situ hybridization of COX-
lb and COX-2 in the gills of the sculpin. C) Expression of COX-lb and COX-2
mRNA in the gills of the sculpin after 24 and 72 hours following salinity transfers to
100% and 20% seawater. Expression is normalized to the ribosomal gene L8.











A)


hCOX

18S-


.\lllisejise ''

S.-.. .o -..., .
,".'*''\,i ^



I .w ^'^ <^ ;


Sense


Sense


Figure 3-3. Atlantic hagfish (Myxine glutinosa) COX (hCOX) mRNA expression following
procedures outlined in Choe et al. (2006). A) COX mRNA expression in various
tissues in the hagfish. Expression was constant across tissues for the ribosomal
protein 18S. COX (hCOX) expression was most abundant in the gills (labeled as G),
followed by the heart (H), kidney (K), stomach (S), intestine (I), muscle (M), and
brain (B). B) In situ hybridization of hCOX in the gills of the hagfish from two
contrary gill cross-sections. Arrows highlight staining, indicating hCOX mRNA
expression. Staining for antisense and sense (control) probes is shown.


G M B H S I K

i i i i






















1COX---

18S--


Figure 3-4. Sea lamprey (Petromyzon marinus) COX (1COX) mRNA expression in various
tissues following procedures outlined in Choe et al. (2006). Expression was constant
across tissues for the ribosomal protein 18S. COX (1COX) expression was most
abundant in the heart (labeled as H), followed by the intestine (I), stomach (S), gills
(G), kidney (K), muscle (M), and brain (B)


G M


B H S I K









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BIOGRAPHICAL SKETCH

Justin Chase Havird was born in 1984, in Valdosta, Georgia to Dawn and Kurt

Havird. Justin has one younger brother, Joshua. The Havirds soon moved to Lake City,

Florida, where Justin spent the majority of his childhood and adolescence. Justin

attended Columbia High School in Lake City and graduated with top honors in 2002.

Justin then attended the University of Florida in Gainesville where he majored in zoology

after an initial interest in aerospace engineering. While at UF, Justin was heavily

involved in the university's band program, playing trumpet in the marching, basketball,

volleyball, concert, and symphonic bands for several years. As an undergraduate, Justin

worked in the laboratory of Dr. David H. Evans for a number of years, investigating ion

transporters in the gills of fishes. His senior thesis was titled "Neuronal Nitric Oxide

Synthase in the Gill of the Euryhaline Killifish, Fundulus heteroclitus." Justin graduated

summa cum laude from UF in 2006 with a B.S. in zoology.

In 2006, Justin entered graduate school at the University of Florida in the

Department of Zoology. Continuing to work with Dr. Evans, Justin spent three summers

at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine where he

collaborated with other researchers investigating fish gill physiology. During his

graduate studies, Justin has earned several awards and grants, including a GIAR from

Sigma Xi. Justin has also presented his research at scientific meetings and conferences.

While earning his M.S., Justin also designed experiments and instructed students in a

senior level animal physiology course for 2 years. Justin has also collaborated with other

scientists on a wide range of marine life science projects, most notably a revision of the

cobitid genus Leid,11/ I'h1/li /////\ in SE Asia with Dr. Larry Page. After earning his









M.S., Justin plans to continue his academic career by completing a Ph.D. in a related

marine science field.





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EVOLUTION OF CYCLOOXYGENASE IN THE CHORDATES By JUSTIN CHASE HAVIRD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORI DA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Justin Chase Havird 2

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To the teachers who inspired, directed, and challenged me. My high school marine biology teacher, Larry Joye, first introduced me to the fa scinating world of life in the water and since then my endeavors have never strayed far from it. Dr. David Evans enthusiastically welcomed me into his lab as an undergraduate, although I ha d no former research expe rience. What started as a part-time project soon evolved into an academic career as I was given the opportunity to conduct original research. During this time, Dr. Keith Choe was an invaluable asset and inspiration. Growing from this original partnership, I conti nued working with David on my masters research and he has always supported my academic and research endeavors; I cannot envision a better advisor. 3

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ACKNOWLEDGMENTS I most of all thank my supervisory comm ittee chair, Dr. David H. Evans. Although David was nearing retirement and had resolved to not accept any new students, he welcomed me as a graduate student without any hesitation. He has guided me in developing research strategies, implementing those stra tegies, and interpreting the resu lts. He has always supported my projects and none of them would have been accomplished without his enthusiasm. I also thank my supervisory committee memb ers for their commitment to my research and their willingness to share thei r expertise. Dr. Mi chael Miyamoto contri buted heavily to the phylogenetic aspects of my project and encouraged me to be thorough in my analysis. When I started this project I knew very little about generating meaningful evolutionary relationships. Michael selflessly took the time necessary to make sure I understood how to develop a biologically relevant reconstr uction of evolutionary change s that took place over 600 million years ago. He also put his own projects aside while I used his computers for months at a time. Finally, his guidance was absolutely necessary in interpreting the conflicting results of my analyses. Dr. Marty Cohn was a great influence on my research and prompted me to think in an integrative and comparative framework. He gener ously allowed me to use his lab space. His expertise working with evolutionarily interestin g chordates was invaluable. His students also graciously helped me learn many of the techniques used in this research. I am also indebted to seve ral colleagues for thei r assistance. Dr. Keith Choe pioneered and taught me many of the molecu lar techniques now commonly used in the Evans lab that were critical to this research. He spent coun tless hours with me in multiple laboratories troubleshooting problems, explaining theory, and developing this re search. He also encouraged me to work with the hagfish, lamprey, and amph ioxus and continually helped me develop new ideas and strategies. Dr. Kelly Hyndman was also constantly supportive of this research. She 4

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taught me the localization techniqu es used here and also helped with many aspects of retrieving and analyzing genetic data. Finally, she was al ways receptive to critical discussions of my research and tackled every circumstance in a friendly, often contagious manner. I am very thankful to my family and friends for their continued support of my education and research. My entire family has been very interested in the resear ch I have undertaken and when they were not interested they feigned inte rest in order to make me happy. My parents, Kurt and Dawn Havird, have encouraged my desire to be a scientist, and have been overwhelmingly supportive during my academic caree r; often in the form of food, clean laundry, or money. My friends have also been very suppor tive of my research interests: fellow scientists have empathized with the typical problems asso ciated with research a nd non-scientists have given me the confidence to persevere. I also thank the following pe ople for providing help with this project in particular, other related projects, or the daily support needed to become a successful student: Dr. James B. Claiborne, Rachel Rose, Dr. GuangJun Zhang, Donovan German, Justin Catches, Andrew Diamanduros, Sara Takeuchi, Jim Stidham, Curtis Lanier, Julia Curtis-Burnes, Oriana GalardiEste, Rebecca Kreh, Dr. Susan Edwards, Gini Luchini, Makesha Foster, Emily Cornwell, Dr. Larry Page, Pete Ryschkewitsch, Frank Davis, Di ana Davis, Cathy Moore, Karen Pallone, Vitrell Sherif, Dr. David Reed, Dr. Paul Lewis, and th e laboratories of Dr. Lou Guillette and Dr. David Julian. My research was primarily funded by Na tional Science Foundation grants IBN-0089943 and IOB-0519579 to David Evans. A grant-in-aid of research to Justin Havird from Sigma Xi also supported a portion of this research. Fina lly, initial support was provided in part by a University Scholars award to Justin Havird. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAPTER 1 GENERAL INTRODUCTION..............................................................................................13 Introduction................................................................................................................... ..........13 Cyclooxygenase Evolution.....................................................................................................16 Mammals.........................................................................................................................17 Fishes...............................................................................................................................18 Other Vertebrates.............................................................................................................19 Other Chordates...............................................................................................................19 Cyclooxygenase Function in Mammals..................................................................................20 Gastrointestinal Tract......................................................................................................20 The Kidney......................................................................................................................21 Platelets............................................................................................................................23 Reproduction...................................................................................................................23 Nervous System...............................................................................................................25 Cyclooxygenase Function in Non-Mammals.........................................................................27 Fishes...............................................................................................................................27 Osmoregulation........................................................................................................27 Reproduction............................................................................................................29 Differential functions of novel forms.......................................................................30 Other functions.........................................................................................................30 Amphibians..................................................................................................................... .31 Reptiles............................................................................................................................31 Birds................................................................................................................................32 Study Overview................................................................................................................. .....33 2 CYCLOOXYGENASES IN THE ANCE STRAL CHORDATES: SEQUENCE AND PHYLOGENETIC ANALYSES............................................................................................39 Introduction................................................................................................................... ..........39 Materials and Methods...........................................................................................................41 Animals and Holding Conditions....................................................................................41 Tissue Collection.............................................................................................................42 Reverse Transcription, Primer Desi gn, PCR, Cloning, and Sequencing.........................43 5 and 3 Rapid Amplification of cDNA Ends (RACE)..................................................45 6

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Sequence Analysis...........................................................................................................45 Phylogenetic Analysis.....................................................................................................46 Results.....................................................................................................................................48 Molecular Identification of Cyclooxygenases.................................................................48 Branchiostoma .........................................................................................................48 Myxine ......................................................................................................................48 Petromyzon ...............................................................................................................48 Fundulus ...................................................................................................................49 Myoxocephalus .........................................................................................................49 Sequence Analyses..........................................................................................................50 Teleosts COX-1a, COX-1b, and COX-2..................................................................50 Lamprey, hagfish, and amphioxus COX forms........................................................52 Phylogenetic analyses..............................................................................................53 Discussion...............................................................................................................................56 Novel COX Forms...........................................................................................................57 Phylogenetic Analyses.....................................................................................................58 Summary..........................................................................................................................62 3 SUMMARY AND FUTURE DIRECTIONS.......................................................................105 Summary...............................................................................................................................105 Novel COX Sequences..................................................................................................105 Phylogenetic Analyses...................................................................................................106 Future Directions..................................................................................................................106 Completion of Novel Sequences...................................................................................107 Additional Phylogenetic Analysis.................................................................................107 Additional Sequences....................................................................................................108 Additional Taxa.............................................................................................................109 Function of Cyclooxygenases........................................................................................110 LIST OF REFERENCES.............................................................................................................117 BIOGRAPHICAL SKETCH.......................................................................................................130 7

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LIST OF TABLES Table page 2-1. Degenerate primers used in PCR.......................................................................................64 2-2. Amphioxus COX specific primers used in PCR................................................................65 2-3. Hagfish COX specific primers used in PCR......................................................................66 2-4. Lamprey COX specific primers used in PCR....................................................................67 2-5. Killifish COX specific primers used in PCR.....................................................................68 2-6. Sculpin COX specific primers used in PCR......................................................................69 2-7. Sequences included in phylogenetic analyses....................................................................70 2-8. LRTs between amino acid based models of COX evolution.............................................72 2-9. LRTs between tree topologies with altered positions of hCOX and lCOX.......................73 8

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LIST OF FIGURES Figure page 1-1. Cascade of reactions depicting the co nversion of arachidonic acid to the primary prostaglandins via catalyzation with either cyclooxygenase (COX) form........................35 1-2. General phylogeny representing the different COX forms in the fishes...........................36 1-3. Accepted evolution of ancestral chordates (Pough, et. al 2005).......................................37 1-4. Evolution of COX forms in th e chordates, based on Jarving et al (2004)........................38 2-1. Amino acid alignment of the forms of COX sequenced from the gills of the euryhaline killifish (Fundulus heteroclitus ).......................................................................74 2-2. Amino acid alignment of the three forms of COX sequenced from the gills of the longhorn sculpin ( Myoxocephalus octodecemspinosus ): COX-1a, COX-1b, and COX-2................................................................................................................................76 2-3. Amino acid alignment of the forms of COX sequenced from the gills of the lamprey ( Petromyzon marinus ), hagfish ( Myxine glutinosa ), and amphioxus ( Branchiostoma lancelolatum ), along with sCOX from the dogfish ( Squalus acanthias Accession #AAL37727)......................................................................................................................78 2-4. Amino acid alignment of lCOX sequenced from the gills of the lamprey ( Petromyzon marinus ), with COX-1 and COX-2 forms fr om vertebrates (Accession # NP_032995, NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942).............................80 2-5. Amino acid alignment of lCOX sequenced from the gills of the lamprey ( Petromyzon marinus ), with COX-1 and COX-2 forms fr om vertebrates (Accession # NP_032995, NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942).............................82 2-6. Amino acid alignment of COXc a nd COXd sequences from the lancelet ( Branchiostoma lancelolatum ) with COXa and COXb sequences from the sea squirt ( Ciona intestinalis ).............................................................................................................85 2-7. Evolutionary history of COX sequen ces inferred using the minimum evolution method with a distance optimality criterion.......................................................................87 2-8. Evolutionary history of COX seque nces excluding hagfish (hCOX) and lamprey (lCOX) sequences inferred using the mi nimum evolution method with a distance optimality criterion........................................................................................................... ..89 2-9. Evolutionary history of COX se quences inferred using a maximum liklihood optimality criterion........................................................................................................... ..90 9

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10 2-10. Evolutionary history of COX sequen ces excluding sequences from the hagfish (hCOX) and lamprey (lCOX) inferred using a maximum liklihood optimality criterion..............................................................................................................................92 2-11. Alternate likelihood ratio test (aLRT) scores for branch support of COX sequences.......94 2-12. Alternate likelihood ratio te st (aLRT) scores for branch support of COX sequences excluding hagfish (hCOX) and lamprey (lCOX) sequences..............................................96 2-13. Evolutionary history of COX sequences inferred using a Bayesian optimality criterion..............................................................................................................................97 2-14. Evolutionary history of COX sequen ces excluding hagfish (hCOX) and lamprey (lCOX) sequences inferred using a Bayesian optimality criterion....................................99 2-15. The 21 alternate tree topologies generated to test the effects of relocating the hagfish (hCOX) and lamprey (lCOX) sequences on the likelihood score (Ln L )........................100 2-16. Unrooted phylogeny showing the most likely scenario for COX evolution in the chordates based on several other analyses.......................................................................104 3-1. Euryhaline killifish ( Fundulus heteroclitus ) COX-1b mRNA expression following procedures outlined in Choe et al (2006)........................................................................113 3-2. Longhorn sculpin ( Myoxocephalus octodecemspinosus ) COX-1a and COX-2 mRNA expression following procedures outlined in Choe et al (2006).....................................114 3-3. Atlantic hagfish ( Myxine glutinosa ) COX (hCOX) mRNA expression following procedures outlined in Choe et al (2006) .......................................................................115 3-4. Sea lamprey ( Petromyzon marinus ) COX (lCOX) mRNA ex pression in various tissues following procedures outlined in Choe et al (2006)...........................................116

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVOLUTION OF CYCLOOXYGENASE IN THE CHORDATES By Justin Chase Havird May 2008 Chair: David Evans Major: Zoology Cyclooxygenase (COX) is the enzyme found in animals responsible for converting arachidonic acid into prostaglandins. These pros taglandins can then perform multiple functions including regulation of inflammation respons es, changes in vascular tone, and ion transport/osmoregulation. Before this thesis only two main forms of cyclooxygenase (COX-1 and COX-2) and one ancestral form ( Ciona COX) were known from the chordates. I sequenced COX genes from several hitherto uninvestigated chordates in order to determine a more comprehensive scenario for the evolutionary history of cyclooxygenase in the chordates. After designing primers, partial COX sequences were obtained from the Sea Lamprey ( Petromyzon marinus ) and amphioxus (Branchiostoma lancelolatum ). Complete COX sequences were obtained from the Atlantic Hagfish ( Myxine glutinosa ), euryhaline killifish ( Fundulus heteroclitus), and longhorn sculpin ( Myoxocephalus octodecemspinosus). These novel sequences along with other known COX sequences were then subjected to a variety of phylogenetic analyses using standa rd techniques. The results of these analyses suggest a complex history for the evolution of cyclooxygenase in the chordates. There appears to have been at least 3 evol utionary independent origins of COX in the chordates. The forms from the sea squirt ( Ciona intestinalis ) (COXa and COXb) represent the 11

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12 most ancestral forms, the forms from amphioxus represent another independent origin (COXc and COXd), and the vertebrate forms (COX-1 and COX-2) represent yet another origin, with the hagfish and lamprey as likely basal members. Fu rthermore, the teleosts appear to have duplicate COX-1 (COX-1a and COX-1b) or COX-2 (COX-2a and COX-2b) forms. These results agree with previous studies and reveal novel informa tion about COX forms in the chordates. More intense sampling of other chorda tes could reveal other novel orig ins of COX or contribute to a more robust understanding of COX evolution. Finally, the function of most non-mammalian COX forms remains to be investigated.

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CHAPTER 1 GENERAL INTRODUCTION Introduction Cyclooxygenase (COX) is the enzyme responsible for the rate limiting step that converts arachidonic acid to Prostaglandin G2 and Prostaglandin H2. Arachidonic acid is an essential fatty acid required by most mammals and can be obtaine d by the conversion of linoleic acid found in plants (occurring in herbivores). Some animals cannot convert linol eic acid into arachidonic acid and therefore must obtain it from other animals (obligate carnivores). COX oxidizes arachidonic acid to make the hydroperoxy endoperoxide Prostaglandin G2, which is then reduced to form the hydroxy endoperoxide Prostaglandin H2. Prostaglandin H2 can then be convert ed to the primary prostanoids (Pro staglandin I2, Prostaglandin D2, Prostaglandin E2, Prostaglandin F2 and Thromboxane A2) by a variety of enzymatic and non-en zymatic pathways. Therefore, the expression of COX can directly in fluence the amount of prostagla ndins that are synthesized due to the arachidonic ac id cascade (Vane et al ., 1998, Fig. 1-1). Prostaglandins are found in vertebrates, some invertebrates, and po ssibly even in plants (Rowley et al ., 2005; Ryan, 2000). In mammals, they are expressed in virtua lly all tissues and have a myriad of physiological functions, but they mainly regulate vascular tone in smooth muscle by acting as vasodilators and vasoconstr ictors. In addition, prostaglandins are key regulators of such diverse processes as salt and water homeostasis in the mammalian kidney (Harris and Breyer, 2001), protecti on of stomach lining (Kargman et al ., 1996), platelet aggregation in blood (F rster and Parratt, 1996), birth (Challis et al. 2002), and central nervous system function (Hopkins, 2007). They also may play prominent roles in pathological states such as fever, inflammation, and cancer (Vane et al ., 1998). 13

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Prostaglandins were first extracted from se men, prostate (hence the name prostaglandin), and seminal vesicles by Goldblatt and von Euler in the 1930s (von Euler, 1936) and were shown to affect blood pressure. The reac tion that catalyzes Prostaglandin G2 synthesis was first characterized in the 1970s (Ham berg and Samuelsson, 1973; Hamberg et al ., 1974), but it wasnt until 1988 that cyclooxygenase (COX) was first described as the enzyme responsible for endoperoxide synthesis by three separa te groups (DeWitt and Smith, 1988; Merlie et al ., 1988; Yokoyama et al ., 1988), all of which described a COX from sheep seminal vesicles which was later named COX-1. However, before COX-1 wa s isolated there was early evidence that multiple COX forms existed, based on prostaglandin activity in the rat brain and spleen (Flower and Vane, 1972). In the early 1990s, a sec ond COX form was cloned from mouse and chicken fibroblast cell cultures a nd was named COX-2 (Kujubu et al ., 1991; Xie et al ., 1991; O'Banion et al ., 1992). This discovery would later prove to be of major importance in the field of prostaglandin biology due to the development of dr ugs that could selectivel y inhibit the different forms of cyclooxygenase. Both forms catalyze the reactions that form prostaglandins from arachidonic acid, but early studies suggested that COX-1 was consti tutively expressed in many tissues, while COX-2 was an inducible isoform that was expressed in response to p hysiological stimuli (Funk, 2001). Because basal expression of COX-2 was low in some cell types and COX-2 was found to be expressed much more so than COX-1 in response to mitogens and cytokines, it was assumed that COX-1 was constitutively expressed at basal le vels and served a housekeeping function and that COX-2 was inducibly expressed to serve in infla mmatory functions (Kujubu et al ., 1991; Xie et al ., 1991; O'Banion et al ., 1992). However, this was later sh own to be an oversimplification (Funk, 2001). Notably, it was shown that COX-2 is constitutively expressed in the mammalian 14

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kidney, where it regulates blood flow and ion tran sport in the Loop of He nle (Harris and Breyer, 2001) and plays a role in cell su rvival in medullary interstiti al cells during dehydration (Yang et al ., 2002; Yang, 2003). Despite their potential functional differences in different tissues, COX-1 and COX-2 are biochemically very similar. Both are inhi bited by nonsteroid an ti-inflammatory drugs (NSAIDs), such as aspirin, due to competition with arachidonic acid for the active sites of the enzymes or irreversible acetyla tion (Vane, 1971). Both proteins in humans are ~600 amino acids and share about 63% sequence similarity with each other. However, the proteins originate from different genes on different chromosomes and en code different mRNA transcripts. COX-1 comes from a much larger gene (22 kilobases) than C OX-2 (8 kilobases). The amino acid residues that form the binding and catalytic sites (the functi onally important sites) as well as the adjacent amino acids are all identical in human COX-1 and COX-2, except for two substitutions. At positions 434 and 523, valine is substituted for isoleucine in COX-2 when compared to COX-1. These substitutions have been implicated as the most likely reason behind the biochemical differences between COX-1 and CO X-2. COX-1 appears to be more selective in terms of the fatty acids it will accept as substrates than COX-2 (Otto and Smith, 1995). COX-2 selective inhibitors have offered an insight into the bi ochemistry behind the different forms. These inhibitors can differentiate between COX1 and COX-2 with over 1000 fold specificity (Griswold and Adams, 1996). The rationale behind these selective inhibitors is that the smaller size of valine on position 523 in COX-2 allows th e inhibitor to bind to a side pocket in COX-2, but the larger isoleucine on position 523 of COX-1 appears to prev ent this binding. When this position is altered in both forms to the alternat e amino acid, the selectivity for inhibitors is reversed (Wong et al ., 1997; Gierse et al ., 1996; Guo et al ., 1996), suggesting th at this residue 15

PAGE 16

plays a critical role in differing biochemical activ ity. COX-2 selective inhi bitors were thought to be a major medical breakthrough due to the ability to stop disease induced inflammation without affecting normal prostaglandin levels produced by COX-1 and therefore causing minimal side effects. However, due to the constitutive expres sion of COX-2 in certain tissues (especially the kidney), COX-2 selective inhibitors may not be as affective as once thought. It is important to remember that COX-1 and COX-2 are very structur ally similar and that their main difference is due to expression. As outlined above, COX form and function have been extensively studied extensively in mammals, especially humans. However, non-ma mmalian COX forms and their functions have not been examined in great detail in other ch ordates. The goal of this research was to characterize COX evolution in the chordates, with specific attention on the more evolutionarily ancestral chordates. Describing ancestral COX form s is the first step in elucidating the evolution of COX function, which may lead to a better understanding of overall COX function, and even the development of new drugs. In this chapter, I give a brief overvie w of COX evolution and function in the chordates, concluding with a br ief summary of the research described in the following chapters. Cyclooxygenase Evolution Like mammals, most vertebrates appear to have two homologous copies of cyclooxygenase named COX-1 and COX-2. However, other C OX forms exist throughout the chordates, including variants of COX-1 and CO X-2 as well as forms representing independent origins of COX that are not closely related to COX-1 or COX-2. There are also COX forms in corals and other invertebrates (Jarving et al ., 2004), but the focus of this section is to review the forms found in the chordates. 16

