Atypicat molecular evolution of afrotherian and xenarthran B-globin cluster genes with insights into the B-globin cluster gene organization of stem eutherians. By ANGELA M. SLOAN A thesis submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology University of Manitoba Winnipeg, Manitoba, Canada @ Angela M. Sloan, July 2005 TIIE I]MVERSITY OF' MANITOBA FACULTY OF GRADUATE STT]DIES ***** - COPYRIGHTPERMISSION ] . Atypical molecular evolution of afrotherian and xenarthran fslobin cluster genes with insights into thefglobin cluster gene organization òf stem eutherians. BY Angela M. Sloan A ThesislPracticum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfill¡nent of the requirement of the degree of Master of Science Angela M. Sloan @ 2005 Permission has been granted to the Library of the University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the fiIm, and to University Microfïlms Inc. to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright ownér. ABSTRACT Our understanding of p-globin gene cluster evolutionlwithin eutherian mammals .is based solely upon data collected from species in the two most derived eutherian superorders, Laurasiatheria and Euarchontoglires. Ifence, nothing is known regarding_the gene composition and evolution of this cluster within afrotherian (elephants, sea cows, hyraxes, aardvarks, elephant shrews, tenrecs and golden moles) and xenarthran (sloths, anteaters and armadillos) mammals. To address this shortcoming, I sequenced and classified atotal of 24 'þ-llke' globin genes from 1 I afrotherian and Z xenarthran species, and conducted a comprehensive analysis on the molecular evolution of p-globin cluster genes within these two basal eutherian clades. Analyses of the 'adult expressed' F- atrd ò-globin products suggested that the ô- globin locus of paenungulate (and presumably all afrotherian) mammals encodes the only 'f3-type' chain component of their post-natal hemoglobin. This finding challenges recent views on the evolution of the eutherian p-globin cluster, and provides the first documented evidence of a dispensable plocus within Mammalia. It appears that the ò- locus of stem afrotherians gained a functional advantage by means of a capable promoter region, donated by the neighbouring p gene via gene conversion, after which p was silenced through nonfunctionalization. Hyraxes were also found to possess a second putatively functional ô-globin gene (òH), which may or may not be expressed. A ML analysis of ò-globin IVS2 sequences grouped elephants and sirenians into a monophyly to the exclusion of hyraxes supporting the 'Tethytheria' hypothesis, and placed the tree hyrax as the most basal hyrax species. Analyses of the 'embronically expressed' r-, y- and q-globin genes revealed the presence of both Y- and e-loci within members of the *p"T.9:: Afrotheri4 providing compelling evidence that the ancestral eutherian p-globin cluster ðontained at least four genes (u, ò, p) T, by the time this clade diverged from other eutherians -107 mya. Earlier studies have revealed that hemoglobin of 5-month old elephant fetuses and manatee calves contains two 'F-type' chains. My data suggests these distinct p-chain components are encoded by the Y- and ôloci in these species, thus providing only the second example of developmentally delayed expression of a y locus within mammals. Sequence analyses of the armadillo B-globin cluster further indicated that the eutherian p-cluster contained five (e, ò, p) loci Y, ï, by the time xenarthrans diverged from eutherians -102 mya. Remarkably, of the five armadillo loci, only the two outermost genes (e and p) appeared capable of transcribing a polypeptide. Together with earlier studies that demonstrated adult armadillos express two distinct p-type chains, this finding implies that these xenarthrans may uniquely extend expression of the e-locus into post-natal growth stages or, like marsupials, possess an unlinked 'pJike' gene outside their p-globin cluster. A comparative assessment of evolution rates indicated that, with few exceptions, descendants of the'e-like' loci diverged more slowly than the 'p-like' loci in both coding and non-coding gene regions within species of all four eutherian superorders. These findings signify that rates of evolution for the parlogous genes within eutherian p-clusters are a function of selection pressure, contradicting the molecular clock theory that all genes evolve at an equal rate. ACKNOWLEDGEMENTS First and foremost, I wpuld like to thank mV acad-e]1i,c advijor, Dr. Kevin Campbell, for giving me the opporhrnity to gain invaluable research experience, and devising a thesis project to which I am proud to attach my name. I am also very grateful for all your adVice, patience, and