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University of Ulm Department of Human Genetics Prof. Dr. med. Walther Vogel Cytogenetic and Molecular Characterization of the Macro- and Micro-inversions, which Distinguish the Human and the Chimpanzee Karyotypes - from Speciation to Polymorphism Thesis Applying for the Degree of Doctor of Human Biology (Dr. hum. biol.) Faculty of Medicine University of Ulm Presented by Justyna Monika Szamalek from Wrze śnia in Poland 2006 Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin 1. Berichterstatter: Prof. Dr. med. Horst Hameister 2. Berichterstatter: Prof. Dr. med. Konstanze Döhner Tag der Promotion: 28.07.2006 Content Content 1. Introduction ...................................................................................................................7 1.1. Primate phylogeny........................................................................................................7 1.2. Africa as the place of human origin and the living area of the present-day chimpanzee populations .................................................................9 1.3. Cytogenetic and molecular differences between human and chimpanzee genomes.............................................................................................10 1.4. Cytogenetic and molecular differences between common chimpanzee and bonobo genomes................................................................................17 1.5. Theory of speciation .....................................................................................................18 1.6. Theory of selection .......................................................................................................20 1.7. Aims of the work presented in this thesis.....................................................................21 2. Materials and Methods .................................................................................................22 2.1. Materials .......................................................................................................................22 2.2. Methods ........................................................................................................................36 3. Results .............................................................................................................................47 3.1. Characterization of the chimpanzee lineage-specific pericentric inversion of chromosome 5 .........................................................................47 3.2. Characterization of the human lineage-specific pericentric inversion of chromosome 1 .........................................................................54 3.3. Verification of identity of the chimpanzee-specific pericentric inversions between Pan troglodytes and Pan paniscus ..............................66 3.4. Determination of the human-chimpanzee DNA divergence rate within inverted and collinear regions..................................................71 3.5. Genome-wide search for micro-inversions by gene order comparison between human and chimpanzee .......................................75 4. Discussion .......................................................................................................................84 4.1. Comparison of the pericentric inversions of chromosomes 1 and 5.............................84 4.2. Determination of the time interval within which the chimpanzee lineage-specific inversions became introduced ..........................................................89 4.3. Testing of the chromosomal speciation theory.............................................................90 3 Content 4.4. Micro-inversions as a newly discovered class of human-chimpanzee divergence ....................................................................................94 4.5. Inversions and their influence on neighbouring genes .................................................97 4.6. Conclusions ..................................................................................................................99 5. Summary ..................................................................................................................... 101 6. References .................................................................................................................... 103 7. Appendix ..................................................................................................................... 118 8. Acknowledgements ..................................................................................................... 131 9. Curriculum vitae ........................................................................................................ 132 4 Abbreviations Abbreviations aCGH Array based comparative genomic hybridization AIDS Acquired immune deficiency syndrome Alu Short stretch of DNA characterized by the action of the Alu restriction endonuclease isolated from Arthrobacter luteus BAC F-factor-based bacterial artificial chromosome bp Base pair BSA Bovine serum albumin CCD Charge-coupled device CND Copy number difference CNV Copy number variation CNP Copy number polymorphism DAPI 4,6 Diamidino-2-phenylindole DC Duplication cluster DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate dUTP Deoxyuridine triphosphate EBV Epstein-Barr virus EDTA Ethylene diaminetetraacetic acid ERV Endogenous retroviral sequences F1 First filial generation, in Mendelian genetics means the hybrid offspring of purebred parental generation FCS Fetal calf serum FISH Florescence in situ hybridization FITC Fluorescein isothiocyanate GGO Gorilla gorilla (Gorilla) HSA Homo sapiens (Human) HERV-K Human endogenous retrovirus K ISCN International System for Human Cytogenetic Nomenclature kb Kilobase L1 Family of long transposable sequences belonging to LINEs LCR Low copy repeat LINE Long interspersed nuclear elements 5 Abbreviations LTR Long terminal repeat Mb Megabase MFA Macaca fascicularis (Macaque) Mya Million years ago NAHR Non-allelic homologous recombination NHEJ Non-homologous end joining PAC P1-derived artificial chromosome PBS Phosphate buffered saline solution PCR Polymerase chain reaction PHA Phytohemagglutinin, an initiator of mitosis PPA Pan paniscus (Bonobo) PPY Pongo pygmaeus (Orangutan) PtERV Pan troglodytes endogenous retrovirus PTR Pan troglodytes (Common chimpanzee) RT Room temperature SD Segmental duplication SDS Sodium dodecyl sulfate SINE Short interspersed nuclear elements SSC Saline sodium citrate solution TE Tris-EDTA Tris Tris (hydroxymethyl) aminomethane 6 Introduction 1. Introduction It was postulated by Goodman in 1999 that, if the taxonomic classification were based exclusively upon genomic DNA sequence similarity, the nearly 99% identity of human, chimpanzee and bonobo genomes would require a reclassification of the latter two into the genus Homo . However, whereas there are many similarities in the biology, life history, and the behaviour of humans and great apes there are also many striking variations that need to be explained. The mostly recognized and often mentioned differences are the bipedalism and the developed speech, which are exclusive to humans, but also differences in susceptibility to diseases such as AIDS or malaria, and above all the large brain size and extended cognitive capacities that unique human characteristics (reviewed in Olson and Varki 2003; Carroll 2003; Varki and Altheide 2006). 1.1. Primate phylogeny More than 300 species of over 60 genera were classified as extant members of the order Primates. The data obtained from comparative sequencing approaches suggest that Primates divide into the Strepsirrhini (e.g. lemurs, lorises), the Tarsiiformes (tarsiers) and the Anthropoidea . The letter ones split into the Platyrrhini , which are represented by New World monkeys, and the Catarrhini that consist of Old World monkeys and apes (Hominoidea ) (Goodman et al. 2005; Figures 1 and 2). The story of the hominoid taxonomy is one of gradual demotion of humans from a special position in the taxonomy to being one branch among many. Although according to the modern taxonomy, based on the genetic similarities, humans are included in the group of apes, in this work I will refer to humans and great apes or African great apes separately, in the now colloquial sense (Figure 1). The close evolutionary relationship of humans with the African great apes i.e. chimpanzees and gorillas was posited already more than 100 years ago (Darwin 1871; Huxley 1863). In the traditional theory based on anatomical and mental differences between these species, the chimpanzees and the gorillas were grouped closer to the orangutans rather then to humans. In fact, there is already convincing DNA sequence evidence, that not only are chimpanzees and gorillas more closely related to humans than to orangutans but also chimpanzees and humans are more closely related to each other than either is to gorillas (Goodman et al. 2005). Despite the cladistics nuisances, there is general agreement that human ( Homo sapiens ) shared a common ancestor with chimpanzee ( Pan troglodytes ) and bonobo (Pan paniscus ) ~5-7 million years ago (Mya), with gorilla 7 Introduction (Gorilla gorilla ) ~7-8 Mya, and with orangutan (Pongo pygmeus ) ~12-13 Mya (Yoder and Yang 2000; Chen and Li 2001; Brunet et al. 2002). PRIMATES Prosimii Anthropoidea Catarrhini Orangutans Gorilla Bonobo Common Human chimpanzee chimpanzee AFRICAN GREAT APES Strepsirrhini Tarsiiformes Platyrrhini GREAT APES Malagasy Lorises & Tarsiers New World Old World Gibbons & lemurs galagos monkeys monkeys
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