PAGE 17

Mammals COX-1 and COX-2 sequences have been either cloned or derived from genomic sequences for most of the classic mammalian model species, includ ing mouse (Accession# NP_032995 and NP_035328), rat (Accession# NP _058739 and NP_058928), dog (Accession# NP_001003023 and NP_001003354), cow (Accession# XP_869575 and NP_776870), sheep (Accession# NP_001009476 and NP_001009432), a nd rabbit (Accession# NP_001076150 and NP_001075857). COX sequences also exist for rhesus monkey, guinea pig, pig, horse, and mink, but these are either partial sequences or are not available for both forms. A third COX variant has been proposed to occur in mammals (Flower and Vane, 1972) based on inhibitory studies (see below). This form is known as COX-3 or COX-1b because of its close similarity to COX-1. Recently, a potential COX-3 (Accession# AY547265) was cloned from mouse (Kis et al ., 2006) and rat (Accession# AY523672, Snipes et al ., 2005). However, these forms likely do not participate in prostaglandin synthesis (Kis et al ., 2006) and are not a separate COX form. Furthermore, a similar study using an artificially created human COX-1b found no inhibitory similarity to th e proposed COX-3 enzyme (Censarek et al ., 2006). Therefore, while a third, genuinely different COX variant may exist in mammals, its sequence remains elusive. Analysis of the recently completed gray short-tailed opossum genome yielded the predicted COX-1 and COX-2 forms (A ccession# XP_001370514 and XP_001375945), as well as two additional COX-1 forms, named COX-1b and COX-1b2 (Accession# XP_001370542 and XP_001370595). These forms are complete and di ffer in amino acid composition and length. These are the first COX-1 variants in a mammal that do not represent modified versions of normal COX-1 and although is has not been sugges ted, it is possible that one of these forms 17

PAGE 18

represents the elusive COX-3 va riant. Functional studies to determine how these forms differ from normal COX have not been published to date. Fishes Cyclooxygenase has been sequenced in several fishes, including the rainbow trout ( Oncorhynchus mykiss) (Zou et al ., 1999), brook trout ( Salvelinus fontinalis ) (Roberts et al ., 2000), Atlantic croaker ( Micropogonias undulates ), and zebrafish ( Danio rerio) (Grosser et al ., 2002). These fishes, along with genomic sequence data from the stickleback ( Gasterosteus aculeatus), green spotted puffer ( Tetraodon nigroviridis ), pufferfish ( Takifugu rubripes), and Japanese medaka ( Oryzias latipes ) suggest that fishes posses both COX-1 and COX-2 forms (Jarving et al ., 2004). Furthermore, COX-2 has recently been sequenced from European sea bass ( Dicentrarchus labrax) (Buonocore et al ., 2005) and euryhali ne killifish ( Fundulus heteroclitus ) (Choe et al ., 2006), supporting this prediction. A cyclooxygenase has also been sequen ced from the spiny dogfish shark ( Squalus acanthias ) (Yang et al ., 2002). However, this form was not designated as COX-1 or COX-2 because of its near equal identity to both forms (it is only slightly more similar to COX-1). This form was named sCOX by the authors and may repr esent an evolutionarily distinct form of cyclooxygenase found in the elasmobranchs. Ishikawa and colleagues have recently fu rther characterized COX evolution in the teleosts by demonstrating that both the zebrafish (Ishikawa et al ., 2007) and rainbow trout (Ishikawa and Herschman, 2007) have two functional COX-2 genes (named COX-2a and COX2b). In addition, by analyzing sequence databases, they concluded that not all teleosts have two COX-2 forms, but that the stickleback, green sp otted puffer, pufferfish, and Japanese medaka have two COX-1 forms instead (nam ed COX-1a and COX-1b) (Ishikawa et al ., 2007). They concluded that a genome duplication before the te leosts, and a subsequent loss of either one 18

PAGE 19

COX-1 or COX-2 form characterizes the spec ies mentioned (Ishikawa and Herschman 2007) (Fig. 1-2). This is supported by the apparent ge nome duplication events in teleosts (Crollius and Wessenbach, 2005). Other Vertebrates Although functional studies on prostagla ndin roles and cyclooxygenase are not uncommon in other vertebrates, cyclooxygenase sequences from non-mammalian and nonteleost vertebrates are lacking. Cyclooxygenase forms have been predicted from the chicken genome ( Gallus gallus ), suggesting that birds have both COX-1 and COX-2 forms (Urick and Johnson, 2006). Both forms have also been found in the African clawed frog ( Xenopus laevis ) and COX-2 has been found in the western clawed frog ( Xenopus tropicalis ), suggesting that both forms occur in amphibians (Klein et al ., 2002). Functional studies sugg est COX exists in reptiles as well (Seebacher and Franklin, 2003) and the recently completed Anolis carolinensis genome contains both COX forms. Taken together, the presence of COX-1 a nd COX-2 forms in the mammals, teleosts, birds, and amphibians suggest that both forms orig inated in the early vertebrates, possibly from the duplication of an ancestral COX gene. The presence of sCOX in the dogfish indicates that COX forms in the early vertebrate s may not have diverged (in sequence or possibly in function) to the extent that allows them to be labeled as COX-1 or COX-2, although it is predicted that two COX forms exist in all vertebra tes, since sCOX groups with COX1 and is not ancestral to the COX-1/COX-2 split (Fig. 1-2, unpublished phyl ogeny generated using the online program PHYML) (Guindon and Gascuel, 2003; Guindon et al ., 2005). Other Chordates Although the subphylum Vertebrata includes the majority of species in the phylum Chordata, two other subphyla are included in the chordates: the Urocho rdata (sea squirts or 19

PAGE 20

tunicates) and the Cephalochordata (amphioxus or lancelets). These ot her subphyla are basal to the vertebrates and represent evolutionarily dist inct monophyletic groups (Fig. 1-3). There is some debate as to whether the urochordates or cephalochordates represent the base of the chordate phylogeny, although urochordates have been generally been considered basal (Pough et al ., 2005). Interestingly, the genomes of sea squirts ( Ciona intestinalis and Ciona savignyi ) predict COX genes that are not homol ogous to COX-1 and COX-2. These forms (named COXa and COXb) are basal to vertebrate COX forms and indi cate an evolutionarily distinct branch in COX evolution (Jarving et al 2004) (Fig. 1-4). This separate bran ch is situated between the COX of corals and the vertebrate COX fo rms (Fig. 1-4). This suggests that the COX-1 and COX-2 forms originated sometime after the origin of the sea squirts but before the origin of the elasmobranchs (around 550-600 mya). Cyclooxygenase Function in Mammals Cyclooxygenase was first described from mamm als, and due to its medical importance, its function has been intensel y examined in mammals, specifically humans. Since COX stimulates the production of pros taglandins, COX function is intrin sically linked to the roles that prostaglandins play in everythi ng from water balance to reproduc tion. Here, I review the major functions of COX-1 and COX-2 in various ma mmalian physiological systems (modeled after Vane et al ., 1998), but it should be noted that ma mmalian COX studies are ongoing and that COX likely plays many roles not described here. Gastrointestinal Tract In the stomach, prostaglandins play a cy toprotective role, guarding the stomach lining against digestive acids and enzymes. The mech anism behind this protection is an increased mucosal blood flow caused by the vasodilating properties of prostagl andins (specifically 20

PAGE 21

Prostaglandin I2). It has generally been accepted that these prostaglandins are synthesized by COX-1, although COX-2 is present in small amounts (Kargman et al ., 1996). This is supported by studies showing that irradi ated mice have further redu ced numbers of crypt cells (cytoprotective mucosal cells) when indome thacin (a non-specific COX inhibitor) was administered, but not when a COX-2 sele ctive inhibitor was administered (Cohn et al ., 1997). COX-2 expression in the esopha gus of patients with gastro esophageal reflux disease was upregulated in response to acid e xposure, suggesting a protective function in the distal esophagus (Lurje et al ., 2007). This may indicate th at throughout the gastrointe stinal (GI) track, COX-2 is inducibly expressed in response to inflammation responses. Furthermore, throughout the GI track, inflam matory mediators such as COX may also serve to repair damaged mucosa (Martin and Wallace, 2006). While it seems that COX-1 is responsible for baseline prostagl andin expression in the GI tr act, COX-2 and not COX-1 seems to be more involved in response to GI tract cancers (Ristimki et al ., 1997). However, recent studies on patients with gastric cancer from Dalian, China show ed that COX-2 expression was infrequent in gastric cancers due to hypermethylation and that a COX-2 focused strategy for treatment would likely be ineffective (Huang et al ., 2006). The Kidney Prostaglandins can modulate blood flow in the kidney, as in other organs (Vane and Botting, 1994). In addition, COX-2 has been shown to play a major role in ion regulation and water balance in the mammalian kidney (Harris et al ., 1994). It has also been shown that the COX-2 selective inhibitor SC-58236 and seve ral non-selective COXinhibiting NSAIDs (including sulindac, ibuprofen, a nd indomethacin) cause medullary interstitial cell (MIC) death in the kidney, but the COX-1 selective inhib itor SC-58560 was 100-fold less potent for inducing MIC death (Hao et al ., 1999). COX-2 knockout mice also develop terminal nephropathy 21

PAGE 22

(abnormal kidney function) which results in death by the third month (Morham et al ., 1995). These results suggest that COX-2, and not COX1, plays a critical role in kidney function via MIC cell survival. This is contrary to the or iginal description of COX-1 as the constitutively expressed form responsible for maintaining hom eostasis and COX-2 as the inducible form responsible for inflammatory responses. In the kidney, COX-2 is present in a subset of tubular epithelial cells located in the cortex and outer medulla (Vio et al ., 1997). These cells ar e part of the medullary thick ascending limb (MTAL), the part of the nephron that regulates extracellular fluid vol ume by establishing the osmotic gradient in the medulla that concentrates urine (Hebert et al ., 1981; Hebert and Andrioli, 1984). In vitro MTAL preparations have demonstrat ed high levels of Prostaglandin E2 (PGE2) (Ferreri et al ., 1984; Hirano et al ., 1986; Lear et al ., 1990) which has been shown to inhibit basolateral Na+/K+-ATPase (NKA) and apical Na+/K+-2Clcotransporter (NKCC) in the MTAL (Kaji et al ., 1996; Stokes and Kokko, 1976; Wald et al ., 1990). These proteins are critical for NaCl reabsorption in the MTAL. Furthermore, the COX-2 selective inhibitor NS-398 has been shown to prevent tumor necrosis factor (TNF) mediated PGE2 production in cultured MTAL cells (Ferreri et al ., 1999). TNF is a cytokine expressed in the MTAL that may be an important mediator of ion transport by inhibiting ion uptake via a prostanoid dependant mechanism (Escalante et al ., 1994; van-Lanschot et al ., 1991). Taken as a whole these results show that COX-2 may influence ion uptake via TNF mediated prostaglandi n production in the MTAL. Therefore, COX-2 is involved in cell survival and ion transport in the mammalian kidney. This finding is critical for treatment prot ocols using COX inhibitors. NSAIDs are the most common drugs used to treat pain and inflammation with over 30 million uses per day worldwide (Singh and Triadafilopoulos, 1999) Although the negative side effects 22

PAGE 23

(hypertension, renal ischemia, a nd GI toxicity) associated with chronic NSAID use have been known for some time, the discovery of COX-2 se lective inhibitors sugge sted a treatment for inflammation without causing disruption of nor mal COX function (thought to be mediated by COX-1). The role of COX-2 in the kidney has reduced the supposed power of these new drugs and negative side effects associated with C OX-2 selective inhibitors are now well documented (see Harris and Breyer, 2006 for a review). Furthermore, in 2005 the Food and Drug Administration (FDA) required additional warning la beling of celecoxib to highlight side effects and required Pfizer to remove valdecoxib from the market, both of which are selective COX-2 inhibitors. Platelets Interestingly, COX-1 seems to be the onl y form expressed in mammalian platelets. COX-2 was not detected in human platelets using western blotting techniques (Reiter et al ., 2001) or in canine platelets using northe rn blotting techniques (Kay-Mugford et al ., 2000). In the platelet, COX-1 is involved in platelet aggregation (clott ing) through the production of Thromboxane A2 (TXA2). When inhibited with aspiri n, COX-1 mediated production of TXA2 is irreversibly inhibited, causing redu ced platelet aggregation (blood th inning). This effect occurs about an hour after oral ingestion of even low doses of aspirin and can inhibit platelet function for several days after a dose (Vane et al., 1998; Vane, 1971). Inhibiti on of COX-1 by resveratrol and other components of red wine is responsible for the anti-plate let activity associated with the cardiovascular benefits of red wine consumption (Szewczuk and Penning, 2004). Reproduction Cyclooxygenase and the prostaglandins it produc es play critical roles in almost every aspect of reproduction in mammals, although its ro le in pregnancy has been studied the most 23

PAGE 24

intensely. Both isoforms are likely involved in reproductive processes, with COX-1 expression constitutive and COX-2 expres sion inducible (Chakraborty et al ., 1996). In a recent study, COX-2 was shown to contri bute to fertility in male mice (Balaji et al ., 2007). COX-2 expression was intense in the epithe lial cells of mice vas deferens and the COX-2 specific inhibitor nimesulide was shown to decrea se sperm motility six hours after administration (Balaji et al ., 2007). Interestingly, although prostagl andin levels initially dropped after nimesulide administration, they tended to ri se after sustained C OX-2 inhibition (Balaji et al ., 2007). This suggests that COX-1 expression may recover COX func tion or at least contributes heavily to prostaglandin production in the vas defere ns. A similar situation exists with respect to human female fertility, in which COX-1 expres sion can recover function in cases where COX-2 is lost or inhibited (Wang et al ., 2004). It also seems that COX-2 is critical for follicle development and ovulation in women and that COX inhibitors can have se vere effects on female fertility (Sirois et al ., 2004). Prostaglandins are also important during pr egnancy and labor. Generally, COX-1 is expressed more than COX-2 during normal stages of pregnancy, and COX-1 may play an active role in maintaining a healthy pregnancy (Trautman et al ., 1996). COX-1 is expressed at high levels is the fetal brain, kidne ys, heart, and lungs (Gibb and Sun, 1996). However, both COX-1 and COX-2 are expressed in the uterine epitheliu m during early pregnancy and may play roles in implantation and placental formation (Chakraborty et al ., 1996). This was recently confirmed for COX-2 in rats (Diao et al ., 2007). COX-2 may play a domi nant role in inducing labor. During labor, contractions are stimulated by prostaglandins a nd therefore any effect causing increased COX activity will induce labor. Up-regul ation of COX-2 during intrauterine infection may increase prostaglandins to the poin t of causing premature labor (Spaziani et al ., 1996). 24

PAGE 25

Furthermore, a similar up-regulation in sheep may reduce progesterone levels to the point that pregnancy cannot be maintained (McLaren et al ., 1996). However, it was recently shown that during the course of pregnancy there is a gr adual change in COX expression, with COX-1 dominating during early pregnancy and COX-2 dominating in later stages (Johnson et al ., 2006). It was concluded that while COX-2 is expressed at high levels in late st ages of pregnancy, it is not up-regulated suddenly to induce labor (Johnson et al ., 2006). COX inhibitors can halt premature labor by inhibiting pr ostaglandin production (Sawdy et al ., 1997). However, as in treatment strategies that use COX inhibitors for other problems, there are negative side effects associated with the kidneys: re nal clearance and glomerular filtration were reduced during the first weeks of life in babies that were expos ed to COX inhibitors to stop premature birth (Allegaert et al ., 2006). As in other cases, COX-2 selectiv e inhibitors were thought to eliminate these negative side effects but have not proven more effective than non-specific NSAIDs (Olson 2005). Nervous System COX-1 and COX-2 are both expressed in the mammalian nervous system and may participate in nerve transmission, sensory processi ng, thermoregulation, and pain effects. As in other systems, the use of COX inhibitors to tr eat neural diseases (such as Alzheimers) is prevalent, but is accompanied by undesired side effects that are not prevented by using COX-2 selective inhibitors. The brain is one of the few organs that c onstitutively expresses COX-2 and contains high amounts of COX-2 in the cortex, hippocampus hypothalamus, and spinal cord (Breder et al ., 1995), whereas COX-1 is abundantly expressed in the forebrain, where it may be involved in sensory information processing (Yamagata et al ., 1993). COX-2 is expressed in both neuronal 25

PAGE 26

and non-neuronal cells in the brain and is up-regu lated in response to ab normal nerve activity, suggesting a role in nerve transmission (Breder et al ., 1995; Breder and Saper, 1996). COX-2 in the spinal cord has been shown to modulate pain responses (nociception) in both humans and rats due to peri pheral pain stimulation (Martin et al ., 2007; Gardiner et al ., 1997; Willingale et al ., 1997). These results are expected si nce prostaglandins are generated at the ends of sensory neurons during in flammatory pain (Ferreira 1972; Woolf et al ., 1997). However, the up-regulation of COX-2 over COX-1 in the spinal cord during inflammatory pain suggests a role during pain processing in the centr al as well as the peripheral nervous system. This up-regulation has been detected via increases in COX-2 mRNA (Gardiner et al ., 1997; Beiche et al ., 1996) in the spinal cord of rats during inflammatory stimuli. Furthermore, COX-2 selective inhibition also affected the withdrawal reflex of ra ts when stimulated using a noninflammatory, electrical stimulus (Willingale et al ., 1997). This supports the general role of COX-2 in nociception in the spinal cord. COX-2 also plays a thermoregulatory role in the brain by increasing prostaglandin production in the hypothalamus during periods of fever. Injection of bacterial lippopolysaccarides (LPS) into mammals causes a monophasic fever characterized by a single rise in core body temperature. It has long been known that PGE2 levels increase in the hypothalamus during fever and likely mediat e the response (Ivanov and Romanovsky, 2004). Early studies suggested that an up-regulation of COX-2 was the cause of increased PGE2 levels during fever (Cao et al ., 1997). This initial hypothesis has been confirmed, recently in COX-2 deficient mice which do not respond to LPS injec tion, in contrast to CO X-1 deficient mice or control mice (Steiner et al ., 2005). 26

PAGE 27

Cyclooxygenase Function in Non-Mammals Although COX has been extensively studied in mammals due to its role in inflammation and the correlating drug mark et surrounding COX inhibition, studies in non-mammals are lacking. Unfortunately, it seems that alt hough non-mammals posses the mammalian COX-1 and COX-2 forms, these forms may be substantially different (both in structure and function) from their mammalian counterparts (Grosser et al ., 2002) and may also represent independent evolutionary events, such as duplications of an isoform (teleosts) or independent origins (sea squirts). In this section, I review the major studies i nvestigating COX function in nonmammalian chordates. Fishes Cyclooxygenase has likely been studied in the fishes more than other non-mammals due to their status as model organisms. As noted above, COX forms have been found in several fishes and along with genomic sequence data there is a general consensus that fishes possess both COX-1 and COX-2 forms that are homologous to those found in mammals. However, there is recent evidence that teleosts also possess an additional form of COX-1 or COX-2 (Ishikawa et al ., 2007; Ishikawa and Herschman, 2007) and some functional data also exist for these forms. Osmoregulation In fishes, the gills are the dominant site of acid/base regulation, nitrogenous waste secretion, gas exchange, a nd ion transport (Evans et al ., 2005). Several studies have investigated the osmoregulatory role of COX in th e gills of the euryhaline killifish, Fundulus heteroclitus This teleost can withstand instant salinity transfer between full strength seawater and fresh water without any apparent physiol ogical stress (Wood and Laurent, 2003). One study showed that short circuit current across th e opercular epithelium (a ti ssue with known ion transport capabilities) was reduced by using a non-specific COX inhibitor (Evans et al ., 2004). This 27

PAGE 28

suggests that COX-2 plays a role in ion transport in fishes that ma y be similar to its function in the kidneys of mammals. Further studies suppor ted this initial hypothesis by showing that COX2 is expressed most abundantly in the gill, opercu lar epithelium, and kidney of the killifish (Choe et al ., 2006), tissues that are known to be involved in ion transport in fishes. This is supported in the zebrafish, which shows the highest le vels of COX-2 in the gills (Grosser et al ., 2002). Furthermore, COX-2 was localized in mitochondrionrich cells in the gills of killifish, the main sites of ion uptake and secr etion in the gills (Choe et al ., 2006). Finally, COX-2 expression was shown to significantly increas e following either hypotonic or hype rtonic salinity transfers, suggesting COX-2 may play a role in maintaining cell homeostasis or promoting cell survival during periods of osmotic shock (Choe et al ., 2006). Taken together these results strongly suggest that COX-2 in the gills of teleosts acts in the same way as COX-2 in the kidneys of mammals, including regulating ion transpor t and promoting cell survival. Cyclooxygenase has also been shown to play an osmoregulatory role in the rectal gland of the dogfish shark, Squalus acanthias. Sharks rely on the rectal gland rather than the gills for regulation of salt secretion and a COX form (sCOX) has been cloned from the rectal gland (Yang et al ., 2002). This form was expressed most abundan tly in the rectal glan d of the shark, where PGE2 production was also high (Yang et al ., 2002). Finally, using a COX-2 specific inhibitor, vasoactive intestinal peptide mediated chloride se cretion decreased in the rectal gland, but then recovered following removal of the inhibitor (Yang et al ., 2002). Even though sCOX is slightly more similar to COX-1 of mammals than COX-2, th is result can be explained by the presence of valine at position 523 instead of isoleucine (and t hus conferring COX-2 inhibitory properties). This result suggests that sCOX plays a role in ion transport in the rectal glands of sharks that may be similar to the gills of teleosts or the kidneys of mammals. Th e osmoregulatory role of 28

PAGE 29

COX in fishes needs to be examined in greater de tail using other species and diverse techniques. This may be highly feasible because COX seque nces exist for a wide range of teleosts. Reproduction As in mammals, COX has been shown to play a role in reproduction in fishes. By using the non-specific COX inhibitor indomethacin, it was shown in the Atlantic croaker ( Micropogonias undulatus ) that COX pathways may play a ro le in the maturation of ovarian follicles and ovulation through prostaglandin forma tion, although other proteins may play a more dominant role (Patio et al ., 2003). Results from the European sea bass also indicate a similar role for prostaglandins in ovulation, with indom ethacin inhibiting follic le maturation (Sorbera et al ., 2001). However, it has been shown in the brook trout that indomet hacin does not block ovulation (Goetz et al ., 1989), although it does in other fish species (Goetz et al ., 1991). This apparent loss of function in the brook trout may be explained by changes in the levels of COX-1 and COX-2 during ovulation. It was shown that COX-1 levels remain ed constant and high during ovulation but that COX-2 le vels did not increase prior to ovulation as they do in mammals (Roberts et al ., 2000). This is supported by data from the zebrafish which show high levels of COX-1 but not COX-2 in the ovaries (Grosser et al ., 2002). This suggests that COX-2 function in the reproduction of the brook trout (and perhaps other fishes) is different than in other vertebrates. In the Japanese medaka, it was show n that low chronic levels (perhaps comparable to those found in waste water) (Metcalfe et al ., 2003) of the non-specific COX inhibitor ibuprofen causes altered reproduction by decr easing the number of spawning events but increasing the number of eggs per spawning event (Flippin et al ., 2007). This conforms to delayed pregnancies associated with NSAID use in mammals. These results indicate that COX likely plays a role in fish reproduc tion, although it may vary among species. 29