good-natured kidding, which provided me with a pleasant work environment for the entirety of my graduate work. You took me on as a novice to the field of molecular phylogenetics and I thank you for your faith in me - it will forever be appreciated. I also thank my other committee members, Dr. Gunnar Valdimarsson who not only donated essential lab equipment but plentiful advice concerning laboratory procedures, and Dr. Ivan Oresnik who allowed me to partake in an intriguing research project which ultimately boosted my career interest in the flreld of site-directed mutagenesis. I additionally appreciate the helpful feedback they both supplied throughout the course of my thesis project, which proved invaluable to the efficiency of data collection and analyses. Thanks to both for your kind and generous natures, as well as your eyes for detail! For allowing me to work in his lab for more than two years, I extensively thank Dr. Nate Lovejoy, who was more patient and giving than I could have ever hoped for. I also thank the "Lovejoy Lab" graduate students, Kristie Lester, Stuart Willis, and Tommy Sheldon for their friendship and moral support over the years. I must also graciously thank the providers of blood, tissue, and DNA samples. These include Andrew Taylor at the University of Pretoria in South Africa, Brenda McDonald at the University of Adelaide in Australia, Christophe Douady at Université 111 Claude Bernard Lyon I in France, Gabi fürlach at the Marine Biological Laboratory in Woods Hole, Massachusetts, Gary Bronner at the University of Cape Town in South Africa, Galen Rathbun at the California Academy of Sciencei irr Cam6ri4 California, Gordon Glover at the Assiniboine Park Zoo in Winnipeg, Manitob4 Robert Bonde at the Florida Integrated Science Centre in Gainseville, Florida, Sarit¿ Maree at the University of Pretoria in South Africa, Terry Robinson at the University of Stellenbosch in Cape Town, South Africa, and Wendy Korver at the Bowmanville Zoo in Ontario, Canada. Without the generous donations of these skilled academics, this study could not have been the complete molecular analyses I dreamed it would be. I could not forget my mother, Merle, and father, Gary, to whom I thank for everything they have ever given me, be it advice, support, friendship, and love. These are the best parents anyone could ask for and I owe them everything I also thank my sister, Jennifer, for a precious friendship and an immense amount of moral support and encouragement when I needed it most. Thanks as well to Amrit Singh for all the talks and advice on lab procedures, and to Octavio Orellana for providing me with analytical equipment and helping me become a stronger and more confident individual. I love you all. Last but not leas! I would like to thank the organizations that funded my research, including the University of Manitoba Graduate Fellowship association (providing financial support to myself), and the Natural Sciences and Engineering Research Council of Canada (NSERC), The Winnipeg Foundation and the University of Manitoba Research Grants Program (tlRGP) (providing supporr to Kevin Campbell) 1V TABLE OF CONTENTS Page Abstract Acknowledgements 111 Table of Contents List of Figures List of Tables xll Abbreviations xlv General Introduction Chapter I Atypical molecular evolution of afrotherian and xenarthran 'B-like' globin genes ....... 7 Introduction 8 Materials and Methods l3 Results 19 Discussion 38 Chapter II Molecular evolution of the eutherian p-globin cluster infened from afrotherian and xenarthran gene sequences . 45 Introduction ... ... .. 46 Materials and Methods ... .. 49 Results . ... ... 52 Discussion ... ..... 69 Literature Cited 77 Appendix I t-1 95 Primers used in lhe (A) amplification and @) sequencing reactions of "p-like'globin genes. T-2 96 DNA sequences of the 'BJike' gfobin genes amplifred in this study. 1-3 125 Lengths of exons and introns for sequenced 'p-like' globin genes. r-4 726 Lengths of additional sequence data obtained for the 5' external region of sequenced 'p-like' globin genes, and positions of upstream features relative to the putative CAp site. 1-5 127 Lengths of additional sequence data obtained for the 3' external region of sequenced 'p-like' globin genes, and positions of poly- adenylation signals (AATAAA) relative to the termination codon. Appendix 2 2-l 128 Primers used in the (A) amplification and (B) sequencing reactions of "e-like' globin genes. 2-2 tze DNA sequences of the 'e-like' sì"ui' g"n", ;*piir,; ir rr,ir' sh:dy. 2-3 t42 Lengths of exons and intons for 'e-like' globin genes 2-4 t43 Lengths of sequence data obtained for the 5' external region of 'e-like' globin genes, and positions of upstream features relative to the putative Cap site.