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THE TYPE I ANTIFREEZE PROTEIN GENE FAMILY IN PLEURONECTIDAE by Kyra Keiko Nabeta A thesis submitted to the Department of Biochemistry in conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada (January, 2009) Copyright © Kyra Keiko Nabeta, 2009 Abstract Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, Platichthys stellatus , has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis. ii Acknowledgements I'd like to thank my supervisor Dr. Peter Davies for the opportunity to gain insight into the true nature of research. Special thanks to Dr. Laurie Graham for teaching me how to write in Science and for her patience and support and guidance through all that sequence. I would also like to thank Sherry Gauthier and the rest of the Davies lab for their technical assistance and moral support. Thanks to my family and friends for their proofreading skills, encouragement and general positivity. Here's to new future directions! iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ............................................................................................................................ iv List of Figures ................................................................................................................................. vi List of Tables ................................................................................................................................. vii List of Abbreviations .................................................................................................................... viii Chapter 1 Introduction ..................................................................................................................... 1 1.1 Flounder type I AFPs ............................................................................................................. 3 1.1.1 Liver isoform ................................................................................................................... 3 1.1.2 Skin isoform .................................................................................................................... 7 1.1.3 Hyperactive isoform ...................................................................................................... 10 1.2 Type I AFP in the Pleuronectidae family ............................................................................. 12 1.2.1 Yellowtail flounder ....................................................................................................... 15 1.2.2 American plaice ............................................................................................................. 16 1.2.3 Other Pleuronectidae ..................................................................................................... 17 1.3 Selective pressure: Shaping evolution .................................................................................. 18 1.4 Goals and Objectives ............................................................................................................ 23 Chapter 2 Materials and Methods .................................................................................................. 24 2.1 Isolation of genomic DNA ................................................................................................... 24 2.2 Southern blotting .................................................................................................................. 25 2.3 Library preparation and amplification .................................................................................. 26 2.4 Library screening.................................................................................................................. 26 2.5 Phage isolation ..................................................................................................................... 27 2.6 DNA sequencing .................................................................................................................. 28 2.7 PCR analysis ........................................................................................................................ 29 2.8 Bioinformatic analysis .......................................................................................................... 32 Chapter 3 Results ........................................................................................................................... 34 3.1 Preface .................................................................................................................................. 34 3.2 Genomic DNA samples were of variable quality ................................................................. 34 3.3 Genomic Southern blot showed strong liver AFP gene signal in starry flounder ................ 35 3.4 Genomic Southern blot shows strong hyperactive AFP gene signal .................................... 38 iv 3.5 Genes for starry flounder liver and skin AFPs are closely linked ........................................ 39 3.6 Starry flounder liver and skin AFPs are homologous to their winter flounder counterparts 43 3.6.1 Liver isoform ................................................................................................................. 43 3.6.2 Skin isoform .................................................................................................................. 48 3.6.3 Regulatory elements ...................................................................................................... 51 3.7 The starry flounder has multiple variants of the liver and skin AFPs .................................. 52 3.7.1 Liver variants ................................................................................................................. 54 3.7.2 Skin variants .................................................................................................................. 57 3.8 The American plaice has multiple variants of the skin AFPs .............................................. 60 3.9 The starry flounder has multiple variants of the hyperactive AFPs ..................................... 62 3.10 Genes for starry flounder hyperactive and skin AFPs are closely linked ........................... 65 3.11 Regulatory elements are conserved in stfs-AFP8 ............................................................... 69 3.12 Starry flounder hyperactive AFP is homologous to its winter flounder counterpart .......... 70 3.13 Starry flounder-specific probes do not alter Southern blot banding patterns ..................... 71 Chapter 4 Discussion ...................................................................................................................... 73 4.1 Conclusions .......................................................................................................................... 80 References ...................................................................................................................................... 82 Appendix A DNA alignment of winter and starry flounder liver AFPs ......................................... 91 Appendix B DNA alignment of winter and starry flounder skin AFPs.........................................