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Differential functions of novel forms Recently, the discovery of novel COX2 forms in the zebrafish (Ishikawa et al ., 2007) and rainbow trout (Ishikawa and Herschman, 200 7) and analyses of genomic sequence data indicates that all teleosts ma y posses at least three COX form s: either COX-1a, COX-1b, and COX-2 or COX-1, COX-2a, and COX-2b. Prelim inary data on the described COX-2 forms suggests that they are functionally different fr om each other. In the zebrafish, COX-2a and COX-2b are expressed at differe nt levels in the gill, kidney, and other tissues and respond differently to 12-O-tetradecanoylphorbol-13-ace tate (TPA, which is known to induce COX-2 expression in mouse) (Ishikawa et al ., 2007). A similar result was found in the rainbow trout, with differential expression of the two forms due to TPA (Ishikawa and Herschman, 2007). Interestingly, COX-2a and not COX-2b was upregulated in response to lippopolysaccarides (Ishikawa and Herschman, 2007). Clearly, the discovery of these new forms indicates a new chapter in studying COX function and further studies are needed to de termine the functional differences among the teleosts forms of COX. Other functions Cyclooxygenase has also been implicated in determining body plan development during embryonic stages in the zebrafish (Grosser et al ., 2002; Cha et al ., 2005; Cha et al., 2006a; Cha et al ., 2006b; Yeh and Wang, 2006). Briefly, COX may function to signal cell motility during gastrulation (Cha et al ., 2006b), and COX-1 function has been s hown to participate in vascular tube formation during development (Cha et al ., 2005). COX-2 expression has also been shown to increase in response to lippopolysaccarid es in the rainbow trout (Brubacher et al ., 2000; Holland et al ., 2002), suggesting an immunoregulatory role for COX-2 in fish as in mammals ( e.g ., fever induction). Clearly, the function of COX in fishes is just beginning to be explored, 30

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and further studies will likely indicate more sim ilarities and differences with COX functions in mammals. Amphibians As in other vertebrates, there is evid ence that COX is involved in ovulation and reproduction in amphibians. Indomethacin was shown to inhibit oocyte ovulation in the frog ( Rana temporaria ), while Prostaglandin F2 was shown to promote ovulation (Skoblina et al ., 1997). This suggests another medi ator may be influencing PGF2 production in the frog, but this contrast between seemingly relate d proteins has not been confirme d in other species. COX may also function in testosterone synthesis via PGE2 in the crested newt, Triturus carnifex (Gobbetti and Zerani, 2002). Ion and water transport have also been studied and non-specific COX inhibitors reduce short circuit current and voltage potential across the frog skin, bladder, and corneal epithelium (Shakhmatova et al ., 1997; Carrasquer and Li, 2002). As in reptiles, fever and thermoregulatory changes are behaviorally induced in amphibian s. Injection of lippopolysaccarides (LPS) normally causes a behavior ally induced fever in toads. However, when indomethacin is administered, the fever indu ced by LPS is completely blocked in the toad ( Bufo paracnemis) (Bicego et al ., 2002). This indicates that the COX pathway plays a role in the behaviorally induced fever caused by LPS and that this response has an ancient origin that may be conserved in all tetrapods. Reptiles Cyclooxygenase has not been st udied extensively in the lizar ds, crocodilians, snakes, and turtles although some data suggest prominen t roles in thermoregulation. Reptiles undergo behavioral thermoregulation to adjust their body temperatures, and this process is amplified by changes in heart rate. Specifically, an increase in heart rate (heart-rate hysteresis) accompanies an increase in temperature. Prostaglandins and cyclooxygenase were shown to play a role in this 31

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process in the lizard Pogona vitticeps Inhibition of COX-1 and COX2 resulted in no increased heart rate during heating (Seebacher and Fra nklin, 2003). Furthermore, administration of Prostaglandin F2 and Prostaglandin I2, but not Thromboxane B2, caused an increased heart rate (Seebacher and Franklin, 2003). However, this effect was not seen in crocodiles ( Crocodylus porosus ) (Seebacher and Frank lin, 2004) or the lizard Phrynocephalus przewalskii (Liu et al ., 2006), suggesting that thermore gulatory functions of COX via changes in heart rates may vary between closely related species. Interestingly, COX was also implicated in tail regeneration in th e house lizard. Twenty days after tail removal (the time period associated with tissue differentiation), an increase in cyclooxygenase activity occurred along with the appearance of endogenous Prostaglandin E2, which may signal a cascade resulting in tissue differentiation (Jayadeep et al ., 1995). Finally, indomethacin was used in the turtle bladder to sh ow that prostaglandins likely do not play a role in the inhibition of Na+ or H+ transport due to high intracellu lar calcium (Arruda, 1982). COX function in reptiles obviously needs further stud y as many functions associated with COX in mammals have yet to be investigated in re ptiles and there have been no COX targeted sequencing efforts. Birds Cyclooxygenase and prostaglandin function have been studied somewhat in model avian species, specifically chickens, wh ere there is support that COX is involved in similar processes as in mammals, ( e.g., reproduction). COX-2 ( but not COX-1) and prostaglandin activity was shown to increase with administration of transfor ming growth factor in granulosa cells of white leghorn hen follicles, suggesting that COX-2 a nd not COX-1 plays a role in granulosa cell proliferation during follicular development (Li et al ., 1996). It was also shown that indomethacin reduced the prolifer ation of granulosa cells (Jin et al ., 2006). When using COX-1 32

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and COX-2 specific inhibitors, it was found that both types reduced granulosa cell proliferation, but that COX-2 specific inhibitor showed a strong er effect, supporting orig inal studies that COX2 is the dominant form in granulosa cell proliferation (Jin et al ., 2007). However, COX-1 was found to be expressed highly in the brain and seminal vesicle of the chicken (Reed et al ., 1996). COX-1 (but not COX-2) is also up-regulated in ovarian cancers occurring in hens, suggesting a target for treatment (Urick and Johnson, 2006). C OX inhibitors, both specific and non-specific, were shown to decrease sperm motility in the domes tic turkey, demonstrating a further role for COX in bird reproduction (Kennedy et al ., 2003). As in mammals, COX-2 has been implicated with the detecti on and persistence of peripheral inflammatory pain in chickens. The number of C OX-2 containing neurons increased significantly in laminae under inflammatory c onditions, but not under control conditions, 12-24 hours after injection of Fr eunds adjunvant (Yamada et al ., 2006). This suggests that the numbers of COX-2 containing neurons are re lated to inflammatory pain as in mammals. It appears that COX may not play a thermoregulator y role in birds as it does in mammals because indomethacin did not cause any change in rectal temperat ure during heat stress in chickens (Furlan et al ., 1998). However, COX and its prostaglandins may play a role in memory retention in birds, because inhibitors produce amnesic effects (Hlsc her, 1995). Finally, COX-2 is expressed in the kidneys during development in chickens (Mathonnet et al ., 2001) and at high levels in the kidneys of adults (Reed et al ., 1996). Study Overview As outlined above, the family of cyclooxygenase genes has an interesting and functionally important history in the vertebrates. Drugs deri ved from cyclooxygenase function are among the most important and widely used tr eatment methods in medicine. However, COX function and evolution is not fully understood in the ancestral chor dates, specifically the teleosts, 33

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sharks, hagfish, lampreys, cephalochordates, a nd urochordates. Indee d, the origin of the mammalian forms of COX is unclear since the sea squirt possesses evolutionarily distinct COX forms. Resolving the evolution of COX in the chordates could prove invaluable in determining new targets for drug development (Searls, 2003). In Chapter 2, I describe the cloning and characterization of 9 new COX genes in the chordates. These include COX-1 and COX-2 form s from teleosts as well as novel forms from the hagfish, lamprey, and amphioxus. In phylogenet ic analyses, these forms indicate that several origins of COX occurred during chordate e volution, with the mammalian COX-1 and COX-2 forms likely originating with the hagfish (craniates). Data from the teleosts supports the findings of Ishikawa and colleges that teleosts in the Acanthopterygii posses two COX-1 forms and one COX-2 form, whereas earlier teleosts such as the zebrafish and rainbow trout posses two COX-2 forms and one COX-1 form. Furthermore, analyses of protein alignments from novel sequences indicate conserved and derived functional resi dues and areas between novel COX forms and the COX-1 and COX-2 forms of mammals. In Chapter 3, I summarize the findings of th is research and suggest future avenues of research based on the results presented here. I also present preliminary data investigating the possible function of cyclooxygenase in these ancestral chordates. 34

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Arachidonic Acid Prostaglandin G2 COX-1 or COX-2 Prostaglandin H2 Prostaglandin D2 Prostaglandin E2 Thromboxane A2 Thromboxane B2 Prostaglandin I2 6-keto-Prostaglandin F1 COX-1 or COX-2 Prostaglandin F2 Figure 1-1. Cascade of reactions depicting th e conversion of arachidonic acid to the primary prostaglandins via catalyzation with either cyclooxygenase (COX) form. Arachidonic acid is obtained from the conversion of linolet ic acid found in plants or directly from other animals. It is then oxidi zed by COX to produce Prostaglandin G2 which is subsequently reduced by COX to form Prostaglandin H2. Prostaglandin H2 can then be converted into the other prostaglandi ns via other enzymes (Vane et al., 1998). 35

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COX-2a Danio COX-2a Oncorhynchus COX-2b Danio COX-2b Oncorhynchus COX-2 Acanthopterygians COX-1a Acanthopterygians COX-1b Acanthopterygians COX-1 Danio COX-1 Oncorhynchus sCOXSqualus Figure 1-2 General phylogeny repr esenting the different COX forms in the fishes. Based on recent studies, it appears that teleosts have multiple forms of COX-1 or COX-2 (Ishikawa et al ., 2007; Ishikawa and Herschman, 2007). It is hypothesized that with the origin of the teleosts, COX-1 and C OX-2 underwent duplication, but that in one lineage (represented by the zebrafish and rainbow trout) one COX-1 form was lost and in another lineage (represented by the Acanthopterygians) one COX-2 form was lost. Also included is the form cloned from the spiny dogfish (Yang et al ., 2002) which loosely groups with COX-1 forms, although it shares near equal identity with COX-1 and COX-2. This phylogeny was simplified from an unpublished phylogeny generated using the online program P HYML (Guindon and Gascuel, 2003; Guindon et al ., 2005). Abbreviations: Danio = Danio rerio (zebrafish), Oncorhynchus = Oncorhynchus mykiss (rainbow trout), Squalus = Squalus acanthias (spiny dogfish). The Acanthopterygians clade is represented by the stickleback ( Gasterosteus aculeatus), green spotted puffer ( Tetraodon nigroviridis ), pufferfish ( Takifugu rubripes ), and Japanese medaka ( Oryzias latipes ). 36

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Figure 1-3 Accepted evolution of ancestral chordates (Pough, et. al 2005). Black lines show relationships only; they do not indicat e times of divergence nor the unrecorded presence of taxa in the fossil record. Ba rs shaded red indicate ranges of time when the taxon is known to be present. Th e subphylum Vertebrata includes the Myxinoidea (hagfishes), Petromyzontoidea (lampreys), Teleoste i (teleosts), and tetrapods. There is some controversy over wh ether the Urochordates (sea squirts) or the Cephalochordates (lancelets) represent the most basal chordate lineage. Also, it has historically been suggested that the hagfish and lamprey constitute a monophyletic group (the Agnathans or Cyclostomes). 37

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38 Invertebrate COX forms COXb Urochordates COXa Urochordates COX-2 Vertebrates COX-1 Vertebrates Figure 1-4 Evolution of COX forms in the chordates, based on Jarving et al (2004). The Urochordates (represented by Ciona intestinalis and Ciona savignyi ) have COX forms that represent an evolutionary dis tinct branch ancestral to the COX-1 and COX-2 forms of the vertebrates. In this tree, the vertebrates are represented by the teleosts, mammals, Xenopus, and Gallus

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CHAPTER 2 CYCLOOXYGENASES IN THE ANCEST RAL CHORDATES: SEQUENCE AND PHYLOGENETIC ANALYSES Introduction Cyclooxygenase (COX) is the enzyme that catalyzes the oxida tion and subsequent reduction of arachidonic acid to form Prostaglandin G2 and Prostaglandin H2 (PGH2). PGH2 can then undergo additional reactions to produce the primary prostagla ndins, which act as vascular tone regulators in the verteb rates. Prostaglandins and C OX participate in a variety of physiological functions in the vertebrates, incl uding inducing fever, maintaining pregnancy, and regulating ion transport in the kidneys (Steiner et al ., 2005; McLaren et al ., 1996; Harris et al ., 1994). These functions have been ex tensively studied in mammals (Vane et al ., 1998), but comparatively little functional data exist for ot her animals. However, COX has been sequenced in several evolutionarily more ancestral chordates, particularly the teleosts (Zou et al ., 1999; Roberts et al ., 2000; Choe et al ., 2006). Based on functional studies of COX in the teleosts, it seems that some functions are conserved (Choe et al ., 2006; Sorbera et al ., 2001; Brubacher et al ., 2000; Holland et al ., 2003) while others may be altered in some species (Goetz et al ., 1989) or novel (Cha et al ., 2006b). In mammals there are two main forms of cyclooxygenase. The first form was isolated from sheep seminal vesicles in 1988 and later named COX-1 (DeWitt and Smith, 1988; Merlie et al ., 1988; Yokoyama et al ., 1988). A second form was isol ated from mouse and chicken fibroblast cell cultures in the early 1990s and named COX-2 (Kujubu et al ., 1991; Xie et al ., 1991; O'Banion et al ., 1992). Originally, COX-1 was considered to be a constitutive form that maintained normal cell functions and COX-2 was c onsidered to be an inducible form that was up-regulated in inflammatory res ponses (Funk, 2001). However, stud ies have shown that this is an oversimplification and COX-2 is expressed constitutively in the brain (Breder et al ., 1995) 39

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and kidneys (Harris and Breyer, 2001) of mammals. This has led to the abandonment of COX-2 selective inhibitors ( e.g., celecoxib and valdecoxib) which were thought to treat inflammatory pain without the negative side effects associ ated with non-selective COX inhibition. Although structurally and biochemically similar, COX-1 a nd COX-2 vary in expression in the vertebrates and participate in different functions. For example, COX-2 but not COX-1 plays a role in granulosa cell proliferation in chicken follicles (Li et al ., 1996; Jin et al ., 2007). Although the amino acid sequences of COX-1 and COX-2 share about 63% similarity, the presence of valine in COX-2 at position 523 instead of isoleucine is thought to be re sponsible for their differences in substrate selectivity and sensitivity to specific inhibito rs (Otto and Smith, 1995). Inhibitory studies in mammals have suggested a COX-3 form also exists, but it has not been characterized (Kis et al ., 2006; Censarek et al ., 2006). Analyses of genomic sequence and targeted cloning efforts have demonstrated that, like mammals, other vertebrates have COX-1 and COX-2 forms (Jarving et al ., 2004). However, some variation exists in the COX-1 and COX-2 dichotomy, notably in the more evolutionarily ancestral chordates. Ishikawa and colleagues (Ishikawa et al ., 2007; Ishikawa and Herschman, 2007) have recently shown that teleosts posse s three forms of COX. The zebrafish ( Danio rerio) and the rainbow trout ( Onchorhynchus mykiss ) both posses two COX-2 forms (named COX-2a and COX-2b) and one COX-1 form whereas the stickleback (Gasterosteus aculeatus), green spotted puffer ( Tetraodon nigroviridis ), pufferfish ( Takifugu rubripes ), and Japanese medaka ( Oryzias latipes) posses two COX-1 forms (named COX-1a and COX-1b) and one COX-2 form. They also showed that COX-2a and COX-2b ar e functionally different in the zebrafish and rainbow trout. Also, a COX form (named sC OX) has been cloned from the spiny dogfish ( Squalus acanthias ) and groups with COX-1 in phylogene tic analyses, but shares strong 40

PAGE 41

sequence identity to COX-2 as well (Yang et al ., 2002). Furthermore, sea squirts (subphylum Urochordata) possess two forms of COX (named COXa and COXb) that do not correspond to the COX-1 or COX-2 of vertebrate s and represent an ancestral or igin of COX in the chordates (Jarving et al ., 2004). The current view of the evolution of cyclooxygenase in the chordates represents an interesting but incomplete account of this gene family, both from a phylogenetic and functional standpoint. Therefore, the first goal of this study was to pinpoint the orig in of COX-1 and COX2 in the chordates by searching for COX forms in the sea lamprey ( Petromyzon marinus), Atlantic hagfish ( Myxine glutinosa ), and amphioxus ( Branchiostoma lancelolatum ). A second goal was to confirm the findings of Ishikawa and colleagues by searching for the 3 forms of COX in the euryhaline killifish ( Fundulus heteroclitus ) and longhorn sculpin ( Myoxocephalus octodecemspinosus ). It was hypothesized that both te leosts would posses COX-1a, COX-1b, and COX-2 since both are in the Acanthopterygii clade of Teleostei and all other acanthopterygiians posses COX-1a, COX-1b, and COX-2. Here, I re port the cloning of 9 novel COX sequences from the species mentioned above. Sequence an d phylogenetic analyses s uggest that ancestral COX forms may have had similar functions as COX-1 and COX-2 in mammals, which likely originated with the origin of the vertebrates, although the hagf ish and lamprey may have COX-1 and COX-2 forms that re present novel lineages. Materials and Methods Animals and Holding Conditions All procedures were approved prior to be ginning the experiment by the University of Florida Institutional Animal Care and Use Committee (IACUC). Euryhaline killifish ( Fundulus heteroclitus ) were captured from Northeast Creek n ear the Mount Desert Island Biological Laboratory (MDIBL), Salisbury Cove Maine using minnow traps. Th ey were transported to the 41

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MDIBL where they were kept in fiberglass ta nks containing 100%, flowi ng seawater from the Gulf of Maine. The tanks were expose d to natural conditions. Longhorn sculpin ( Myoxocephalus octodecemspinosus ) were purchased from fishermen and were housed in a similar way at the MDIBL. Atlantic hagfish ( Myxine glutinosa ) were also purchased from fishermen and were housed at the MDIBL in 100% seawater and maintained on a 12 h light/dark cycle. Female, non-migratory lampreys ( Petromyzon marinus ) were a generous gift from the USGS Great Lakes Science Center at the Ha mmond Bay Biological Stat ion in Millersburg, Michigan and were dissected there. Lancelets ( Branchiostoma lancelolatum ) were purchased from Gulf Marine Specimens (Panacea, FL) and were kept in the bags they were shipped in for a few days before they were sacrificed. All housed animals were fed to satiation regularly. Some killifish were shipped to the Univers ity of Florida (Gainesv ille, FL) for further experiments. These killifish were housed as described previously (Choe et al ., 2006). Some hagfish were also transported to the Universi ty of Florida (Gainesville, FL) for further experiments. These hagfish were housed for a few days in a large Rubbermaid tank in 100% seawater at 4 C before they were killed. Tissue Collection After initial anaesthetiza tion with MS-222 (~600 mg L-1), killifish, sculpin, and lampreys were pithed and/or decapitated. The gill arches (1st and 2nd arches for lampreys, 2nd and 3rd arches for teleosts) were then removed using ster ile, RNAse free dissecting tools. Hagfish were decapitated and all gill baskets were then rem oved using sterile, RNAse free tools. Lancelets were cut in half with sterile, RNAse free tools. After removal, tissues were immediately placed in liquid nitrogen and stored at -80 C for 2 weeks to 4 months before further processing. 42

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Reverse Transcription, Primer Design, PCR, Cloning, and Sequencing Reverse transcription, PCR, cloning, and sequencing were performed as described previously (Choe et al ., 2006) with modifications. Total R NA was isolated from the gills of killifish, lampreys, sculpin, and hagfish as well as the anterior half of lancelets using TRI-reagent (Sigma, St. Louis, MO), and revers e transcribed with a SuperscriptTM II or SuperscriptTM III reverse transcriptase kit (Invi trogen, Carlsbad, CA) following th e manufacturers protocol and using oligo-dT as a primer. The resulting cDNA was stored at -20 C until used for PCR. Degenerate primers (those labe led as CH COX, Table 2-1) were first designed to amplify non-specific cyclooxygenases in the hagfish, lamp rey, and lancelet based on conserved amino acid sequences between COX-1 and COX-2 of Mus musculus, COX-2 of Fundulus heteroclitus sCOX of Squalus acanthias (GenBank accession numbers: NP_032995, NP_035328, and AAS21313), and predicted COXa and COXb of Ciona intestinalis based on genomic data. These primers were also later used to amplify COX-2 in sculpin. Another set of degenerate primers (those labeled as CH COX-1 in Table 2-1) were designed to amplify COX-1 forms specifically over COX-2 forms in teleosts ba sed on conserved amino acid sequences between COX-1 in Oncorhynchus mykiss Danio rerio, Salvelinus fontanalis (GenBank accession numbers: CAC10360, NP_705942, and AAF14529) and a predicted COX-1 of Tetraodon nigroviridis based on genomic data. These primers firs t amplified a COX-1a form in sculpin and a COX-1b form in killifish (see below). A further set of degenerate primers (those labeled as CH COX-1a in Table 2-1) were designed to amp lify COX-1a forms specifically over COX-1b in teleosts based on conserved amino acid seque nces between predicted COX-1a forms in Gasterosteus aculeatus Oryzias latipes and Tetraodon nigroviridis based on genomic data and a COX-1a found in sculpin earlier in this study (see below). A fi nal set of degenerate primers (those labeled as CH COX-1b in Table 2-1) were designed to amplify COX-1b forms specifically 43

