Zonadhesin-like genes in three fish species: Atlantic salmon (Salmo salar), Puffer fish (Tahyugu rubripes) and Zebrafish (Danio rerio) Peter Nicholas Darien Hunt B. Sc., University of Victoria, 2002 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE In the Department of Biology We accept this thesis as conforming to the standard required. O Peter Nicholas Darien Hunt, 2004 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopying or other means, without permission of the author. Supervisor: Dr. Ben F. Koop Abstract The sperm membrane protein zonadhesin (ZAN) has been characterized in mammals and has been implicated in species-specific egg-sperm binding interactions. Zonadhesin is the only protein that contains MAM, mucin and von Willebrand D domains. A zonadhesin-like transcript was obtained fi-om an Atlantic salmon gut tissue cDNA library. This transcript and the corresponding genomic locus were sequenced. A zonadhesin-like gene (ZLG) with a predicted open reading fi-ame of 4,5 18 nucleotides that encodes MAM, mucin and VWD domains was characterized. Although the predicted protein contains the same major domains as zonadhesin, the domain order is altered. ZLG is expressed in the liver and the gut but not in the testis as a typical zonadhesin. This gene is unlikely to be directly involved in reproduction, because it has a unique domain structure and expression, but is likely to have a novel function. In contrast, zonadhesin-like genes with the same domain content and order as mammalian zonadhesins can be deduced from puffer fish and zebrafish genomic sequencing projects. Zonadhesin-like genes from these three fish species are compared and evolutionary relationships are explored. Efaminers: // Contents Abstract ii Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgements X Introduction 1 Results 33 3.1 BAC Identification and primary sequence.-----------------------------------------------.------- 33 3.2 Repeats -------------------------------------------------------------------<------------------ - ......................... 34 3.3 cDNA primary sequence and protein prediction -.---------------------------.--------.-------37 3.4 Expression----------------------------------------*---------------------.---------------------------.--------.------- 4 1 3.5 Puffer fish zonadhesin stl-lxture .------------------------------------------------------.----------------43 3.6 Zebrafish zonadhesin stnlcture--.-----------------.------------------------------------.---------------- 46 3.7 Cross-species comparisons --------.----------** ......................................................... --5 2 Discussion 57 4.1 Zonadhesin genes in fish ---------- * --------------.-----------------* -------------.----------------- -------7- 57 4.2 Puffer fish zmadhesin-------------- ** ------------------------. * ------------------------------.--------------- 5 8 4.3 Zebrafish zonadhesins---.-----------.------------------------------------------------------------------------ 59 4.4 The Atlantic salmon zonadhesin-like gene---------------------------------------.--------------- 60 4.5 p~ssiblelink to the Imcin genes 63 4.6 Evolution from a zonadhesin --------.--------* ............................ ............................... 66 4.7 Repeat ~~~~entsin the ZLG 10cus - ........................ 69 4.8 Furtherstudy ------------------------.--* --------------------------------------*---------------------- - --------.----72 4.9 Concluding remarks 73 74 References -----------------------------------------------------------.-------.-----------7-- - ---------- Appendices-----------------.-----------------------------------------.------------------------------------- - ---- ----------- ---- 8 1 A. Amino acid alignment of MAM hnains----------------.------------- - ------------ - ------------------- 8 1 B. Amino acid alignment of VWD domains-----------------.---------------------------------------------- 82 List of Tables Table 1. pl-km sequences.----------------------------------------- * ---------20 Table 2 PCR reaction parameters ---------------------------------.--------------------------.------.---2 1 Table 3. Summary of repeat elements found in 722P 12.................................... 35 Table 4. Atlantic salmon exon sizes and ~hses 38 Table 5. h.lffer fish exon sizes and ~hses-------- - -----------------.--------------------------.---44 Table 6. zebrafish ZAN1 exon sizes and ~hsses--.----------- - ---------------.---------------- 47 Table 7. zebrafish ZAN2 exon sizes and chsses ----------------.------------------------------5 0 vii List of Figures Figure 1. Domain structures of zonadhesins and related proteins ...................... 