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over COX-1b in teleosts based on conserved am ino acid sequences between predicted COX-1b forms in Gasterosteus aculeatus Oryzias latipes and Tetraodon nigroviridis based on genomic data and a COX-1b found in killif ish earlier in this study (see be low). All degenerate primers were designed using the online program Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) (Rose et al ., 1998). Initial PCR reactions were performed on 1/20th of a reverse transcri ptase reaction with a TaKaRa Ex Taq Hot Start DNA Polymerase Kit (Takara Bio Inc., Japan) in a PCR Express thermocycler (ThermoHybaid, Franklin, MA) with the following parameters: initial denaturing at 95 C for 5 minutes, then 35 cycles of 30 seconds at 95 C, 30 seconds at 60 C, and 1.5 minutes at 72 C. A final elongation step of 7 minutes at 72 C was performed for each PCR and then the reaction was held at 4 C. Initial PCR products were visualized by ethidi um bromide staining on 1-2% agarose gels and then ligated into PCR4-TOPO vectors and transformed into TOP10 chemically competent cells using a TOPO TA Cloning Kit (Invitrogen) following the manufacturers protocol. Plasmids were then isolated using a High Pure Plasmid Isolation Kit (Roche, Germany) following the manufacturers protocol and plasmid DNA was sequenced in both directions at the Marine DNA Se quencing Center at the MDIBL. After initial fragments were sequenced, Pr imer Express software (Applied Biosystems, Foster City, CA) was used to design specific prim ers based on the initial fragments (Tables 2-2, 2-3, 2-4, 2-5, and 2-6). These specific primers, along with the original de generate primers, were used to extend COX sequences. PCR was comple ted as above with slight modifications to annealing temperature and elongation time for each reaction, as well as nested PCR reactions in some cases to increase specificity. Products were visualized, cloned, and sequenced as above. 44

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5 and 3 Rapid Amplific ation of cDNA Ends (RACE) After initial sequences were extended with sp ecific primers, the 5 and 3 ends were sequenced using 5 and 3 RACE. Following th e manufacturers protocols, a Generacer Kit (Invitrogen) was used to make 5 and 3 cDNA for hagfish, k illifish, and scul pin. Initial sequence data was used to make 5 and 3 RACE specific primers for each species (those labeled with 5 or 3 in Tables 2-2, 2-3, 2-4, 2-5, and 2-6). These primers, along with kit primers that anneal to the ends of the 5 and 3 cDNA, we re used in touchdown PCR reactions with the following parameters: 5 cycles of 30 seconds at 94 C and 1.5 minutes at 72 C; 5 cycles of 30 seconds at 94 C and 1.5 minutes at 70 C; and 25 cycles of 30 seconds at 94 C, 30 seconds at 60 C, and 1.5 minutes at 72 C. As above, a fi nal elongation step of 7 minutes at 72 C terminated the reaction, which was held at 4 C. For some reactions, annealing temperatures and elongation times were slightly altered and nest ed PCR reactions were performed for some reactions using specific primers (Tables 2-2, 23, 2-4, 2-5, and 2-6) to increase specificity. Products were visualized, cloned, and sequenced as above. Sequence Analysis Initial sequences from dege nerate primers, sequence exte nsions, and 5 and 3 RACE were assembled using GeneTools software (BioT ools Inc., Edmonton, Alberta) and the resulting sequences were searched for open reading frames. The predicted amino acids were aligned with other COX proteins using PepTools software (BioTools Inc.) for phylogenetic analysis. Multiple alignments were also generated using ClustalW to search for conserved protein domains across COX sequences. Alignments were manually adju sted and the N and C-terminal areas were realigned using Microsoft Word based on previous COX alignments (Ishikawa et al ., 2007; Yang et al ., 2002; Kulmacz et al ., 2003). For the novel sequences, COX forms were aligned with 45

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other relevant COX proteins and annotate d using functional amino acids and domains highlighted from Ishikawa et al (2007), Yang et al (2002), and Kulmacz et al (2003). Phylogenetic Analysis To investigate the evolutionary relationshi ps of the different forms of COX in the chordates, phylogenies were ge nerated using the novel amino acid sequences generated in this study and several other COX protein sequences from GenBank and genome databases. All novel protein sequences were included in the data set. COX sequences from teleosts were included in the analysis only if all 3 forms of COX were av ailable. These included the zebrafish, rainbow trout, green spotted puffer, stickleback, eur yhaline killifish, and longhorn sculpin. COX sequences from non-teleosts were included in the analys is if at least two forms of COX were available and both sequences were designated as Reference Sequences (RefSeqs) in GenBank. These included the chicken, frog, mouse, rat, human, rabbit, dog, cow, sheep, and opossum. COX sequences were also included if they represented an evolutionarily interesting group. These included the dogfish shark an d the sea squirt. In total, 50 protein sequences were included in the analyses (Table 2-7). Models of evolution to be used in phylogenetic analyses were evaluated to account for amino acid substitutions, among-site variation, and invariable sites. Using likelihood ratio tests, a model using the WAG rate matrix (W helan and Goldman, 2001) and the gamma ( ) distribution for among-site varia tion was chosen (Table 2-8). A distance phylogeny was first generated using Molecular Evolutionary Genetics Analysis 4 (MEGA) software (Tamura et al ., 2007) with a minimum evolution (ME) analysis and 1000 bootstrap replicates. In this analysis the parameter was fixed to the value estimated from maximum likelihood (ML) analysis ( = 0.737, see below). Additionally, a JTT rate matrix (Jones et al ., 1992) was used instead of a WAG matrix because a W AG matrix is not available 46

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for MEGA 4. A complete deletion option was also selected. After comparing results between ML, ME, and Bayesian Phylogenetics (BP), lampre y and hagfish sequences were identified as possible rouges and another distance analysis with identical parameters (but with = 0.742) was performed excluding these two sequences. A maximum likelihood phylogeny (ML) wa s generated using the program PhyML (Guindon and Gascuel, 2003). An initial phylogeny was generated using WAG analysis, 1000 bootstrap replicates, 8 substitution rate categor ies, and a gamma shape parameter with an estimated Again, the analysis was repeated with the rouge lamprey and hagfish sequences removed. To evaluate support for monophyletic groups, an approximate likelihood ratio test (aLRT, Anisimova and Gascuel, 2006) was pe rformed both including and excluding the lamprey and hagfish sequences. This test used the mi nimum of Chi-Squared and Shimodaira-Hasegawa (SH) support values. Finally, A Bayesian phylogenetics (BP) analys is was also performed using an initial run with 2 million generations and a final run with 10 million generations using the program MrBayes 3.1.2. Once again, analyses were perf ormed with and without hagfish and lamprey sequences. Each run consisted of on e cold and three heated chains ( T = 0.2). The WAG + model was used with an estimated After discarding the firs t 10% of each run as burnin, posterior probabilities were calculated based on trees sa mpled every 500 generations. Due to inconsistencies in tree topologie s between the three methods used above, alternative topologies were ge nerated in which the positions of the hagfish and lamprey sequences were forced to be included in diffe rent clades (Figure 2-15). These alternative topologies were generated and viewed using TreeView 1.6.6. Alternative topologies were 47

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evaluated using Ln L scores generated by maximum likelihoo d criterion with online execution in PhyML (Guindon et al ., 2005). Results Molecular Identification of Cyclooxygenases Branchiostoma Initial PCR reactions using the degenerate primers CH COX F3 and CH COX R2 (Table 2-1) amplified two 532 bp products from the lancelet ( Branchiostoma lancelolatum ). These two products (referred to hereafter as COXc and COXd ) were found to share the most identity with COXa and COXb from the tunicat e. COXc was 50.9% identical to COXa from the tunicate and COXd was 58.8% identical to COXb from the tuni cate. All attempts to extend these sequences using specific primers designed against these se quences (Table 2-2) we re unsuccessful. After searching for open reading frames two putative 177 amino acid prot eins were predicted from the lancelet. Myxine Initial PCR reactions using the degenerate primers CH COX F1, CH COX F2, CH COX R1, and CH COX R2 (Table 2-1) were used to amplify a single 1454 bp product from the hagfish ( Myxine glutinosa ). This product (hereafte r referred to as hCOX) was found to be 63.8% identical to COX-1 of the zebrafish ( Danio rerio). RACE primers (5 and 3, Table 2-3) were designed against this initial pr oduct and used with kit primers to completely sequence the 2416 bp product. Searching for open reading frames yi elded a putative 610 amino acid protein. Petromyzon Initial PCR reactions using degenerate primers CH COX F3 and CH COX R1 (Table 2-1) amplified a single 541 bp product from gill tissue of the lamprey ( Petromyzon marinus). This product (hereafter referred to as lCOX) was later extended using degenerate primers (Table 2-1) 48

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and specific primers designed agains t the initial 541 bp product (Table 2-4). Despite all attempts to amplify the complete lCOX product (including RACE) the sequence was only extended to 860 bp. A 286 amino acid open reading frame was pred icted from this product. Fundulus Because the COX-2 sequence fo r the euryhaline killifish ( Fundulus heteroclitus ) was previously completed (Choe et al ., 2006), the degenerate primer CH COX-1 F2 (Table 2-1) designed to amplify COX-1 forms over COX-2 form s was used with the degenerate primer CH COX R1 (Table 2-1) to initially amplify a non-COX-2, 1335 bp product (hereafter referred to as COX-1b Fundulus ) from killifish gill tissue. This product was then extended and completed using specific primers and RACE primers (Table 2-5). The completed 2226 bp sequence contains a 598 amino acid open reading frame that was nested inside the teleost COX-1b clade in all phylogenetic analyses (see be low), prompting the design of degenerate primers (Table 2-1) that were used to amplify COX-1a forms in teleosts over COX-1b forms. Using the degenerate primers CH COX-1a F1 and CH COX-1a R1 (Table 2-1), a 759 bp product was initially amplified in PCR reactions. Using RACE primer s designed to complete this sequence (Table 25), the 3 end of this product was completed, resulting in a 1655 bp product (hereafter referred to as COX-1a Fundulus) that contains a 452 amino acid reading fr ame. However, the 5 end of this product was not sequenced despite multiple attemp ts. This putative protein grouped with the COX-1a sequences of other teleos ts in all phylogenetic analyses (see below), leading to the conclusion that the COX-1a, COX-1b, and COX-2 sequences included in the analyses all represent different COX products expressed in the gill tissu e of the euryhaline killifish. Myoxocephalus Using the degenerate primers CH COX F1 and CH COX R2 (Table 2-1), a 977 bp product was initially amplified in PCR reactions using cDNA from the gills of the longhorn 49

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sculpin ( Myoxocephalus octodecemspinosus). This product (hereafter referred to as COX-2 Myoxocephalus) was then extended and completed usi ng RACE and specific primers (Table 26), resulting in a 2618 bp product that contains a 605 amino aci d open reading frame. This sequence grouped with COX-2 forms from other te leosts in all phylogenetic analyses, inciting the use of COX-1 degenerate primers (Table 2-1) CH COX-1 F2 and CH COX-1 R2 in PCR reactions to amplify a 1000 bp product (hereafter referred to as COX-1a Myoxocephalus ). This product was then extended and completed using RA CE primers (Table 2-6), resulting in a 2633 bp product encoding a 622 amino acid open reading frame. This product grouped with COX-1a forms from other teleosts in all phylogenetic analyses, prompting the design of degenerate primers used to amplify COX-1b forms in teleosts over COX-1a forms (Table 2-1). Using the degenerate primers CH COX-1b F1 and CH CO X-1b R0 (Table 2-1), an initial 895 bp product (hereafter referred to as COX-1b Myoxocephalus ) was amplified in PCR reactions. This product was extended and completed using specific RACE primers (Table 2-6), resulting in a 2332 bp product containing a 600 amino acid open readin g frame. This product grouped with other teleost COX-1b forms in all phylogenetic analyses, suggesting that the COX-2, COX-1a, and COX-1b forms sequenced represent 3 different C OX forms expressed in the gills of the longhorn sculpin. Sequence Analyses Teleosts COX-1a, COX-1b, and COX-2 Three different COX forms are now available from the teleosts studied here: the COX-2 sequenced from the euryhaline killifish (Choe et al ., 2006), the COX-2 sequenced here from the longhorn sculpin, and two the COX-1 forms sequenced here from both the killifish and sculpin, named as COX-1a and COX-1b (following Ishikawa and Herschman, 2007). COX-1a and COX1b predicted proteins from the killifish were more similar to Mus COX-1 than COX-2 (70% 50

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versus 64% for COX-1a and 68% versus 59% for COX-1b, respectively). Similarly, COX-2 from the sculpin was more similar to Mus COX-2 than Mus COX-1 (70% versus 60%) and COX-1a and COX-1b were more similar to Mus COX-1 than Mus COX-2 (53% versus 47% for COX-1a and 67% versus 57% for COX-1b, respectively). An alignment of COX forms from the killifis h (Figure 2-1) shows that all the important amino acid residues for cyclooxygenase function are conserved in all forms. However, because COX-1a is incomplete it is unknown whether the Nterminal characteristics are conserved in this form. These include an active site tyrosine (Tyr-385, using ovine COX-1 numbering), haembinding histidines (His-207 and His-338), and the aspirin acetyla tion site (Ser530) (Ishikawa et al ., 2007; Ishikawa and Herschman, 2007; Yang et al ., 2002; Kulmacz et al ., 2003). In addition the COX-2 form contains a C-terminal amino aci d insertion and the COX-1 forms contain an Nterminal amino acid insertion. This is consis tent with the COX-1 and COX-2 forms of other vertebrates (Herschman et al ., 2003; Kulmacz et al ., 2003), although killifish COX-2 seems to have a shorter than average in sertion. In mammals, the ami no acid residues thought to give COX-1 and COX-2 their differing affinities for su bstrates are positions 513 (His in COX-1 and Arg in Cox-2) and 523 (Ile in COX-1 and Val in COX-2) (Guo et al ., 1996). However, as previously noted in other teleosts (Grosser et al ., 2002; Choe et al ., 2006; Ishikawa et al ., 2007) these differences appear to be absent in teleosts, with Arg an d Val present in positions 513 and 523, respectively, in both teleost COX-1 and COX-2. This is also the case in the killifish with Arg in position 513 and Val in position 523 in all COX forms reported here. As in the killifish, alignment of the three COX forms sequenced from the sculpin (Figure 2-2) shows conserved amino acid re sidues characteristic of COX func tion in all three forms. The characteristic terminal amino acid insertions f ound in mammalian COX forms are also present in 51

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their respective sculpin counterpa rts. However, COX-1b from th e sculpin seems to have both N and C-terminal insertions. Disulfide bonds near th e n-terminus of sculpin COX-1a also seem to be altered due to the substitution of other amino acids for cysteine. It also appears that some functional amino acid residues near the C-terminus of sculpin COX-1a are not conserved with the other forms. These include the substitution of Leu for Arg at position 513, Leu for Val at position 523, and Phe for Ser at position 530. Lamprey, hagfish, and amphioxus COX forms The one incomplete putative COX protein obtained from the gills of the lamprey shares near equal identity (63%) with Mus COX-1 and COX-2. The gene, referred to here as lamprey COX (lCOX) (Yang et al ., 2002) shares the most identity (70 %) with a form from the lancelet (COXc), but shares notable identi ty (64%) with the form from the hagfish as well. Based on an alignment of COX forms sequenced from select non-mammalian and non-teleost chordates (Figure 2-3), the partial COX form sequenced fro m the gills of the lamprey has the predicted histidine site (His-207) critical for peroxidase activity as well as conserved haem-binding domains. However, the membrane binding regi ons near the Arg-277 loop that differ between COX-1 and COX-2 forms appear to be different from both COX-1 and COX-2 in the lamprey form. This is seen when the lamprey form is aligned with COX-1 and COX-2 forms from vertebrates (Figure 2-4). The putative COX protein from the gills of the hagfish shares slightly more identity with Mus COX-2 than Mus COX-1 (63% versus 60%). The gene, referred to here as hCOX, shares the most identity (> 60%) with vertebrate COX-2 forms, but also shares notable identity with lCOX (64%) and forms from amphioxus (> 60%). An alignment of hCOX with other nonmammalian, non-teleost COX proteins (Figure 2-3) shows that all important amino acid residues and binding sites critical to cycl ooxygenase activity are conserved in hCOX. An alignment of 52

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hCOX with COX-1 and COX-2 protei ns from various vertebrates (Figure 2-5) shows that hCOX has an insertion of amino acids near the C-terminal; characteristic of COX-2 proteins in vertebrates. However, there are several re gions where COX-1 differs from COX-2 but hCOX shows no identity to COX-1 or COX-2 amino acids (indicated as sites with a + over them in Figure 2-5). Two putative incomplete COX sequences were obtained from amphioxus. These sequences, referred to here as COXc and COXd, are most identical (81%) to each other, but also share notable identity with hCOX (66%), lC OX (67%), vertebrate COX-1 (> 60%), and vertebrate COX-2 (> 60%). Th ey are the least identical to Ciona COXa (48%) and COXb (55%). Based on an alignment of non-mammalia n, non-teleost COX proteins (Figure 2-3), it appears that amino acids critical to COX functi on are conserved in COXc and COXd. However, the first haem-binding domain seems to be very di fferent in the amphioxus forms. This is also the case for Ciona COX forms and COXa, COXb, COXc, and COXd all share notable identity in this region as shown by alignment (Figure 2-6). Phylogenetic analyses A phylogeny (Figure 2-7) generated under a distance optimality criterion with 1000 bootstrap replicates using MEGA4 shows predic ted COX clades. COX-1a and COX-1b forms from teleosts form monophyletic sister clades The novel COX-1 forms from the killifish and sculpin group together with othe r teleosts in these clades as expected based on their designation here. Eutherian COX-1 forms group together in a monophyletic clade sister to the teleost COX-1 clade. The COX-1 proteins from Gallus, Monodelphis, and Xenopus are located at the base of this monophyletic vertebrate COX-1 clade, with sCOX being the most basal. Vertebrate COX-2 forms group together in another monophyletic clade that is sister to the vertebrate COX-1 clade. There are two monophyletic clades within the COX-2 group: a teleos t clade and a tetrapod clade. 53

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The forms from lamprey and hagfish (lCOX and hC OX) are basal to the vertebrate COX clade, while the forms from amphioxus (COXc and C OXd) form a monophyletic group basal to these forms. The forms from Ciona (COXa and COXb) form another monophyletic group that is basal to all other COX forms. Boot strap values give robust support for vertebrate clades, the amphioxus clade, and the Ciona clade, but give weak support for placement of hCOX, lCOX, and sCOX. Based on these re sults and other generated phyl ogenies (see below), hCOX and lCOX were identified as possible rogue sequences due to their inconsistent positions depending on the type of analysis used and another distan ce analysis was complete d without including these sequences (Figure 2-8). The t opology of this phylogeny is very similar to the complete COX phylogeny. However, sCOX is basal to the vert ebrate COX clade and not included with the other vertebrate COX-1 forms. Additionally, bootstrap values for most clades are considerably more robust than when hagfish and la mprey sequences are included. To further test relationships between COX proteins, a phyloge ny (Figure 2-9) was generated using the maximum likelihood criter ion, 1000 bootstrap replicates, and the program PhyML (Guindon and Gascuel, 2003). The topology of this tree is very similar to the one generated using distance criteri on. Briefly, it contains well supported, monophyletic clades for COX-1 (teleost COX-1a, teleost COX-1b, tele ost COX-1, mammalian COX-1, and vertebrate COX-1 with sCOX as the most basal sequence) and COX-2 (teleost COX-2, mammalian COX-2, and vertebrate COX-2). The lamprey (lCOX) is also basal to the vertebrate COX clade, the amphioxus COXc and COXd form a basal monophyletic clade, and the most basal clade in the tree is a monophyletic COXa and C OXb from the sea squirt. However, in the ML analysis, the hagfish (hCOX) sequence groups at the base of the vertebrate COX-2 clade instead of being basal to the vertebrate COX clade. Again, th e analysis was repeated without hCOX and lCOX 54

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(Figure 2-10), causing bootstrap values to incr ease dramatically. Also, the estimated gamma shape parameter increased slightly from 0.737 to 0.742. To further evaluate support for COX clades, an aLRT analysis (Anisimova and Gascuel, 2006) was performed. This type of analysis prov ides values of support similar to Felsensteins bootstrap support values but is much faster because bootstrap sampling requires many (1000 in the previous analyses) runs wh ile aLRT is run only once. The fundamental difference between the two is that bootstrap support values are based on repeatability whereas the aLRT is a measure of the likelihood gain of including a branch versus collapsing it (Anisimova and Gascuel, 2006). The resulting aLRT statistics can be interpreted using parametric Chi-Squa red distributions or a non-parametric Shimodaira-Hasegawa-like (SH) pr ocedure (Anisimova and Gascuel, 2006). The former seems to be more liberal (giving values similar to Bayesian posterior probabilities) and the later more conservative (Anisimova and Gascuel, 2006). Here, the most conservative approach was used and values represent the minimum of Chi-Squared and Shimodaira-Hasegawa (SH) support values. When all sp ecies are included (Figure 2-11) clades of interest have robust support (> 0.90) except for the vertebrate COX-2 clade (contai ning hCOX, 0.683) and the clade containing lCOX (0.87). When hCOX and lCOX ar e excluded from the analysis (Figure 2-12), support values generally increase, with a notable incr ease in the vertebrate C OX-2 clade (0.883). A Basesian phylogenetics (BP) analysis wa s also performed (Figure 2-13) using the program MrBayes 3.1.2, with one initial 2 million generation run, and one final 10 million generation run. The topology of the consensus tree (with burnin = 10%) is similar to those described above, with well-supporte d aforementioned vertebrate COX clades. However, in BP analysis lCOX groups at the base of the amphioxus COXc/COXd clade instead of being the basal member of the vertebrate COX clade. This grouping is well-supported by posterior probability 55

PAGE 56

scores (0.93). As in ML analysis, hCOX groups w ith vertebrate COX-2 prot eins at the base of the clade. Also similar to previous analys es, when lCOX and hCOX are excluded (Figure 2-14) scores improve and all clades have robust support. Due to the contrasting topologies genera ted using distance, ML, and BP methods, 21 different tree topologies (Figure 2-15) were generated by using th e most likely tree from ML analysis (Figure 2-9) and reloca ting hCOX and/or lCOX sequences to other branches of the tree. Positions of all other sequences were not altered from the most likely tree. A Ln likelihood score (Ln L ) was generated for each topology using onlin e execution of the program PhyML (Guindon et al ., 2005). Likelihood scores were compared to th e most likely tree. These tests indicate that lCOX and hCOX can be moved some what freely around the base of the tree without resulting in a noticeably different Ln L than the most likely topology (Table 2-9). It appears that moving lCOX to the base of the COX-1 clade causes the smallest change in Ln L whereas moving hCOX or lCOX deeply into COX-1 or COX-2 clades causes the largest changes in Ln L Discussion Here, I present the first forms of the en zyme cyclooxygenase (COX) sequenced from the hagfish, lamprey, and amphioxus. I also show that the killifish and sculpin conform to the current hypothesis that teleos ts posses additional COX-1 and COX-2 forms. The phylogenetic relationships among the various COX forms are stil l not definite, although the results presented here allow some conclusions to be drawn and pred ictions to be made. Specifically, it appears that COX sequences in the chordates form at least 3 well supported phylogenetic groups: the urochordata, cephalochordata, a nd vertebrata. Hagfish and lamprey positions are variable depending upon the analysis used, but likelihood scores seems to favor placement within the vertebrata clade. 56

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Novel COX Forms Although the two COX sequences from amphioxus were not able to be extended, they aligned readily with other COX se quences. They show a first haem-binding domain that shares little conservation with vertebrate forms (20%), as in the urochordates. However, the second haem-binding domain is completely conserved in th ese forms, as is histidine-207 which is crucial for peroxidase activity. The arginine-277 loop region does no t resemble COX-1 or COX-2 forms. Due to the truncated nature of these proteins, it is difficult to determine if COX in the cephalochordates plays similar roles as in the vertebrates, but some functions are likely conserved. Following the nomenclature used by Jarving et al (2004) these forms are named COXc and COXd. The COX sequence found in th e gills of the hagfish was extended to completion and readily aligned wi th other COX forms. It ha s conserved amino acids, binding domains, and bonds critical for COX function. Ther efore, it likely plays a similar role in the hagfish. The hagfish COX has an N-terminus deletion and valine-523 char acteristic of COX-2 forms, but there are multiple sites where it shares equal or no homology with COX-1 and COX-2 of vertebrates. Therefore, following the nomenclature of Yang et al (2002) this form is named hCOX. Although incomplete, the one COX sequen ce cloned from the gills of the lamprey readily aligns with other COX sequences and contains amino acids and regions critical to COX function. However, like the hagfish sequence it shares no biased homology with COX-1 or COX-2 forms. This form is therefore na med lCOX. As predicted based on phylogenetic position, COX-1a, COX-1b, and COX-2 were found in th e gills of the euryhaline killifish and the longhorn sculpin. With the exception of COX-1a in the sculpin, all COX forms from these teleosts contain conserved regions critical to COX function. As has been reported in other teleosts (Ishikawa et al ., 2007), these forms all contain va line at position 523, unlike in mammals. COX-1a from the sculpin does not have several amino acids critical for COX 57

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function located near the C and N terminus of the sequence. These include disruption of disulfide bonds near the N terminus due to cy stine replacements, little conserved homology in the membrane binding domain, and alternate residues for arginie-513 (leucine), valine-523 (leucine), and serine-530 (phenylalanine). This is not the case in the other acanthopterygiians, where all COX forms have these conserved regions. This may indicate th at in the sculpin COX1a has partially lost function, and COX-1b or COX-2 may therefore have additional responsibilities. Phylogenetic Analyses As has been seen when tracing the evolutionary histories of other gene families in evolutionary deep time (Abbasi and Grzeschik, 2007; Goudet et al ., 2007), the pedigree of the COX family is littered with duplications, losse s, and uncertainty. Despite utilizing a multifaceted approach to depict COX evolution in the chordates, the results indicate that COX has a complicated and under-examined history of evoluti on in the chordates. This is not surprising, considering the complex functions COX has been s hown to have in the chordates. Because of these complex functions, duplications, partitioning of roles, differential expression, and multiple forms with multiple functions are to be expected. In all phylogenetic analyses, the more recent history of COX evolution is more clear and consistent than the relationships of the ancestral taxa. The teleosts consistently form monophyletic COX clades nested within the gna thostomes, as expected. The gnathostomes consistently form two COX sister clades in phylogenetic analyses: COX-1 and COX-2. This is expected based on previous phylogenetic analyses in the vertebrates (Gu, 2001; Gu, 2006). The COX-1 clade is consistently rooted with the sequence from the dogfish (sCOX), although support values are not robust fo r this relationship when hagf ish and lamprey sequences are included. In distance analysis excluding hagfish and lamprey sequences, sCOX groups outside 58

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the COX-1 and COX-2 clades, representing anot her COX gnathostome lineage. These groupings are as expected, based on Yang et al .s (2002) original descripti on of sCOX as neither a COX-1 nor COX-2. These are the first phylogenetic analyses of COX to include sCOX and it is concluded that sCOX is most likely a ba sal member of the COX-1 clade. In all analyses, the COX-1 clade contains robustly supported, m onophyletic teleost and eutherian mammal clades. In the teleost cl ade, COX-1b and COX-1a form well-supported, monophyletic clades. Although this is expected based on initial studies (Ishikawa et al ., 2007; Ishikawa and Herachmann, 2007), this is the fi rst study to use sophisticated tree-building techniques to show the monophyly and legitimacy of the COX-1 forms in teleosts. It is also shown in each analysis that the non-duplicate COX-1 forms from the zebrafish and trout are more related to COX-1b forms from the acanthopte rygiians, grouping with them at the base of a monophyletic COX-1/COX-1b clade. The mammalian COX-1 group is not as robustl y supported using all t ypes of analyses. The expected relationships (based on assumed ev olutionary relationships in the tetrapods) are shown and supported with some confidence in th e maximum likelihood anal ysis (ML) when the hagfish and lamprey sequences are excluded. In this anlaysis, the tetr apods form a monophyletic group with the frog and chicken at the base of the clade, followed by the opossum sequences, and then the eutherians. Even in this analys is, the frog and chicken sequences group together, contrary to what is biologically expected (the frog should be more basal, representing an evolutionary ancient non-amniote lineage, and the chicke n should be a sister to the mammals). However, this grouping is not well-supported in any analysis. When hagfish and lamprey are included in ML analysis, the chicken/frog clade occurs at the base of the mammalian/teleost clade, contrary to biological predictions. Howeve r, this relationship is poorly supported. These 59

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low support values are confirmed in the aLRT anal ysis. In Bayesian a nd distance analyses, the frog is basal to the chicken, but these sequences are again at the ba se of a biologically irrelevant teleost/mammal clade. However, unlike dist ance and ML methods, Bayesian posterior probabilities strongly support this relationship. Bayesian posterior probabilities traditionally overestimate support values for a variety of reasons (Cummings et al ., 2003: Lewis, pers. comm..) and can give incorrect clades strong poste rior probabilities. This may explain why this biologically unexpected relationship is strongly supported only in the Bayesian trees. Therefore, ML analyses (supported by bootstrap and aLRT sc ores) show the most probable scenario for COX-1 evolution in the gnathostomes: a basa l sCOX, followed by a teleost COX-1 clade (as described above) sister to a tetrapod COX-1 clade. The COX-2 groups within the gnathostomes mi rrors the same general trends as the COX1 clade. Teleosts consistently form a well-supported, monophyletic group. The COX-2a sequences from zebrafish and trout group together to form the most basal members of this clade. The COX-2b sequences group with the non-duplicate COX-2 sequences from the acanthopterygiians to form the other teleost COX-2 clade. Sister to the teleosts, the tetrapods form another COX-2 clade in the gnathostomes. In this tetrapod COX-2 clade, frog and chicken sequences form the most basal (although not well-s upported) clade, with the mammals as a sister clade. This biologically rele vant relationship between COX-2 sequences is predicted in all analyses. Basal to the COX-1 and COX-2 clades in the gnathostomes are two clades that are consistently and robustly supporte d in all phylogenetic analyses: a urochordate COX clade and a cephalochordate COX clade. The cephalochorda te clade consists of the two COX sequences obtained in this study (COXc and COXd) and th e urochordate clade consists of the two 60

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sequences from Ciona (COXa and COXb). The urochordate COX forms always forms the base of the entire tree and the cepha lochordate COX forms always group as a sister clade to the gnathostome COX clade. This study is the first to show this biologically relevant relationship between COX forms in these ancestral chordates. The hagfish (hCOX) and lamprey (lCOX) sequences have alternate phylogenetic positions depending on the technique used to reconstruct their evolutionary histories. Also, when these sequences are removed from analys is, bootstrap, aLRT, and posterior probability scores supporting COX clades increase. Because of their uncertain placement in COX evolution, these sequences are designated as rogues a nd analyses excluding them likely show a more accurate representation of COX evolution. In distance analysis, both hCOX and lCOX are placed outside the gnathostome COX clade and form two separate COX lineages between the cephalochordate and gnathostome clades. In ML analysis, lCOX remains basal but hCOX is placed at the base of the gnathostome COX-2 clade. In BP analysis, hCOX remains in the COX2 clade but lCOX is placed within the cephaloch ordate clade. Support values for hCOX and lCOX placement are weak in all but BP analyses Clearly, the placement of hCOX and lCOX in COX evolution is not clear and without further evidence, placing them with any degree of certainty becomes difficult. In an attempt to find the most likely placement of these sequences, 21 different phylogenies were generated based on the mo st likely tree with hCOX and lCOX placed in variable positions. The trees were then evaluated in a maximum likelihood framework. It is important to note that although Ln L scores were generate d for each topology, other biological factors may influence which topology or type of topology is actually the most likely. For example, placing hCOX in the COX-2 clade and lCOX at the base of the tree (technically the most likely tree) implies that another COX lin eage exists for all vertebrates (the lineage 61

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represented by lCOX) because the hagfish is biol ogically ancestral to the lamprey. This would mean that dozens of novel COX forms have yet to be discovered in well-examined groups such as the mammals and teleosts. With genomes of se veral of these groups available, it is unlikely that such a vast amount of sequence data have yet to be found. Therefore, the most likely scenarios for COX evolution would imply that ei ther hagfish is basal and lamprey is derived, both are basal, or both are derived. However, placing lCOX, hCOX, or sCOX in the COX-1 or COX-2 clade implies that another COX form has yet to be found in these species. It appears that moving lCOX into the base of the COX-1 clade causes less of a change in Ln L scores than moving hCOX outside the gnathosto me COX clade. However, it is apparent that either sequence can be moved somewhat freely throughout the tr ee without resulting in noticeably worse (~10 Ln L ) tree scores. This leads to th e conclusion that although hCOX and lCOX may represent independent, basal lineages of COX, they are most likely basal members of the COX-2 and COX-1 clades (respectively) in the gna thostomes. If this is the case then at least one other form of COX should exist in the hagf ish and lamprey, as well as in the dogfish shark. Summary Presented here are the first COX sequences from amphioxus, the hagfish, and the lamprey. All predicted COX sequences from the killifish and sculpin are also described. These sequences all likely represent functional forms of COX found in the chordates based on conserved amino acids and domains critical to COX function. For the first time, a sophisticated phylogenetic analysis of COX forms from the majo rity of representative chordate lineages was attempted. Although still not definitive, a gene ral hypothesis for the evol utionary history of COX in the chordates can be made (Figure 2-16). In this scenario, there are three main COX groups corresponding to the three subphyla found in the phylum Chordata: the urochordata, cephalochordata, and vertebrata. Each contains two main COX groups, with the well62

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documented COX-1 and COX-2 forms only found in the vertebrata clade. These forms contain monophyletic groups for the teleosts and the tetr apods, with multiple forms in the teleosts confirming previous hypotheses (Ishikawa et al ., 2007; Ishikawa and Herschman, 2007). Hagfish and lamprey placement is uncertain, but most likely these sequences represent basal members of the vertebrata clade. This does not support the timing of geno mic duplications in the chordates according to the most recent view of the 2R Hypothesis (Kasahara, 2007), indicating two rounds of genome duplication af ter the origin of the urochordate s but prior to th e radiation of the jawed vertebrates. However, several developm entally important proteins have been shown to not support this timing and indicate an earlier duplication (Hughes, 1999). Multiple COX-1 and COX-2 genes in the teleosts do support the hypot hesis that the teleosts are characterized by another round of genome duplication after the origin of the vertebrates (Li et al ., 2007). Clearly, this uncertain period representing some 600 milli on years of evolution is characterized by many losses and duplications and remains to be resolved definitively. 63

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Table 2-1. Degenerate primers used in PCR Name Orientation Nucleotide sequence (5' to 3') CH COX F1 Sense ATG GAC GAC TAC CAG TGY GAY TGY AC CH COX F2 Sense GAT GTT TGC ATT TTT CGC TCA RCA YTT YAC CH COX F2.25 Sense TGG CG T GGA CCT AGG TCA NRT NTA YGG CH COX F2.5 Sense CGA GCT GCG GTT CCA TAA ARA YGG NAA RYT CH COX F3 Sense CCA CTA TA T GGC TGC GGG ARC AYA AYM G CH COX F5 Sense CAT GTG GAA TT C CAT CAC CTG TAY CAY TGG CA CH COX R1 Antisense CCC C GA AGG TGG ATG GYT TCC AVY A CH COX R2 Antisense GCA TCA GCG GGT GCC ART GRT A CH COX R3 Antisense TCC TCT TTT AGT ATG TCG CAG ACT CKR TTR TGY TC CH COX-1 F4 Sense TGC CAG ACA GCA TCC ACA THG AYG GNG A CH COX-1 F5 Sense CAG CAG ACA ATG CGC AGG NCA RAT HGG CH COX-1 R2 Antisense TGG GTG TA T GTT GTG TCC TCC NCC DAT YTG CH COX-1a F1 Sense CCC CAC C AA CCT ACA ATA CCA ART AYG GNT A CH COX-1a F2 Sense AAA GTG CTG ACT GAR MGN TTY TT CH COX-1a F3 Sense GGA CTA ATC TGA TGT TCG CGT TYA TGG CNC A CH COX-1a F4 Sense AGC GGA AGG CGG CTT YAC NAA NGC CH COX-1a F5 Sense CAA ATA GTA AAT GGG GAR AYN TAY CC CH COX-1a R1 Antisense ATA GGG AAT GTC GTC NCC RTC DAT CH COX-1a R2 Antisense GGG GCA CGT TTT CCG GRT ANA YCA T CH COX-1a R3 Antisense ACC TCA GTG ACA GTG GGA GGR TAN RTY TC CH COX-1b F1 Sense CGG AT G GGT GTG GGC TTY ACN AAR GC CH COX-1b R0 Antisense GGA GA A GGG AGC ACC CAT YTC NAC CAT CH COX-1b R3 Antisense CCA GCG TCC GCG CDA TYT CYT C 64

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Table 2-2. Amphioxus COX specific primers used in PCR Name Orientation Nucleo tide sequence (5 to 3) COXa F1 Sense TAC TCC ACC GTG TGG CTG C COXa F2 Sense TGG GAC GAC GAG AGG CTC COXa F2.5 Sense ACC GGT GAG ACC ATC AAC ATC AT COXa F3 Sense CAC CTG AGC GGC TAC AAC TTT COXa F4 Sense GAC CTG TTC TGG GAC CCT GAG COXa F5 Sense CCA GTA CCA GAA CCG CAT CTT C COXa R1 Antisense CGT ACG GCG GGT AGA TCA TG COXa R2 Antisense GCT GCC GCT CCA CGG COXa R3 Antisense CCC GT A GAT ATG GCT CAG GTC A COXa 3 F1 Sense GAG GCT CTT CCA GAC AGC CA COXa 3 F2 Sense CAC CTG AGC GGC TAC AAC TTT COXa 5 R1 Antisense T GG CTG TCT GGA AGA GCC TC COXa 5 R2 Antisense TGG AGT AGA CGA ACA GGC CC COXa 5 R3 Antisense CCC CGT AGA TAT GGC TCA GGT COXb F0 Sense CAG ACA GCT AGA CTC ATT CTT ATC AGT GA COXb F1 Sense TAT CAA CAT CGT CAT TGG AGA GTA TG COXb F1.5 Sense GGC TGG CAA AAA CTT CCA ACT COXb F2 Sense TGC AGT ACC AGA ACA GCA TAT TTG COXb F3 Sense AGC ATA TTT GTG GAG TTT AAC CAC TTG COXb R1 Antisense CGT CCG GAG GGT AAA TCA TGT COXb R2 Antisense ACT TCA GTT TCC CGT CCT GGA COXb R3 Antisense CGT A AA CAT GGC TCA TGT CCA CT COXb 3 F1 Sense GTC CCC GAG AAG AAG CGA TT COXb 3 F2 Sense GAA CG G CTC TAC CAG ACA GCT AG COXb 3 F3 Sense TGT TCT GGG ACC CTG AGC TG COXb 5 R1 Antisense TGG TAG AGC CGT TCG TCG TC COXb 5 R2 Antisense GGA GGG TAA ATC ATG TGT ACG GA COXb 5 R3 Antisense ACA TGG CTC ATG TCC ACT GC 65

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Table 2-3. Hagfish COX speci fic primers used in PCR Name Orientation Nucleo tide sequence (5 to 3) hCOX 3 F1 Sense TTA ACC ACC TGT ACC ACT GGC AC hCOX 3 F2 Sense CTC AAC CAT GGA GTA CGA GGT CT hCOX 3 F3 Sense CAA TGC TAT GGA GTT CTA CCT GGG hCOX 3 F0.8 Sense CAG TAC AGC AAC CGC ATC TCA hCOX 3 F0.6 Sense AAT GGG ACG ACG AGA GAA TCT TT hCOX 3 F0.4 Sense GGT TTG CTG TTG GTC ACG AAG hCOX 3 F0.2 Sense AGT CCA CCC ACA TAC AAT GCC hCOX 3 F0.1 Sense CTC ACG CAC TTT GCT CCC TT hCOX 3 F0 Sense GAA CTG CAC CTA CCC CGA GAC hCOX mid F1 Sense GTT TCG AGT CCA CCC ACA TAC AA hCOX mid F2 Sense CAT ACT CCA ACC TCA GCT ACT TCA CT hCOX mid R1 Antisense CAC ATC GAG TAC GTC ACA CAC G hCOX mid R2 Antisense AAG GGT CCC GTA GAG CAT GA hCOX 5 R1 Antisense TCT CCG AAT AAA GCT CCC TGT C hCOX 5 R2 Antisense TGC TGC CAT TTC CGT TTC TC hCOX 5 R3 Antisense CAT GTT TGA TGG ATG CTG TTG C hCOX 5 R4 Antisense AGG CG A GTG AAG TAG CTG AGG TT hCOX 5 R5 Antisense CCT CCC AGG ACT TGT ACC GTA AT hCOX 5 R6 Antisense GAG GA A GTA ATG CAC GGT ATC TGG 66

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Table 2-4. Lamprey COX speci fic primers used in PCR Name Orientation Nucleo tide sequence (5 to 3) lCOX F1 Sense GAG GAA CGC TTG AGA GAC AAC A lCOX R1 Antisense TCT TG T TGA TGT TT C GAC CGC lCOX R2 Antisense CCA CCG CCA ATC TGG C lCOX R3 Antisense AAT CAG GAA GCT CTC AGG CAT C lCOX 3 F1 Sense GAG TTC CTC TTC AAC CCC GG lCOX 3 F2 Sense TCG AAA CAT CAA CAA GAA CCT CC lCOX 3 F3 Sense TTC ACC TCC TTC CTG GAG CTC lCOX 5 R1 Antisense TGC TGC ACG TAC TCC TCG ATC lCOX 5 R2 Antisense GCA GCT GGT GTT GTC TCT CAA 67

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Table 2-5. Killifish COX specific primers used in PCR Name Orientation Nucleo tide sequence (5 to 3) Fh COX-1b 3 F1 Sense CTT CAC CGA TAG CGA GGA GAT AG Fh COX-1b 3 F2 Sense AGC TCT ACG GTG ACA TCG ACA CT Fh COX-1b 3 F3 Sense GGG TAA CCC CAT ATG TTC TCC AC Fh COX-1b 5 R1 Antisense CCC ATG TGA GAG AGC CTT AGT GA Fh COX-1b 5 R2 Antisense C AG CCT CTC AAA CAA CAC CTG AG Fh COX-1b 5 R3 Antisense GT A GTA GGA TTC CCA GCT GAG GTA GT Fh COX-1a 3 F1 Sens e GGT GTA CCC CGA AGG TTT CC Fh COX-1a 3 F2 Sense GAG TAC GTG CAG CAC CTG AGC Fh COX-1a 3 F3 Sense AGT ACA CCA ATC GCA TCG CC Fh COX-1a 5 R1 Antisense GAT GAT AAG TCT GGC GGT CTG G Fh COX-1a 5 R2 Antisense GAT GGT GGC ATA CAC GGT GA Fh COX-1a 5 R3 Antisense TTT ATG AAG CCG AAG CTG GTG 68

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Table 2-6. Sculpin COX speci fic primers used in PCR Name Orientation Nucleo tide sequence (5 to 3) Mo COX-2 3 F1 Sense CCC CGG TCT GAT GAT GTA CG Mo COX-2 3 F2 Sense ACC GAG TGT GTG ACG TGT TGA Mo COX-2 3 F3 Sense TC G AGG ACT ACG TGC AGC AC Mo COX-2 3 F4 Sense CAG GTC TAA CGC AAT CTT TGG G Mo COX-2 3 F5 Sense CCT CAA GGG CTT AAT GGG AAA C Mo COX-2 3 F6 Sense AAC ATC GTC AAC ACC GCC TC Mo COX-2 mid F1 Sense AC A GTT CAT TCC GGA TCC ACA Mo COX-2 mid F2 Sense CA C CAG CCT GAT GTT TGC ATT Mo COX-2 mid R1 Antisense CGA AGC GGT GAG ATT CAG GA Mo COX-2 mid R2 Antisense CTC CAT CCA GGA CCT GAT ATT TAA G Mo COX-2 5 R1 Antisense TCC GGA ATG AAC TGT CTT CTC A Mo COX-2 5 R2 Antisense AGG TCG GAG GAC TAT CAA TCA AGT Mo COX-2 5 R3 Antisense GAA GGA GAT GGA GTT AAT GAT GTT CC Mo COX-1a 3 F1 Sense CA T CAC CTC CGG CTT CAT AAA Mo COX-1a 3 F2 Sense GAG GGA GCA TAA CAG ACT CTG TGA Mo COX-1a 3 F3 Sense CTG GAG TTC TGC CAC CTC TAC C Mo COX-1a 3 F4 Sense GC A GCC CTT CAA TGA ATA CAG G Mo COX-1a 3 F5 Sense CAT GCT GGA GAA GAC CCT TCC Mo COX-1a 3 F6 Sense GAG TAT GTT GGA GAT GGG TGC TC Mo COX-1a 5 R1 Antisense GCC GAA ATG TCT TCC TCC TAA A Mo COX-1a 5 R2 Antisense CAT CGG TAA AGG GCA GTC CTC Mo COX-1a 5 R3 Antisense TGA CCG TCA GCA CTA ATC TCA TG Mo COX-1b 3 F1 Sense TCA CAC ACT ACG GCA TCG AGA Mo COX-1b 3 F2 Sense TTG GCG GTG GCT TTA ACA TC Mo COX-1b 3 F3 Sens e CAG CCC TTC AAC GAG TAC AGG Mo COX-1b 5 R1 Antisense GAG CGA TAC GGT TCC CAT ACT G Mo COX-1b 5 R2 Antisense GTT GTG CTC CCT GAG CCA GA Mo COX-1b 5 R3 Antisense AAT AGC CAT CTG AGC CTC AGG A 69