4 Figure 2. Figure 3. Atlantic salmon ZLG exons and surrounding repeat elements -----------35 Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Dot plot of zebrafish ZAN 1 and human proteins .---------.----------------------48 Figure 10. Figure 1 1. Figure 12. Neighbour-joining trees of fish and human MAM domains --------------5 3 Figure 13. Figure 14. Figure 15. Dot plot of salmon ZLG and human ZAN proteins-------.---------.----------- 62 .. Vlll List of Abbreviations % percent "C degrees centigrade Pg microgram PL microliter 3' three prime 5' five prime ACHE acetylcholine esterase ARS2 arsenite resistance 2 BAC bacterial artificial chromosome bp base pair BSA bovine serum albumin cDNA copy deoxyribonucleic acid cfil colony forming units C-terminal carboxy-terminal DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate dT deoxythymidine DTT dithiothntol E. coli Escherichia coli EDTA ethylenediamine tetra-acetic acid EtOH ethanol IPTG isopropyl-P-D-thiogalactopyranoside LB Luria-Bertani M molar mL milliliter mM millimolar mol mole MYA million years ago NaOAc sodium acetate NEB New England Biolabs ng nanogram nrn nanometer nt nucleotide N-terminal amino-terminal OD optical density PCR polymerase chain reaction P pic0 poly(A) pol yadenylate psi pounds per square inch rcf relative centrifugal force RNA ribonucleic acid RNase ribonuclease rI'm revolutions per minute RT room temperature SDS sodium dodecyl sulphate SSC sodium citrate and sodium chloride solution TBE tris borate EDTA TE tris-EDTA UTR untranslated region uv ultraviolet X-Gal 5-bromo-4-chloro-3-indoyl-P-D-galactopyranoside ZAN zonadhesin ZLG zonadhesin-like gene Acknowledgements Thank you to my supervisor, Ben Koop, for the opportunity to work in his lab with excellent people on exciting projects. I am grateful to the many people who gave their time to assist me with technical issues, to point me in the right direction and help me maintain my sanity. To thank the following people for individual reasons would fill a book and would still not do them justice, so for a mountain of reasons, thank you: Roberto Alberto, Marianne Beetz-Sargent, Gord Brown, Maura Busby, Melanie Conrad, Glenn Cooper, Aura Danby, Ross Gibbs, Sally Goldes, Nathanael Kuipers, Linda McKinnell, Melanie Mawer, Stephen Phillips, Matthew Rise, Kris von Schalburg and Michael Wilson. Thank you to Simon Jones for supplying me with salmon tissue. I would also like to thank my committee members Nancy Shenvood and John Taylor for their constructive criticism of this work. And of course a big thank you to my friends and family for their support. Introduction 1. Overview The Genome Research on Atlantic Salmon Project (GRASP) was initiated to better understand the genome of this economically important fish and apply this knowledge to other closely related salmonid species. Study of the Atlantic salmon (Salmo salar) is important not only to maximize their economic value as farmed fish but to evaluate their ecological effect on wild stocks. The interaction between farmed and wild Atlantic salmon has been hotly debated, and genetic techniques have been used in attempts to clarify the effects the salmon farming industry has on wild stocks. Population analysis using genetic data has, with some success, quantified genetic flow between wild and farmed salmon of the same species (Clifford, McGinnity et al. 1998; O'Reilly, Hamilton et al. 1996). Understanding the genetic elements that control the interaction between gametes, for example genes coding for gamete adhesion and recognition proteins, will advance our knowledge of how populations, wild or farmed, interact. It is these genes that ultimately dictate the flow of genetic information. GRASP has reported the sequencing of 80,388 salmonid ESTs (Rise, von Schalburg et al. 2004), one of which was identified as having similarity to a gene involved in reproduction in mammals. The purpose of this study was to characterize this potential zonadhesin gene in Atlantic salmon. Although this gene was found to have domain content similar to mammalian zonadhesins, it had a different tissue expression pattern. In an attempt to clarify the evolutionary origin of this zonadhesin-like gene (ZLG) it was compared to zonadhesins of mammals and putative zonadhesin genes of puffer fish (Takifugu rubripes) and zebrafish (Danio rerio). Other evolutionary origins were explored, including a possible link to the mucin gene family. 1.2 Zonadhesin Zonadhesin (ZAN) is a multi-domain sperm protein that plays a role in the species-specific binding of egg and sperm. Porcine (Sus scrofa) ZAN was first described by Hardy et al. (1 995) as a protein expressed by developing sperm that would bind to the zona pellucida of the egg. Since its