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Table 2-7. Sequences included in phylogenetic analyses # Name Organism Size (AA) 1 COX1a Fundulus Fundulus heteroclitus (killifish) 452 2 COX1b Fundulus Fundulus heteroclitus (killifish) 598 3 COX2 Fundulus Fundulus heteroclitus (killifish) 610 4 COX1a Myoxocephalus Myoxocephalus octodecimspinosus (lh. Sculpin) 622 5 COX1b Myoxocephalus Myoxocephalus octodecimspinosus (lh. Sculpin) 600 6 COX2 Myoxocephalus Myoxocephalus octodecimspinosus (lh. Sculpin) 605 7 COXa Ciona Ciona intestinalis (sea squirt) 653 8 COXb Ciona Ciona intestinalis (sea squirt) 600 9 COXc Branchiostoma Branchiostoma lancelolatum (amphioxus) 177 10 COXd Branchiostoma Branchiostoma lancelolatum (amphioxus) 177 11 hCOX Myxine Myxine glutinosa (Atlantic Hagfish) 610 12 lCOX Petromyzon Petromyzon marinus (Sea Lamprey) 286 13 COX1a Oryzias Oryzias latipes (Japanese medaka) 605 14 COX1b Oryzias Oryzias latipes (Japanese medaka) 606 15 COX2 Oryzias Oryzias latipes (Japanese medaka) 609 16 COX1a Gasterosteus Gasterosteus aculeatus (stickleback) 598 17 COX1b Gasterosteus Gasterosteus aculeatus (stickleback) 598 18 COX2 Gasterosteus Gasterosteus aculeatus (stickleback) 620 19 COX1a Tetraodon Tetraodon nigroviridis (Green Spotted Puffer) 1023 20 COX1b Tetraodon Tetraodon nigroviridis (Green Spotted Puffer) 589 21 COX2 Tetraodon Tetraodon nigroviridis (Green Spotted Puffer) 456 22 COX1 Oncorhynchus Oncorhynchus mykiss (rainbow trout) 624 23 COX2a Oncorhynchus Oncorhynchus mykiss (rainbow trout) 607 24 COX2b Oncorhynchus Oncorhynchus mykiss (rainbow trout) 609 25 COX1 Danio Danio rerio (zebrafish) 597 26 COX2a Danio Danio rerio (zebrafish) 601 27 COX2b Danio Danio rerio (zebrafish) 606 28 COX1 Monodelphis Monodelphis domestica (gr. S.t. opossum) 625 29 COX1b Monodelphis Monodelphis domestica (gr. S.t. opossum) 627 30 COX1b2 Monodelphis Monodelphis domestica (gr. S.t. opossum) 729 31 COX2 Monodelphis Monodelphis domestica (gr. S.t. opossum) 608 32 COX1 Gallus Gallus gallus (rd. jungle fowl) 649 33 COX2 Gallus Gallus gallus (rd. jungle fowl) 603 34 COX1 Bos Bos taurus (cattle) 600 35 COX2 Bos Bos taurus (cattle) 604 36 COX1 Xenopus l Xenopus laevis (Afr. Claw. Frog) 587 37 COX2 Xenopus l Xenopus laevis (Afr. Claw. Frog) 604 38 COX1 Canis Canis lupus familiaris (dog) 633 39 COX2 Canis Canis lupus familiaris (dog) 604 40 COX1 Homo Homo sapiens (human) 599 41 COX2 Homo Homo sapiens (human) 604 42 COX1 Rattus Rattus norvegicus (norway rat) 602 43 COX2 Rattus Rattus norvegicus (norway rat) 604 44 COX1 Mus Mus musculus (house mouse) 602 70

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Table 2-7. Continued. # Name Organism Size (AA) 45 COX2 Mus Mus musculus (house mouse) 604 46 COX1 Oryctolagus Oryctolagus cuniculus (rabbit) 606 47 COX2 Oryctolagus Oryctolagus cuniculus (rabbit) 604 48 COX1 Ovis Ovis aries (sheep) 600 49 COX2 Ovis Ovis aries (sheep) 603 50 sCOX Squalus Squalus acanthias (spiny dogfish) 593 71

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Table 2-8. LRTs between amino acid based models of COX evolution Evolutionary Model* Ln liklihood (Ln L ) Score (Ln L1LnL2) d.f.@ P# WAG -26632 WAG + I -26315 317 1 < 0.01 WAG + -25294 1021 1 < 0.01 WAG + I + -25294 0 2 > 0.05 *Evolutionary models are arranged from the most simple to the most complex. Only models using the WAG matrix were evaluated. I repr esents an invariable sites parameter and represents an among site variation parameter @ d.f. indicates the difference in free paramete rs between the complex and simple models #P values represent the comparison of simple to complex models and were obtained using a chisquared distribution test 72

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Table 2-9. LRTs between tree topologies with altered positions of hCOX and lCOX Tree Topology Name* Ln liklihood (Ln L ) Score Estimated @ (Ln L1LnL2)# COX2 Hag (most likely) -25294.22 0.737 COX1Lamp/COX2Hag -25295.57 0.737 2.70 COX2 Agnatha -25299.09 0.737 9.74 Lamp/Amp -25299.27 0.737 10.1 COX2 Vert -25299.61 0.736 10.7 COX1 Hag -25300.92 0.735 13.4 COX1 Vert -25301.22 0.735 14.0 Ancestral Hag -25301.56 0.735 14.6 COX1 Agnatha -25301.78 0.735 15.1 COX1Hag/COX2Lamp -25302.19 0.735 15.9 Ancestral agnatha -25302.55 0.735 16.7 COX1 Lamp -25303.25 0.735 18.1 COX2 Lamp -25303.85 0.735 19.3 Agnatha/Amp -25308.82 0.735 29.2 Hag/Amp -25308.90 0.735 29.4 Hag Internal -25321.15 0.737 53.9 Lamp Internal -25322.77 0.736 57.1 Lamp Mammal -25341.41 0.732 94.4 Lamp Teleost -25344.68 0.735 101 Hag Teleost -25350.30 0.734 112 Hag mammal -25408.58 0.731 229 *Each tree has the same topology as the most likely tree (Figure 2-9) but with alternative placements of hagfish (hCOX) and lamprey (l COX) sequences as noted in Figure 2-15. @ represents the shape value under the model #Where Ln1 is the most likely tree score and Ln2 is the tree score of the topology in question 73

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COX-1b MRASVLGSVCALLVLLREPGCQGDEVTTSTVNPCCYLPCKHWSVCVRYGEDKYECDC 57 COX-2 MNRITFAVFLLALC--------FSFHKEVLGNACCSEPCQNRGVCTAMGSDSYECDC 49 *N68 COX-1b THTGYYGENCSIPELWTRVRQFLKPSPDVVHYILTHFHWLWDIINN-TFLRNVLMRL 113 COX-2 TRTGYRGQNCTTPEFLTWIKISLKPTPNTVHYILTHFKGFWNIINNISFLRDAIMKY 106 *R120 *N144 COX-1a NLSYYTRLLPPVPKDCPLPMGTKGKPVLP 29 COX-1b VITARSNLIPSPPTFNSKYNYLSWESYYNLSYYTRILPPVPEDCPTPLGVKGRNGLP 170 COX-2 VLTSRSHMIDSPPTYNADYGYKSWEAYSNLSYYTRALPPVPEDCPTPMGVVGKKELP 163 Q203/H207 COX-1a DIKEISERYFKRTEFRPDPQGTNLMFAFMAQHFTHQFFKTSHKVDAGFTKALGHGVD 86 COX-1b DPQVLFERLLKRRTFRPDPQGSNIMFAFFAQHFTHQFFKTYNRMGLGFTKALSHGVD 227 COX-2 DVKVLAEKLLVRRRFIPDPQGTSLMFAFFAQHFTHQFFKSDMKNGPAFTVAKGHGVD 220 *R277 COX-1a ASNIYGEELERQHQLRLHKDGKLKYQLINGEMYPPTVSEVPVHMVYPEGFPAEQRLA 143 COX-1b AGHIYGDSLERQHLLRLFRDGKLKYQLIDGEVYPPSVTDAPVRMSYPPGIPVEKQMA 284 COX-2 LGHIYGENLEKQHKLRLFKDGKLKYTMVNGEVYPPLVKDVGVEMHYPPHVPDSQRFA 277 COX-1a IGQEVFGLVPGLTVYATIWLREHNRVCDILKGEHPTWDDEQLFQTARLIIIGEIINI 200 COX-1b IGQEVFGLLPGLSLYATLWLREHNRVCDILKAEHPTWDDEQLFQTTRLIIIGETIRI 341 COX-2 VGHEAFGLVPGLLMYATIWLREHNRVCDVLKGVHPDWDDERLFQTTRLILIGETIKI 334 Y385/H388 COX-1a IIEEYVQHLSGYYLKLKYDPSLLFGVRFQYTNRIALEFCYLYHWHPLMPDSFLIDGD 257 COX-1b VIEEYVQHLSGYLLQLKFDPTLLFNSNFQYGNRIALEFSQLYHWHPLMPDSFHISGD 398 COX-2 VIEDYVQHLSGYHFKLKFDPELLFNQRFQYQNRIASEFNTLYHWHPLMPDSFHIEEK 391 *N410 COX-1a ELPYSQFLYNTSILMHYGVEKLVDAFSRQPAGQIGGGRNIHQAVLRVAEMVIRDSRA 314 COX-1b ELSYSQFLFNTSVLTHYGVEKLVDAFSRQAAGQIGGGHNINAVITKVIVGTIEESRQ 455 COX-2 DYSYKEFVFNTSVVTEHGINNLVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRK 448 *R513 COX-1a ARLQPFNQYRKRFNLKPYSSFYELTGDEEMARGLEELYGDIDALEFYPGLLLEKTRP 371 COX-1b LRIQPFNEYRKRFNLEPYTSFRDFTDSEEIASTLEELYGDIDTLEFYPGLLLEKTRP 512 COX-2 MRYQSLNAYRKRFSMKPYTSFEDLTGEKEMAAILEELYGDVDAVELYPGLLVEKPRT 505 *V523 *S530 COX-1a SSIFGESMVEMGAPFSLKGLLGNPINSPEYWKPSTFGGETGFNIIKTSTLKKLVCLN 428 COX-1b GAIFGESMVEMGAPFSLKGLLGNPICSPQYWKPSTFGGKVGFDIVNSASLKKLVCLN 569 COX-2 NAIFGETMVEMGAPFSLKGLMGNPICSPEYWKPSTFGGTAGFDIVNTASLQRLVCNN 562 Figure 2-1. Amino acid alignment of the form s of COX sequenced from the gills of the euryhaline killifish (Fundulus heteroclitus ). The COX-2 sequence is from GenBank (Accession #AAS21313) and the others were obta ined in this study. Amino acids that share at least 60% identity across sequences are highlighted. *I ndicates amino acids (numbered based on ovine COX-1) involved in COX function: active site of COX (tyrosine-385, histidine-388, and serine-530) substrate binding site (arginine-120), N glycosylation sites (asparagine-68, 144, and 410), sites for peroxidase activity (glutamine-203 and histidine-207), and the two sites which define conformational differences in channels between COX-1 and COX-2 in mammals (arginine-513 and valine-523) (Ishikawa et al ., 2007). The black boxes indi cate the two domains that define the haem-binding sites and the da shed box indicates the membrane binding domain (Kulmacz et al ., 2003; Ishikawa et al ., 2007). The solid lines represent disulfide bonds, with the beginning and end of the l ongest bond represented by arrows (Kulmacz et al ., 2003). The dashed line represents the arginine-277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003). The numbers to the right of the sequences indicate the amino acid number of each sequence. 74

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COX-1a TK-WCPYVDFHVPRNEEERKPS-----------------------TEL 452 COX-1b SR-TCPYVAFSVPAEEEPGRNDGKERS------------------SEL 598 COX-2 VRGPCPVASFYVPDVKETGSMTINSSTSHSRDSNINPTVILKERTSEL 610 Figure 2-1. Continued. 75

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COX-1a MKRANLSNYFLETNHCEQNISDNYHFITLITIL-SRES--KSLASFPSGLTFCCWGA 54 COX-1b MRSPVLGPVCALLLL-LREPAC------------------RGDEVTSDTVNPCCY-36 COX-2 MYRFTFA-VF---LLALGVLVC-------------------------EGGNPCC--25 *N68 COX-1a HVLLTQVLIPVVITLVRTQECVCDSVQIT-TNVTALALASMETTALSRSSGPEFVRS 110 COX-1b --FPCQHWGVCVRYGEDKYECDCTRTGYTGGNCTVPEFWSRVHQFL-KPS-PEVLHY 89 COX-2 -SEPCQNRGVCTALGTDNYECDCTRTGYHGHNCTTPEFLTWVKISL-KPS-PNTVHY 79 *R120 COX-1a RTRRSSSSSPTSIGCWHLVNN-SFLRGTVMRLVLTVRSDLIPSPPTYNTKYGYLNWE 166 COX-1b ILTHFN-------WLWDIINH-TFLRDVLMRMVLTVRSNLIPSPPTYNSKYDYLSWE 138 COX-2 ILTHFK-------GFWNIINSISFFRDAIMRYVLTSRSHLIDSPPTFNADYGYKSWE 129 *N144 COX-1a SYYNISYYTRLLPPVPEDCPLPMGTKGRPDLPDPKVLTERFFRRKTFRPDPQGANLM 223 COX-1b SYYNLSYYTRILPPVPKDCPTPLGVKGKAGLPDPELLVERLLKRRTFRPDPQGSNLM 195 COX-2 AYSNLSYYTRTLPPVPEDCPTPMGVVGKKELPDAKLLAEKLFMRRQFIPDPQGTSLM 186 *Q203/H207 COX-1a FAFMAQHFTHQFFKTDHELQGGFTKALGHGVDAGNIYGDNLAKQHHLRLHKDGKLKY 280 COX-1b FAFFAQHFTHQFFKTYNRMGVGFTKALAHGVDAGHVYGDNLQRQLKLRLHKDGKLKY 252 COX-2 FAFFAQHFTHQFFKSDMKKGPAFTLATGHGVDLNHVYGGSMERQHKLRLRQDGKLKY 243 *R277 COX-1a QIVNGETYPPTTSEAPVHMMYPEDVPPEKRLAIGQEVFGLLPGLTMYATIWLREHNR 337 COX-1b QLVDGQIYPPSVVDAPVKMSYPPGVPPEAQMAICQEVYGLLPGLGMFATLWLREHNR 309 COX-2 QVLDGEVYPPTVKEVGADMHYPPHVPESHRFAVGHEAFGLVPGLMMYATIWLREHNR 300 COX-1a LCDILKAEHPTWDDEQLFQTTRLIVIGEIINIIIEEYVQQLSGYQLKLKFDPTLLFN 394 COX-1b VCDILKAEHPTWDDEQLFQTARFVIIGETIKIVIEEYVQQLSGYLLQLKFDPALLFN 366 COX-2 VCDVLKEVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYNFKLKFDPELLFN 357 *Y385/H388 *N410 COX-1a ERFQYSNRIALEFCHLYHWHSLMPDSFLIDGDDIPYSQFFYNTSILMHYGVEKLVDA 451 COX-1b SNFQYGNRIALEFSQLYHWHPLMPESFLINGDELPYKRFLFNNTVLTHYGIENLVTA 423 COX-2 QRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHGIGNLVES 414 Figure 2-2. Amino acid alignment of the three forms of COX sequenced from the gills of the longhorn sculpin ( Myoxocephalus octodecemspinosus ): COX-1a, COX-1b, and COX2. All sequences were obtained during this study. Amino acids that share at least 60% identity across sequences are highlighted. *Indicates amino acids (numbered based on ovine COX-1) involved in COX func tion: active site of COX (tyrosine-385, histidine-388, and serine-530), subs trate binding site (arginine-120), N -glycosylation sites (asparagine-68, 144, and 410), sites fo r peroxidase activity (glutamine-203 and histidine-207), and the two sites which defi ne conformational differences in channels between COX-1 and COX-2 in mammal s (arginine-513 and valine-523) (Yang et al ., 2002; Ishikawa et al ., 2007). The black boxes indicate the two domains that define the haem-binding sites and the dashed box indicates the membra ne binding domain (Kulmacz et al ., 2003; Ishikawa et al ., 2007). The solid lines represent disulfide bonds, with the beginning and end of the longest bond represented by arrows (Kulmacz et al ., 2003). The dashed line repres ents the arginine-277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003; Kulmacz et al ., 2003). The numbers to the right of the sequences indicate the amino acid number of each sequence. 76

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COX-1a FSHQPAGQIGGGHNSHAVVLKVAEMVIRESRETRVQPFNEYRKKFNLQPYTSFYDLT 508 COX-1b FSRQVAGQIGGGFNINAAVTKVSVLTIKESRKLRMQPFNEYRKRFNLKPYTSFREFT 480 COX-2 FTNQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFSMKPYSSFEDMT 471 *R513 *V523 *S530 COX-1a GDIEMAKGLEELYGDIDAVEFYPGLMLEKTLPTRIFGESMLEMGAPLFPERPVGKPH 565 COX-1b DNEEIARELEEFYGDVDALEFYPGLLLERTREGSIFGESMVEMGAPFSLKGLLGNPI 537 COX-2 GEKEMAAILEEFYGHVDAVELYPGLLVEKPRSNAIFGETMVEMGAPFSLKGLMGNPI 528 COX-1a LLPV-LEAQHLWGRDGFQYRNFHSEETGVPQHQVVSIRGL-PCSPKGRRNQTNKSI620 COX-1b CSPVYWKPSTFGGKVGF---DIVNSAT---LKKLVCLNTR-TCSYVAFRVPTEEQLK 587 COX-2 CSPEYWKPSTFGGSEGF---NIVNTAS---LQRLVCNNVQGPCPVASFSGPDVKDSG 579 COX-1a -----------------------YTL 622 COX-1b TGNDDSKTRT-------------DEL 600 COX-2 SMIINSSTSNSDINPTVILKERTTEL 605 Figure 2-2. Continued. 77

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sCOX MEAARIILLLLPLCFLKMADTTAINPCCYYPCQNKGICVNVGKEGYECDCTRT 53 hCOX MTSEVFVVLCVAVVFAGAA--AADDPCCGSPCENKGVCVSVGFEDYECDCTRT 51 *N68 sCOX GYYGVNCTFPFSWSRVHKFLKPSPSSMHHVLTHYKWLWYIINNISFFSDTLMR 106 hCOX GYYGSNCTYPETWTWIVNMLKPLPDTVHYFLTHFAPFWSLVNRAAFLRDRVMR 104 *R120 *N144 sCOX LVLTVRANIIPSPPTYNSDYTYVSWEGYSNISYLTRLLPPVPKDCPTPTGTQG 159 hCOX HVLMSRAHMVSSPPTYNADYGYKSWEAYSNLSYFTRLLPPVPQGCPTPMGRTG 157 *Q203/H207 sCOX YKKLPDSEQLAEEFLLRRKFIPDPQGSNLMFAFFAQHFTHQFFRTDLDRGPGF 212 lCOX HQFFNTDPVRGPAF 14 hCOX KKELPDAQKLAERFLLRRTFIPDPQGSNLLFAFFAQHFTHQFFKTDFKRGPGF 210 COXc HQFFKTDFKKGAGR 14 COXd HQFFKTDFKKGPGR 14 sCOX TKALGHGVDLTHIYGDSLERQHHLRLFKDGKLKYQVVNGEVFPPSVKEAPIQM 265 lCOX TRALGHGVDLNHIYGGTLERQHQLRLFKDGKLKFQMIDGEAYPPVVRDAPVHM 67 hCOX TKALGHGVDLSHIYGDTLDKQHKLRLHNNGKLKFQMIDGEVFPPLVSEAPVDM 263 COXc TW-GDHGVDLSHIYGETVERQHQLRSFTDGKLKFQRVEGEVYPPSLADAPVHM 66 COXd TW-SDHAVDMSHVYGETVERQRQLRSFQDGKLKYQLVDGEAFPPSLQDASVHM 66 *R277 sCOX KYPSTLPEEKRLAIGHDTFGLIPGLMMYATIWLREHNRVCDILKEEHPVWSDE 318 lCOX VYPEHVPASLRFAVGHEVYGLLPGLLVYATVWLREHNRVCDVLHARHPRWDDE 120 hCOX IYPPHVPEAARFAVGHEAFGLVPGLMLYGTLWLREHNRVCDVLDVDHPEWDDE 316 COXc IYPPYVPEGKRFAIGHEFFGLLPGLFVYSTVWLREHNRVCDVMKELHPDWDDE 119 COXd IYPPDVPEKKRFALGHEFFGLLPGLFVWATVWLREHNRVCDVMKDLHPDWDDE 119 sCOX QLFQTTRLILIGETIKIVIEDYVQHLSGYNFRMMFNPELLFTEHFQYSNRIAV 371 lCOX RLFQTARLILTGETMKIVIEEYVQHLSGYNFHLKFDPTLLFGVNFQYSNRMSL 173 hCOX RIFQTARLILIGETIKIVIEDYVQHLSGYHFKLKFDPKLLFGQPFQYSNRISV 369 COXc RLFQTARLILTGETINIIINEYVQHLSGYNFDLFWDPELLFSDQFQYQNRIFV 172 COXd RLYQTARLILISETINIVIGEYVQHLAGKNFQLFWDPELLFEEQXQYQNSIFV 172 Figure 2-3. Amino acid alignment of the forms of COX sequenced from the gills of the lamprey ( Petromyzon marinus ), hagfish ( Myxine glutinosa ), and amphioxus ( Branchiostoma lancelolatum ), along with sCOX from the dogfis h (Squalus acanthias, Accession #AAL37727). Amino acids that share at least 60% identity across sequences are highlighted. *Indicates amino acids (num bered based on ovine COX-1) involved in COX function: active site of COX (tyrosine-385, his tidine-388, and serine-530), substrate binding site (arginine-120), N -glycosylation sites (a sparagine-68, 144, and 410), sites for peroxidase activity (gluta mine-203 and histidine-207), and the two sites which define conformational differen ces in channels between COX-1 and COX2 in mammals (arginine-513 and valine-523) (Yang et al ., 2002; Ishikawa et al ., 2007). The black boxes indicate the two domains that define the haem-binding sites and the dashed box indicates the membrane binding domain (Kulmacz et al ., 2003; Ishikawa et al ., 2007). The solid lines represent disulfide bo nds, with the beginning and end of the longest bond re presented by arrows (Kulmacz et al ., 2003). The dashed line represents the arginine-277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003; Kulmacz et al ., 2003). The numbers to the right of the seque nces indicate the am ino acid number of each sequence. 78

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* *Y385/H388 *N410 sCOX EFDHLYHWHPLMPDSFIVKGQDFSYKDFLFNTDILLNLGVDALVESFSKQIAG 424 lCOX EFNHLYHWHPLMPDSLLIDGRNYSYDEFLFNPGLLADKKLMPLVRSFMRQRAG 226 hCOX EFNHLYHWHGLNPDAFRVGTQEYQYSQFLFNNTILLNHGVRGLLEAFNVQQAG 422 COXc EFNHL 177 COXd EFNHL 177 sCOX RIGGGRNIHQSLLHIAIATIEHGRLLRFQPYNEYRKKLGLTPYKSFQELTGER 477 lCOX TVSGGRNINKNLLHVATSIIEHGRTLRLQSLNQYRHRFNMRPFTSFLELTGDE 279 hCOX RIGGGQNIHGALLHVATASIKHGRKMRFQSLNQYRKQFGLQPYQSFEQLTGET 475 *V523 *S530 sCOX EVAARLEKLYGHIDAMEFYPALLLEAPNKNSIFGESMVEMGAPFSLKGLMGNP 530 lCOX AMAAEME 286 hCOX EMAADLAELYSDINAMEFYLGLMVEKPRQGALFGETMVEAGAPFSLKGLMGNA 528 sCOX ICSPDYWKPSTFGGKTGFDIVNTATFEKLICLNVK-KCPYVGFHVPY-----582 hCOX ICSPEYWKPSTFGGNRGFEIVNSASLRRLVCLNLQGPCPDVAFHVPRDNQQDV 581 sCOX NVDNDYEREKGKP------------STEL 593 hCOX VVNVTGSQGGSDGVTTTPHYVADQQSREL 610 Figure 2-3. Continued. 79

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*H207 COX1 Mus HQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLFKDGKLKYQVLDG 261 COX1 G.gal HQFFKTSGKMGRGFTKALGHGVDLGHLYGDNLQRQHQLRLFQDGKLKFQVVNG 304 COX1 Danio HQFFKTHNRVGLGFTKGLGHGVDAGHIYGDSLDRQLELRLHKDGKLKYQVLNG 259 lCOX HQFFNTDPVRGPAFTRALGHGVDLNHIYGGTLERQHQLRLFKDGKLKFQMIDG 53 COX2 Mus HQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKYQVIGG 245 COX2 G.gal HQFFKTDHKKGPGFTKAYGHGVDLNHIYGETLERQLKLRLRKDGKLKYQMIDG 245 COX2 F.het HQFFKSDMKNGPAFTVAKGHGVDLGHIYGENLEKQHKLRLFKDGKLKYTMVNG 250 *R277 COX1 Mus EVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTIWLREHNRV 314 COX1 G.gal EVYPPSVTEVPVHMVYPPAIPKEKQLAMGQEVFGLLPGLCMYATLWLREHNRV 357 COX1 Danio DIYPPTVLHAQVKMSYPPSVPPEQQLAIGQEVFGLLPGLGMYATLWLREHNRV 312 lCOX EAYPPVVRDAPVHMVYPEHVPASLRFAVGHEVYGLLPGLLVYATVWLREHNRV 106 COX2 Mus EVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHNRV 298 COX2 G.gal EMYPPTVKDTQAEMIYPPHVPEHLQFSVGQEVFGLVPGLMMYATIWLREHNRV 298 COX2 F.het EVYPPLVKDVGVEMHYPPHVPDSQRFAVGHEAFGLVPGLLMYATIWLREHNRV 303 COX1 Mus CDLLKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLSGYFLQLKFDPEL 367 COX1 G.gal CDILKQEHPTWGDEQLFQTARLILIGETIKIVIEDYVQHLSGYFLNLKFDPEL 410 COX1 Danio CEILKQEHPTWGDEQLFQTARLIIIGETIRIVIEEYVQHLSGYRLKLHFDPTL 365 lCOX CDVLHARHPRWDDERLFQTARLILTGETMKIVIEEYVQHLSGYNFHLKFDPTL 159 COX2 Mus CDILKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL 351 COX2 G.gal CDVLKQEHPEWDDEQLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL 351 COX2 F.het CDVLKGVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPEL 356 *Y385/H388 *N410 COX1 Mus LFRAQFQYRNRIAMEFNHLYHWHPLMPNSFQVGSQEYSYEQFLFNTSMLVDYG 420 COX1 G.gal LFEQQFQYRNRIAVEFNQLYHWHALMPDSFTIQGQEYSYEQFLYNTSMLMDYG 463 COX1 Danio LFNSQFQYQNRISVEFNQLYHWHPLMPDSFYIDGDHIQYSKFIFNTSILTHYG 418 lCOX LFGVNFQYSNRMSLEFNHLYHWHPLMPDSLLIDGRNYSYDEFLFNPGLLADKK 212 COX2 Mus LFNQQFQYQNRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHG 404 COX2 G.gal LFNQRFQYQNRIAAEFNTLYHWHPLLPDTFQIHNQEYTFQQFLYNNSIMLEHG 404 COX2 F.het LFNQRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHG 409 COX1 Mus VEALVDAFSRQRAGRIGGGRNFDYHVLHVAVDVIKESREMRLQPFNEYRKRFG 473 COX1 G.gal VEALAESFSMQTAGRIGGGQNINANVLGVAVGVIEESRQLRLQPFNEYRKRFG 516 COX1 Danio LEKLVEAFSIQPAGQIGGGHNIHPVVSGVAERVIVESRELRLQPFNEYRKRFN 471 lCOX LMPLVRSFMRQRAGTVSGGRNINKNLLHVATSIIEHGRTLRLQSLNQYRHRFN 265 COX2 Mus LTQFVESFTRQIAGRVAGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFS 457 COX2 G.gal LSHMVKSFSKQSAGRVAGGKNVPAAVQKVAKASIDQSRQMRYQSLNEYRKRFM 457 COX2 F.het INNLVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFS 462 Figure 2-4. Amino acid alignment lCOX seque nced from the gills of the lamprey ( Petromyzon marinus ), with COX-1 and COX-2 forms fr om vertebrates (Accession # NP_032995, NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942). Amino acids that share at least 60% identity across se quences are highlighte d. *Indicates amino acids (numbered based on ovine COX-1) involved in COX function: active site of COX (tyrosine-385 and histidine-388), N -glycosylation site (asparagine-410), and sites for peroxidase activity (glu tamine-203 and histidine-207) (Yang et al ., 2002; Ishikawa et al ., 2007). The black boxes indicate th e two domains that define the haem-binding sites (Ishikawa et al ., 2007). The dashed line represents the arginine277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003; Kulmacz et al ., 2003). The numbers to the right of the sequences indicate the amino acid number of each sequence. 80

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COX1 Mus LKPYTSFQELTGEKEMAAEL 493 COX1 G.gal LKPYTSFQELTGEEDKAAEL 536 COX1 Danio LKPYTSFAELTGEQEMSKEL 491 lCOX MRPFTSFLELTGDEAMAAEM 285 COX2 Mus LKPYTSFEELTGEKEMAAEL 477 COX2 G.gal LKPFKSFEELTGEKEMAAEL 477 COX2 F.het MKPYTSFEDLTGEKEMAAIL 482 Figure 2-4. Continued. 81

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++ + COX1 Mus MSRRSLSLWFPPLLLLLLPPTPSVLLADPGVPSPVNPCCYYPCQNQGVCVRFG 53 COX1 Danio MRECNFLLKWTVI--LLLSVSFCAGEESPTSSNTANPCCYYPCQNQGICVRYG 51 COX1 Ovis MSRQSISLRFPLL--LLLLSPSPVFSADPGAPAPVNPCCYYPCQHQGICVRFG 51 hCOX MTSEVFVVLCVAVVFA-GAAAAD------------DPCCGSPCENKGVCVSVG 40 COX2 Mus MLFRAVLLCAALGLSQAA-----------------NPCCSNPCQNRGECMSTG 36 COX2 F.het MNRITFAVFLLALCFSFHKEVLG------------NACCSEPCQNRGVCTAMG 41 COX2 Ovis MLARALLLCAAV-VCGAA-----------------NPCCSHPCQNRGVCMSVG 35 + +*N68 + + + + + ++ + COX1 Mus LDNYQCDCTRTGYSGPNCTIPEIWTWLRNSLRPSPSFTHFLLTHGYWLWEFVN 106 COX1 Danio LERYECDCTRTGYYGENCTIPELWTRVYRLLKPSPNVVHYILTHFDWLWDLIN 104 COX1 Ovis LDRYQCDCTRTGYSGPNCTIPEIWTWLRTTLRPSPSFIHFLLTHGRWLWDFVN 104 hCOX FEDYECDCTRTGYYGSNCTYPETWTWIVNMLKPLPDTVHYFLTHFAPFWSLVN 93 COX2 Mus FDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPNTVHYILTHFKGVWNIVN 89 COX2 F.het SDSYECDCTRTGYRGQNCTTPEFLTWIKISLKPTPNTVHYILTHFKGFWNIIN 94 COX2 Ovis FDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPDTVHYILTHFKGVWNIVN 88 + ++ + *R120+ *N144 + COX1 Mus -ATFIREVLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSNVSYYTRILPSVP 158 COX1 Danio R-SFLRDWLMRKVLTVRANLIPSPPTYNSRYDYLNWEAYSNITYYTRILPPVP 156 COX1 Ovis -ATFIRDTLMRLVLTVRSNLIPSPPTYNIAHDYISWESFSNVSYYTRILPSVP 156 hCOX RAAFLRDRVMRHVLMSRAHMVSSPPTYNADYGYKSWEAYSNLSYFTRLLPPVP 146 COX2 Mus NIPFLRSLIMKYVLTSRSYLIDSPPTYNVHYGYKSWEAFSNLSYYTRALPPVA 142 COX2 F.het NISFLRDAIMKYVLTSRSHMIDSPPTYNADYGYKSWEAYSNLSYYTRALPPVP 147 COX2 Ovis KISFLRNMIMRYVLTSRSHLIESPPTYNVHYSYKSWEAFSNLSYYTRALPPVP 141 Figure 2-5. Amino acid alignment of lCOX se quenced from the gills of the lamprey ( Petromyzon marinus ), with COX-1 and COX-2 form s from vertebrates (Accession # NP_032995, NP_035328, XP_425326, XP_422297, AAS21313, and NP_705942). Amino acids that share at least 60% iden tity across sequences are highlighted. *Indicates amino acids (numbered based on ovine COX-1) involved in COX function: active site of COX (tyrosine-385, histidine-388, and serine-530), substrate binding site (arginine-120), N -glycosylation sites (asparagine-68, 144, and 410), sites for peroxidase activity (glutamine-203 a nd histidine-207), and the two sites which define conformational differences in channels between COX-1 and COX-2 in mammals (arginine-513 a nd valine-523) (Yang et al ., 2002; Ishikawa et al ., 2007). +Indicates amino acids unique to hCOX. The black boxes indicate the two domains that define the haem-binding sites and the dashed box indicates the membrane binding domain (Kulmacz et al ., 2003; Ishikawa et al ., 2007). The solid lines represent disulfide bonds, with the beginni ng and end of the longest bond represented by arrows (Kulmacz et al ., 2003). The dashed line repr esents the arginine-277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003; Kulmacz et al ., 2003). The numbers to the right of the sequences indicate the amino acid number of each sequence. 82

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+ + + + *Q203/H207 COX1 Mus KDCPTPMGTKGKKQLPDVQLLAQQLLLRREFIPAPQGTNILFAFFAQHFTHQF 211 COX1 Danio NDCPTPMGTKGKIKLPDPKLLVEKFMLRRNFRLDPQGTNLMFAFFAQHFTHQF 209 COX1 Ovis RDCPTPMDTKGKKQLPDAEFLSRRFLLRRKFIPDPQSTNLMFAFFAQHFTHQF 209 hCOX QGCPTPMGRTGKKELPDAQKLAERFLLRRTFIPDPQGSNLLFAFFAQHFTHQF 199 COX2 Mus DDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMMFAFFAQHFTHQF 195 COX2 F.het EDCPTPMGVVGKKELPDVKVLAEKLLVRRRFIPDPQGTSLMFAFFAQHFTHQF 200 COX2 Ovis DDCPTPMGVKGRKELPDSKEVVKKVLLRRKFIPDPQGTNLMFAFFAQHFTHQF 194 + COX1 Mus FKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLFKDGKLKYQVLDGEVY 264 COX1 Danio FKTHNRVGLGFTKGLGHGVDAGHIYGDSLDRQLELRLHKDGKLKYQVLNGDIY 262 COX1 Ovis FKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYQLRLFKDGKLKYQMLNGEVY 262 hCOX FKTDFKRGPGFTKALGHGVDLSHIYGDTLDKQHKLRLHNNGKLKFQMIDGEVF 252 COX2 Mus FKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDGKLKYQVIGGEVY 248 COX2 F.het FKSDMKNGPAFTVAKGHGVDLGHIYGENLEKQHKLRLFKDGKLKYTMVNGEVY 253 COX2 Ovis FKTDIERGPAFTKGKNHGVDLSHVYGESLERQHNRRLFKDGKMKYQMINGEMY 247 + + *R277++ COX1 Mus PPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTIWLREHNRVCDL 317 COX1 Danio PPTVLHAQVKMSYPPSVPPEQQLAIGQEVFGLLPGLGMYATLWLREHNRVCEI 315 COX1 Ovis PPSVEEAPVLMHYPRGIPPQSQMAVGQEVFGLLPGLMLYATIWLREHNRVCDL 315 hCOX PPLVSEAPVDMIYPPHVPEAARFAVGHEAFGLVPGLMLYGTLWLREHNRVCDV 305 COX2 Mus PPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHNRVCDI 301 COX2 F.het PPLVKDVGVEMHYPPHVPDSQRFAVGHEAFGLVPGLLMYATIWLREHNRVCDV 306 COX2 Ovis PPTVKDTQVEMIYPPHIPEHLKFAVGQEVFGLVPGLMMYATIWLREHNRVCDV 300 + COX1 Mus LKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLSGYFLQLKFDPELLFR 370 COX1 Danio LKQEHPTWGDEQLFQTARLIIIGETIRIVIEEYVQHLSGYRLKLHFDPTLLFN 368 COX1 Ovis LKAEHPTWGDEQLFQTARLILIGETIKIVIEEYVQQLSGYFLQLKFDPELLFG 368 hCOX LDVDHPEWDDERIFQTARLILIGETIKIVIEDYVQHLSGYHFKLKFDPKLLFG 358 COX2 Mus LKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 354 COX2 F.het LKGVHPDWDDERLFQTTRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 359 COX2 Ovis LKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLSGYHFKLKFDPELLFN 353 + *Y385/H388 + *N410 + ++ COX1 Mus AQFQYRNRIAMEFNHLYHWHPLMPNSFQVGSQEYSYEQFLFNTSMLVDYGVEA 423 COX1 Danio SQFQYQNRISVEFNQLYHWHPLMPDSFYIDGDHIQYSKFIFNTSILTHYGLEK 421 COX1 Ovis AQFQYRNRIAMEFNQLYHWHPLMPDSFRVGPQDYSYEQFLFNTSMLVDYGVEA 421 hCOX QPFQYSNRISVEFNHLYHWHGLNPDAFRVGTQEYQYSQFLFNNTILLNHGVRG 411 COX2 Mus QQFQYQNRIASEFNTLYHWHPLLPDTFNIEDQEYSFKQFLYNNSILLEHGLTQ 407 COX2 F.het QRFQYQNRIASEFNTLYHWHPLMPDSFHIEEKDYSYKEFVFNTSVVTEHGINN 412 COX2 Ovis QQFQYQNRIAAEFNTLYHWHPLLPDVFQIDGQEYNYQQFIYNNSVLLEHGVTQ 406 ++ + + + ++ + + COX1 Mus LVDAFSRQRAGRIGGGRNFDYHVLHVAVDVIKESREMRLQPFNEYRKRFGLKP 476 COX1 Danio LVEAFSIQPAGQIGGGHNIHPVVSGVAERVIVESRELRLQPFNEYRKRFNLKP 474 COX1 Ovis LVDAFSRQPAGRIGGGRNIDHHILHVAVDVIKESRVLRLQPFNEYRKRFGMKP 474 hCOX LLEAFNVQQAGRIGGGQNIHGALLHVATASIKHGRKMRFQSLNQYRKQFGLQP 464 COX2 Mus FVESFTRQIAGRVAGGRNVPIAVQAVAKASIDQSREMKYQSLNEYRKRFSLKP 460 COX2 F.het LVDSFSKQIAGRVAGGRNVPGPIMYVAIKSIENSRKMRYQSLNAYRKRFSMKP 465 COX2 Ovis FVESFTRQIAGRVAGRRNLPAAVEKVSKASLDQSREMKYQSFNEYRKRFLLKP 459 *V523 COX1 Mus YTSFQELTGEKEMAAELEELYGDIDALEFYPGLLLEKCQPNSIFGESMIEMGA 529 COX1 Danio YTSFAELTGEQEMSKELEELYGHIDAMEFYPALLLEKTRPGAVFGESMVEMGA 527 COX1 Ovis YTSFQELTGEKEMAAELEELYGDIDALEFYPGLLLEKCHPNSIFGESMIEMGA 527 hCOX YQSFEQLTGETEMAADLAELYSDINAMEFYLGLMVEKPRQGALFGETMVEAGA 517 COX2 Mus YTSFEELTGEKEMAAELKALYSDIDVMELYPALLVEKPRPDAIFGETMVELGA 513 COX2 F.het YTSFEDLTGEKEMAAILEELYGDVDAVELYPGLLVEKPRTNAIFGETMVEMGA 518 COX2 Ovis YESFEELTGEKEMAAELEALYGDIDAMELYPALLVEKPAPDAIFGETMVEAGA 512 Figure 2-5. Continued 83

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*S530 + + + + + COX1 Mus PFSLKGLLGNPICSPEYWKPSTFGGDVGFNLVNTASLKKLVCLNTK-TCPYVS 581 COX1 Danio PFSLKGLMGNPICSPDYWKPSTFGGKTGFDIVNSATLKKLVCLNTK-WCPYVS 579 COX1 Ovis PFSLKGLLGNPICSPEYWKASTFGGEVGFNLVKTATLKKLVCLNTK-TCPYVS 579 hCOX PFSLKGLMGNAICSPEYWKPSTFGGNRGFEIVNSASLRRLVCLNLQGPCPDVA 570 COX2 Mus PFSLKGLMGNPICSPQYWKPSTFGGEVGFKIINTASIQSLICNNVKG-CPFTS 565 COX2 F.het PFSLKGLMGNPICSPEYWKPSTFGGTAGFDIVNTASLQRLVCNNVRGPCPVAS 571 COX2 Ovis PFSLKGLMGNPICSPEYWKPSTFGGEVGFKIINTASIQSLICSNVKG-CPFTS 564 + + + ++ COX1 Mus FR-VPDYPGDDGSVLVRR-------------------STEL 602 COX1 Danio FHTPPSDYKPQRTS-----------------------HGEL 597 COX1 Ovis FH-VPDPRQEDRPGVERP-------------------PTEL 600 hCOX FH-VPRDNQQDVVVNVTGSQGGSDGVTTTPHYVADQQSREL 609 COX2 Mus FN-V-QDPQPTKTATINASASHSRLDDINPTVLIKRRSTEL 603 COX2 F.het FY-VP-DVKETGSMTINSSTSHSRDSNINPTVILKERTSEL 609 COX2 Ovis FS-V-QDAHLTKTVTINASSSHSGLDDINPTVLLKERSTEL 602 Figure 2-5. Continued 84

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*H207 COXa Ciona HMFFKTDPMKGMPYQWGDQLVDLSQIYGHGEKRQHELRSHVNGKLKVSLVDGH 279 COXb Ciona HQFFKTNTIKGMPFQWGEHSVDLSHVYGHTIQRQHELRSHIDGKLKVFETNGE 226 COXc HQFFKTDFKKGAGRTWGDHGVDLSHIYGETVERQHQLRSFTDGKLKFQRVEGE 53 COXd HQFFKTDFKKGPGRTWSDHAVDMSHVYGETVERQRQLRSFQDGKLKYQLVDGE 53 *R277 COXa Ciona EFPPLSNQTTANMSNINLLPQEYQFVFGHQGFSLMPTFLIWSTIWLREHNRIC 332 COXb Ciona VFPPLTESANVTMSGEKLM-RGRKFAIGHPGFGAFPSFFVIATLWLREHNRVC 279 COXc VYPPSLADAPVHMIYPPYVPEGKRFAIGHEFFGLLPGLFVYSTVWLREHNRVC 106 COXd AFPPSLQDASVHMIYPPDVPEKKRFALGHEFFGLLPGLFVWATVWLREHNRVC 106 COXa Ciona DLIKEENPAWDDERIFQTARLVLTGETIKVVIEDYVQHLSGFHYKLLYDPELV 385 COXb Ciona DILKDLHPDWDDERLFQTARLILTGETLKIIVEDYVQHVSGFHFQLSYDPEIL 332 COXc DVMKELHPDWDDERLFQTARLILTGETINIIINEYVQHLSGYNFDLFWDPELL 159 COXd DVMKDLHPDWDDERLYQTARLILISETINIVIGEYVQHLAGKNFQLFWDPELL 159 COXa Ciona QGGSHSFHNQIHVEFQL 402 COXb Ciona HKSTFSYNNQIHAEFHI 349 COXc FSDQFQYQNRIFVEFNH 176 COXd FEEQXQYQNSIFVEFNH 176 Figure 2-6. Amino acid alignment of COXc and COXd sequences from the lancelet ( Branchiostoma lancelolatum ) with COXa and COXb sequences from the sea squirt ( Ciona intestinalis ). Amino acids that share at least 60% identity across sequences are highlighted. *Indicates the amino acid (numbered based on ovine COX-1) involved in peroxidase ac tivity (histidine-207) (Yang et al ., 2002; Ishikawa et al ., 2007). The black boxes indicate the two domains that define the haem-binding sites (Ishikawa et al ., 2007). The dashed line repres ents the arginine-277 loop region, which has been shown to differ between COX-1 and COX-2 forms in mammals (Yang et al ., 2003; Kulmacz et al ., 2003). The numbers to the right of the sequences indicate the amino acid number of each sequence. 85

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Figure 2-7. Evolutionary hist ory of COX sequences inferred using the minimum evolution method with a distance optimality criterion. The optimal tree with the sum of branch length = 6.63322594 is shown. The percentage of replicate trees in which the associated taxa clustered toge ther in the bootstrap test ( 1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutiona ry distances used to infer the phylogenetic tree. The evolutionary distances were co mputed using the JTT matrix-based method (Jones et al ., 1992) and are in the units of the number of amino acid substitutions per site (scale bar = 0.05 amino acid substitutions per site). The rate variation among sites was modeled with a gamma distribution (s hape parameter = 0.737). The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-Joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 147 pos itions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al ., 2007). Sequences enclosed in a box are novel and reported here for the first time. 87

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COX1b Gasterosteus COX1b Myoxocephalus COX1b Oryzias COX1b Fundulus COX1b Tetraodon COX1 Oncorhynchus COX1 Danio COX1a Tetraodon COX1a Fundulus COX1a Oryzias COX1a Gasterosteus COX1a Myoxocephalus COX1 Mus COX1 Rattus COX1 Bos COX1 Ovis COX1 Oryctolagus COX1 Homo COX1 Canis COX1 Gallus COX1 Monodelphis COX1b Monodelphis COX1b2 Monodelphis COX1 Xenopus COX2 Gasterosteus COX2 Myoxocephalus COX2b Oncorhynchus COX2 Oryzias COX2 Fundulus COX2 Tetraodon COX2b Danio COX2a Danio COX2a Oncorhynchus COX2 Xenopus COX2 Bos COX2 Ovis COX2 Gallus COX2 Homo COX2 Mus COX2 Rattus COX2 Monodelphis COX2 Canis COX2 Oryctolagus sCOX Squalus COXc Branchiostoma COXd Branchiostoma COXa Ciona COXb Ciona 100 99 94 71 57 80 44 61 99 62 86 87 34 51 92 98 85 37 99 71 60 26 32 35 91 93 52 82 43 39 41 34 40 70 26 77 94 83 39 20 32 38 41 38 13 0.1 88

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Figure 2-8. Evolutionary hist ory of COX sequences excluding hagfish (hCOX) and lamprey (lCOX) sequences inferred using the mi nimum evolution method with a distance optimality criterion. The optimal tree with the sum of branch length = 6.18990857 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap te st (1000 replicates) is show n next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method (Jones et al ., 1992) and are in the units of the number of amino acid substitutions per site (scale bar = 0.1 amino acid substitutions per site ). The rate variation among sites was modeled with a gamma distribution (sha pe parameter = 0.742). The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-Joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 147 pos itions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al ., 2007). 89

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Figure 2-9. Evolutionary hi story of COX sequences infe rred using a maximum liklihood optimality criterion. The most likely tree with an Ln L score = -25294.222410 is shown. The number of replicate trees in wh ich the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the WAG matrix-b ased method (Whelan and Goldman, 2001) and are in the units of the number of ami no acid substitutions per site (scale bar = 0.1 amino acid substitutions per site). The rate variation among sites was modeled with a gamma distribution (shape parameter estimated = 0.737). Phylogenetic analyses were conducted using PhyML (Guindon and Gascue l, 2003). Sequences enclosed in a rectangle are novel and reported here for the first time. 90

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91

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Figure 2-10. Evolutionary history of COX se quences excluding sequences from the hagfish (hCOX) and lamprey (lCOX) inferred using a maximum liklihood optimality criterion. The most likely tree with an Ln L score = -24242.30059 is shown. The number of replicate trees in which the a ssociated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the sa me units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the WAG matrix-b ased method (Whelan and Goldman, 2001) and are in the units of the number of ami no acid substitutions per site (scale bar = 0.1 amino acid substitutions per site). The rate variation among sites was modeled with a gamma distribution (shape parameter estimated = 0.742). Phylogenetic analyses were conducted using PhyML (Guindon and Ga scuel, 2003). Sequence names are abbreviated from Figure 2-9. 92

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Figure 2-11. Alternate likelihood ra tio test (aLRT) scores for bran ch support of COX sequences. The most likely tree is shown and the topology, branch lengths, and model of evolution are the same as in Figure 2-9. Scores represent the gain in likelihood when collapsing a particular branch and are inde pendent of bootstrap sc ores (Felsenstein, 1985). Scores were interpreted using a c onservative estimate of the minimum of parametric chi-squared and non-parametric Shimodaira-Hasegawa-like (SH) support value interpretations (Anisimova and Gascue l, 2006). Analysis was performed using PhyML (Guindon and Gascuel, 2003). Support va lues of interest to the present study are enclosed in boxes. 94

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95

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Figure 2-12. Alternate likelihood ra tio test (aLRT) scores for br anch support of COX sequences excluding hagfish (hCOX) and lamprey (lCOX) sequences. The most likely tree is shown and the topology, branch lengths, and model of evolution are the same as in Figure 2-11. Scores represent the gain in likelihood when collapsing a particular branch and are independent of bootstrap sc ores (Felsenstein, 1985). Scores were interpreted using a conservative estimate of the minimum of parametric chi-squared and non-parametric Shimodaira-Hasegawa-l ike (SH) support valu e interpretations (Anisimova and Gascuel, 2006). Analys is was performed using PhyML (Guindon and Gascuel, 2003). Support values of intere st to the present study are enclosed in boxes. 96

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Figure 2-13. Evolutionary history of COX se quences inferred using a Bayesian optimality criterion. The consensus tree is shown. The posterior probabilities (10 million generations) are shown next to the branches. Trees were sampled every 500 generations with a burnin of 10% (401 trees). The tree is dr awn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the WAG matrixbased method (Whelan and Goldman, 2001) and are in the units of the number of amino acid substitutions per site (scale bar = 0.1 amino acid substitutions per site). The rate variation among sites was mode led with a gamma distribution (shape parameter estimated = 0.672275). Phylogene tic analyses were conducted using MrBayes 3.1.2. Sequences enclosed in a re ctangle are novel and reported here for the first time. 97

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98

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Figure 2-14. The evolutionary history of COX sequences excluding hagfish (hCOX) and lamprey (lCOX) sequences inferred using a Bayesian optimality criterion. The consensus tree is shown. The posterior probabilities (10 million generations) are shown next to the branches. Trees were sampled every 500 generations with a burnin of 10% (401 trees). The tree is drawn to scal e, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the WAG matrix-based method (Whelan and Goldman, 2001) and are in the units of the number of amino acid substitutions per site (scale bar = 0.1 amino acid substitutio ns per site). The rate variation among sites was modeled with a gamma distri bution (shape parameter estimated = 0.667978). Phylogenetic analyses were conducted using MrBayes 3.1.2. 99

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a) b) c) d) Figure 2-15. The 21 alternate tr ee topologies generated to test the effects of relocating the hagfish (hCOX) and lamprey (lCOX) sequences on the likelihood score (Ln L ). Each topology is simplified, but all relationships remain the same as in the most likely topology (Figure 2-9) with the exception of hCOX and lCOX. Each topology was generated using TreeView1.6.6 based on the most likely topology. Ln L was calculated for each topology online using PhyML (Guindon et al ., 2005), branch lengths and model parameters were estimate d. The topologies are named in Table 2-9 as follows (from most to least likely) : a) COX2 Hag (most likely) b) COX1 Lamp/COX2 Hag c) COX2 Agnatha d) Lamp/Amp e) COX2 Vert f) COX1 Hag g) COX1 Vert h) Ancestral Hag i) COX1 Agnatha j) COX1 Hag/COX2 Lamp k) Ancestral Agnatha l) COX1 Lamp m) COX2 Lamp n) Agntha/Amp o) Hag/Amp p) Hag Internal q) Lamp Internal r) Lamp Mammal s) Lamp Teleost t) Hag Teleost u) Hag Mammal. 100

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e) f) g) h) i) j) Figure 2-15. Continued. 101

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k) l) m) n) o) 2 p) Figure 2-15. Continued. 102

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q) r) s) t) u) Figure 2-15. Continued. 103

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104 Figure 2-16. Unrooted phylogeny showing the mo st likely scenario for COX evolution in the chordates based on several other analyses. Clades of interest are highlighted on the right and sequences that repr esent unclear placements are highlighted in red boxes.

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CHAPTER 3 SUMMARY AND FUTURE DIRECTIONS Summary This thesis is the first attempt to charact erize the evolution of the enzyme cyclooxygenase in the phylum chordata. Traditional molecular protocols such as clon ing and polymerase chain reactions were used to search for cyclooxygenase sequences in a wide ra nge of evolutionarily key chordates. Using these novel sequences an d pre-existing sequences from GenBank and genomic databases, phylogenetic analyses were pe rformed to reveal the identity of the novel sequences and create the most lik ely scenario for the evolutiona ry history of cyclooxygenase in the chordates. Novel COX Sequences Chapter 2 describes the process of obtai ning and characterizing novel cyclooxygenase (COX) sequences in amphioxus ( Branchiostoma lancelolatum ), the Atlantic hagfish (Myxine glutinosa ), the sea lamprey ( Petromyzon marinus ), the euryhaline killifish ( Fundulus heteroclitus ), and the longhorn sculpin ( Myoxocephalus octodecemspinosus ). Total RNA was isolated and reverse transcribed from gill tis sue (or whole animals in amphioxus) and the resulting cDNA was exposed to PCR in the presence of degenerate and/or sp ecific primers. The products were then visualized and cloned. The resulting plasmids were sequenced at the Marine DNA Sequencing Center at the MDIBL. Complete amino acid sequences of putative COX proteins from the hagfish, killifish, and scul pin and incomplete amino acid sequences from amphioxus and the lamprey were predicted based on the sequences found. These putative amino acid sequences were aligned w ith known COX protein sequen ces and conserved domains, regions, and residues critical to COX function were describe d. Based on the novel sequences identity to known COX forms and phylogenetic an alyses (see below), it was concluded that 105

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COX-1a, COX-1b, and COX-2 are found in the sculpin; COX-1a and COX-1b are found in the killifish; a new COX form named lCOX is found in the lamprey; a new COX form named hCOX is found in the hagfish; and two ancestra l forms named COXc and COXd are found in amphioxus. Phylogenetic Analyses Chapter 2 also describes the phylogenetic analyses used to reconstr uct the evolutionary history of cyclooxygenase in the chordates. The nine novel COX protein sequences described here, as well as 41 additional COX protein sequences from GenBank and genomic databases, were subjected to phylogenetic analyses using three optimality criteria: minimum evolution/distance, maximum likelihood, and Baye sian phylogenetics. In each analysis, two clades were well-supported in the gnathostomes: a COX-1 clade and a COX-2 clade. Each of these clades contained a well suppor ted teleost and tetrapod clade. In the teleost clades, clades for COX-1a, COX-1b, COX-2a, and COX-2b were well-supported, confirming the findings of Ishikawa et al (2007) and Ishikawa and Herschman (2007). The placement of the more evolutionarily ancestral chordates was ultim ately uncertain, although it appears that the urochordates, cephalochordates, and vertebrate s (containing COX-1 and COX-2) each represent an independent origin of COX proteins. Th e hagfish and lamprey sequences most likely represent basal members of the COX-2 and COX-1 clades of vertebrates, respectively; however, these forms may belong closer to the root of the tree. Future Directions Although here I report 9 novel cyclooxygena se (COX) sequences from a range of chordates and present a likely scenario for the ev olution of this gene family in the chordates, many questions remain regarding COX evolution a nd function in the chordates. These include determining the true history of C OX evolution in the earliest chor dates, pinpointing the origin of 106

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COX-1 and COX-2, and investigating the history of COX duplication and loss in the teleosts. Finally, the physiological function of COX in the earliest chordate s has only been preliminarily investigated. Completion of novel sequences Several of the novel sequences documented here could not be completed despite multiple efforts using many primer sets and PCR techniqu es. Notably, only 532 bp were obtained of the sequences from amphioxus (COXc and COXd). Co mpared to the complete open reading frame of COX transcripts in other chordates (rangi ng from about 1700-2500 bp), this indicates that about 75% of these sequences remain unknown. Completing the rest of these sequences or thoroughly searching the recently complete genome of Branchiostoma floridae may yield putative proteins that provide novel groupings in phylogenetic analyses. Based on the available sequence data, however, COXc and COXd are we ll-supported as being n ear the base of the chordate COX lineage, sister to the vertebra te COX-1 and COX-2 clade. The lCOX sequence obtained from the lamprey is also only about 40% complete. Completing the open-reading frame of this putative protein or searching genomic data bases could possibly give a more definite view of its identity as this sequ ence groups with different clad es depending on the phylogenetic analysis used. In actuality, th e short size of lCOX compared to the other COX sequences used in the phylogenetic analyses may have contributed to its tendency to a ppear in different clades in different analyses. Finally, the COX-1a sequen ce obtained from the sculpin may warrant further investigation and reaffirmation via additional sequencing since many residues critical to COX function are missing. Additional Phylogenetic Analysis Unfortunately, the evolutionary relationship of the COX proteins in the chordates was not able to be unequivocally resolved, despite the use of three different phylogenetic approaches. 107

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This may be due to the incompleteness of some sequences and a lack of sufficient sampling across taxa or may represent a hi story that is irresolvable und er the current circumstances. However, the phylogenetic techniques used here may also be improved upon in future analyses. Phylogenetically uninformative areas of the se quences (such as near the C and N terminus, which show little homology across sequences) could be removed. Here, protein sequences were used to gain insight into the e volution of COX functionality, but in sights could also be gained by performing similar analyses using DNA sequence data instead of using their inferred protein amino acid sequences. This would allow for gr eater complexity of models to estimate substitution rates and could take additional f actors into account such as codon bias. Using nucleotide versus amino acid would also enable additional search strategies and phylogenetic programs to be used. Choosing the optimum search strategy and computer program may be critical in finding the optimum evolutionary r econstruction. For example, maximum likelihood analyses PhyML (the program used in this study) may find suboptimal likelihood scores when the data-set contains little phylogenetic information (Morrison, 2007). However, programs such as RAxML (Stamatakis, 2006), GARLI (Brauer et al ., 2002), and PAUP* (Rogers and Swafford, 1998) may perform better under the same circum stances (Morrison, 2007). Also, evaluating alternative tree topolog ies using frameworks other th an maximum likelihood may yield alternative results. Additional Sequences Based on the most likely scenario of COX evol ution in the chordates, there are several COX sequences in the chordates examined in this study that remain to be characterized. If hCOX in actuality represents a basal form of CO X-2, then there should also be a COX-1 form in the hagfish. Similarly, if lCOX and sCOX repres ent basal forms of COX-1 in the lamprey and dogfish, then they should also contain versions of COX-2. These additional forms of COX may 108

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be harder to clone and sequence due to accumulated mutations over evolutionary time, which would render the sequences unidentifiable and possi bly non-functional. However, this is not the case in the other vertebrates, as COX-1 and COX2 seem to each have critical functionality. Using Southern blotting techniqu es (Southern, 1975) to identify COX DNA sequences in the DNA of hagfish, lamprey, and dogfish could immedi ately reveal the pres ence of two forms of COX in these animals. If two forms are present, it could be concluded that they likely represent COX-1 and COX-2 forms, and thus the origin of these forms would coincide with the vertebrate lineage (as predicted in the current phylogenetic an alysis). However, if only one form of COX was found the results would suggest a more recent origin of COX-1 and COX-2. This technique could be applied to a wide diversity of chordates to determine how many COX variants they contain. Additional Taxa In this study, a comparativ e evolutionary approach was taken by sampling a wide range of chordates, most of which had no previous record of COX gene s. However, there are still many evolutionarily interesting c hordates that have not been ex amined for COX genes. While COX genes are known from multiple mammals and teleosts, other chordate groups have only been minimally sampled. Most notably is the l ack of any reported COX genes from any reptile. COX has also only been documente d from a single avian species ( Gallus gallus), a single elasmobranch species ( Squalus acanthias ), and a single marsupial ( Monodelphis domestica ). These groups represent major lin eages within the vertebrates and may contain novel forms of COX. The results of this and other recent studies also warran t further investigation within certain chordate groups. For example, evolutio narily ancestral fishes such as the sturgeon, paddlefish, and bowfin could likely contain a single COX-1 and C OX-2 gene, while the teleosts studied here have multiple COX-1 or COX2 genes. Ishikawa and Herschman (2007) 109

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hypothesize that a genome duplication in the tele ost lineage and subsequent loss of either a COX-1 (in the zebrafish/trout lineage) or COX2 gene (in the acanthopterygian lineage) is responsible for this observation. If this is the case, then examining taxa near the base of the teleost lineage may reveal fish with all four variants of COX (COX-1a, COX-1b, COX-2a, and COX-2b). The COX variants from the dogfish shark, lamprey, and hagfish all group weakly with either COX-1 or COX-2. Obtaining C OX sequences from other species of hagfish, lampreys, and elasmobranchs may confirm this in itial relationship or reveal novel groupings. Finally, although support values for the urochorda ta and cephalochordata clades are strong regardless of the phylogenetic technique use d, only one species from each subphylum was included in phylogenetic analyses. Including more species could gi ve a more complete view of COX evolution in the evolutionarily ancestral ch ordates. COX sequences from these additional taxa may be readily available in the near fu ture due to the growing regularity of genome sequencing across many taxa. For example, the recently completed Anolis carolinensis genome contains COX-1 and COX-2 genes. However, due to the length of time required for whole genome projects, the limited number of genomes available, and the difficulty of searching recently completed, non-annotated genomes, studi es that specifically target sequencing COX genes in these taxa are needed. Function of Cyclooxygenases As reviewed in Chapter 1, cyclooxygenase play s several roles in chordates because of its role in prostaglandin production. These functions have been examined in some detail in the mammals and to a lesser degree in the fishes. Ho wever, no studies have examined the functions of the novel COX forms presented here or any no n-vertebrate COX forms in the chordates. Therefore, I have begun preliminary st udies investigating the function of these COX forms. 110

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One preliminary method used to infer possible functions for a gene is to determine where its mRNA transcript or protei n product is expressed in the organism. For example, Choe et al (2006) concluded that COX-2 may play a role in os moregulation in the killif ish partly due to its high level of expression in the gills and its pr esence within mitochondrio n-rich cells. Following similar procedures, I have found that COX-1b mRNA is also expressed highly in the gills of the killifish (Fig. 3-1a). Similar results were also found for COX-1a and COX-2 in the sculpin (Fig. 3-2a, Fig. 3-2b), hCOX in the hagfish (Fig. 3-3a ), and lCOX in the lamprey (Fig. 3-4). To investigate which cells express COX mRNA transcripts, in situ hybridization (ISH) was performed using probes designed against the novel COX sequences described here using previous protocols (Choe et al ., 2006). However, no staining wa s observed for COX transcripts in the gills of the killifish (Fig. 3-1b) or sculpin (Fig. 32b). This is likely due to errors in probe design or hybridization protocol, since COX mRNA was abundant in the gills of both the killifish and sculpin. In the hagfish, probes designed to show hCOX mRNA expression did produce staining patterns in a population of epith elial cells lining gill filaments and lamellae (Fig. 3-3b). This gill structure is similar to those of teleosts but may be functionally different (Evans et al ., 2005) and since ion transport mechanisms in hagfish may not be directly related to those in teleosts (Choe et al ., 1999), it is difficult to assign an osmoregulatory function of hCOX based on this staining pattern. Finally, since COX-2 mRNA expression was shown to increase following salinity transfers using qPCR (Choe et al ., 2006), a similar procedure was used to investigate COX expression after sa linity acclimation in the gills of the killifish (Fig. 3-1c) and sculpin (Fig. 3-2c). The only significant result from this e xperiment was that COX-1b was expressed more abundantly in killifish chronica lly acclimated to fresh water than seawater. However, the same general trend show n for COX-2 in the killifish (Choe et al ., 2006) was also 111

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shown for COX-1b in the killifish: an increase in expression 3 hours af ter salinity transfer (although P = 0.06 in this case) and a return to pr e-transfer expression le vels by 8 hours after transfer. Taken as a whole, these preliminary results suggest that the different COX forms may be playing various roles in osmoregulation in th e killifish, sculpin, lamprey, and hagfish. Clearly, COX function in the non-mammalian chor dates has just begun to be investigated and many questions still remain. Future studies may investigate COX roles in these chordates by examining protein expression patterns usi ng immunohistochemistry, blocking COX function via commercially available COX i nhibitors, or examining COX expression during reproductive events. Of particular interest are the differential roles of the va rious COX forms in the teleosts and the possible partitioning of functionality between COX-1a and COX-1b or COX-2a and COX-2b. 112

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GOB HS I K COX 1b A) B) C) 18S Figure 3-1. Euryhaline killifish ( Fundulus heteroclitus ) COX-1b mRNA expression following procedures outlined in Choe et al (2006). A) COX-1b mRNA expression in various tissues in the killifish. Expression was constant across tissues for the ribosomal protein 18S. COX-1b expression was most abundant in the gills (labeled as G), followed by the heart (H), kidney (K), ope rcular epithelium (O), stomach (S), intestine (I), and brain (B). B) In situ hybridization of COX-1b in the gills of the killifish. C) Expression of COX-1b mRNA in the gills of the killifish following salinity transfers. Expression is normalized to the ribosomal gene L8 and is shown relative to seawater (SW) (FW = fresh water). 113

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GO B H SI K GO B H SIK COX-2 18S COX-1a 18S COX-1a COX-2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 24 hrs 72 hrs TimeExpression 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 hrs 72 hrs TimeExpression Figure 3-2. Longhorn sculpin (Myoxocephalus octodecemspinosus ) COX-1a and COX-2 mRNA expression following procedures outlined in Choe et al (2006). A) COX-1a and COX-2 mRNA expression in various tissues in the sculpin. Expr ession was constant across tissues for the ribosomal protein 18S. COX-1a was most abundant in the opercular epithelium (labeled as O), followe d by the gills (G), ki dney (K), heart (H), brain (B), intestine (I), a nd stomach (S). COX-2 was most abundant in the gills, followed by the opercular epithelium, and kidney. B) In situ hybridization of COX1b and COX-2 in the gills of the sculpin. C) Expression of COX-1b and COX-2 mRNA in the gills of the sculpin after 24 and 72 hours following salinity transfers to 100% and 20% seawater. Expression is normalized to the ribosomal gene L8. Seawater 20% Seawater A) B) C) COX-1a COX-2 114

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GMBH S I K hCOX 18S A) B) Antisense Antisense Sense Sense Figure 3-3. Atlantic hagfish ( Myxine glutinosa ) COX (hCOX) mRNA expression following procedures outlined in Choe et al (2006). A) COX mRNA expression in various tissues in the hagfish. Expression was c onstant across tissues for the ribosomal protein 18S. COX (hCOX) expres sion was most abundant in the gills (labeled as G), followed by the heart (H), kidney (K), stomach (S), intestine (I), muscle (M), and brain (B). B) In situ hybridization of hCOX in the gills of the hagfish from two contrary gill cross-sectio ns. Arrows highlight staining, indicating hCOX mRNA expression. Staining for antisense and sense (control) probes is shown. 115

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116 GM BH S I K lCOX 18S Figure 3-4. Sea lamprey ( Petromyzon marinus ) COX (lCOX) mRNA ex pression in various tissues following procedures outlined in Choe et al. (2006). Expr ession was constant across tissues for the ribosomal protei n 18S. COX (lCOX) expression was most abundant in the heart (labeled as H), follo wed by the intestine (I), stomach (S), gills (G), kidney (K), muscle (M), and brain (B)

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BIOGRAPHICAL SKETCH Justin Chase Havird was born in 1984, in Valdosta, Georgia to Dawn and Kurt Havird. Justin has one younger brother, Jos hua. The Havirds soon moved to Lake City, Florida, where Justin spent the majority of his childhood and adolescence. Justin attended Columbia High School in Lake C ity and graduated with top honors in 2002. Justin then attended the Univer sity of Florida in Gainesville where he majored in zoology after an initial interest in aerospace engineering. While at UF, Justin was heavily involved in the universitys band program, pl aying trumpet in the marching, basketball, volleyball, concert, and symphonic bands for se veral years. As an undergraduate, Justin worked in the laboratory of Dr. David H. Ev ans for a number of years, investigating ion transporters in the gills of fishes. His se nior thesis was titled Neuronal Nitric Oxide Synthase in the Gill of the Euryhaline Killifish, Fundulus heteroclitus. Justin graduated summa cum laude from UF in 2006 with a B.S. in zoology. In 2006, Justin entered graduate school at the University of Florida in the Department of Zoology. Continuing to work w ith Dr. Evans, Justin spent three summers at the Mount Desert Island Biological Laboratory in Salis bury Cove, Maine where he collaborated with other researchers inve stigating fish gill physiology. During his graduate studies, Justin has earned several awards and grants, including a GIAR from Sigma Xi. Justin has also presented his resear ch at scientific meeti ngs and conferences. While earning his M.S., Justin also designed experiments and instructed students in a senior level animal physiology course for 2 years. Justin has also collaborated with other scientists on a wide ra nge of marine life science projec ts, most notably a revision of the cobitid genus Lepidocephalichthys in SE Asia with Dr. Larry Page. After earning his 130

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131 M.S., Justin plans to continue his academi c career by completing a Ph.D. in a related marine science field.