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Pioneering Studies on the Gene Order, DNA Sequence and Evolution of the MHC Class-I Region in the New World Primate Callithrix Jacchus

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Pioneering Studies on the Gene Order, DNA Sequence and Evolution of the MHC Class-I Region in the New World Primate Callithrix Jacchus Pioneering studies on the gene order, DNA sequence and evolution of the MHC class-I region in the new world primate Callithrix jacchus Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von NATALIA REGINA MESA HERRERA aus Medellin (Kolumbien) Göttingen 2007 D 7 Referent: Prof. Dr. rer. nat. Jürgen Wienands Korreferent: Prof. Dr. rer. nat. Ernst A. Wimmer Tag der mündlichen Prüfung: am 5. Juli 2007 ……To my Family and Martin, who always were behind me, …… To my Friends, who encouraged me, …… To my Professors, who believed in me. Table of contents I Table of contents Page 1. Aims and scopes……………………………………………………… 1 2. Introduction……………………………………………………………... 3 2.1 Common marmoset CALLITHRIX JACCHUS……………………… 3 2.2 Taxonomy………………………………………………………………. 4 2.3 The major histocompatibility complex (MHC)………………………. 5 2.3.1 MHC class I molecules………………………………………………... 7 2.3.2 MHC class II molecules……………………………………………….. 9 2.3.3 MHC in Callithrix jacchus and others New World Monkeys (NWM) 10 2.3.4 MHC in other primates ……………………………………………….. 12 2.3.5 Evolution………………………………………………………………… 13 2.4 Framework genes hypothesis………………………………………… 14 2.4.1 ATP6V1G2……………………………………………………………… 15 2.4.2 BAT1…………………………………………………………………….. 15 2.4.3 TCF19…………………………………………………………………… 16 2.4.4 POU5F1………………………………………………………………… 16 2.4.5 TRIM family…………………………………………………………….. 16 2.4.6 TCTEX5……………………………………………………………........ 18 2.4.7 MOG …...…………………………………………………..…………… 18 3 Materials………………………………………………………………… 19 3.1 Chemicals……………………………………………………………… 19 3.2 Buffers and other solutions…………………………………………… 20 3.3 Media............................................................................................... 23 3.4 Commercials kits………………………………………………………. 24 3.5 Enzymes………………………………………………………………… 24 3.6 Oligonucleotides primers for PCR: sequences and annealing temperatures applied………………………………………………….. 25 3.7 DNA ladders and other standards for comparison of molecular size……………………………………………………………………… 26 3.8 Vectors………………………………………………………………….. 27 3.9 BAC-clones used for genomic analysis……………………………… 27 3.10 The gene sequence of Callitrix jacchus used as MHC class I probe……………………………………………………………………. 29 Table of contents II 3.11 Computer programs…………………………………………………… 29 3.12 Plastic articles………………………………………………………….. 29 3.13 Radioactive substrate…………………………………………………. 30 3.14 Lab equipment…………………………………………………………. 30 3.15 Addresses of manufacturers…………………………………………. 32 4 Methods………………………………………………………………… 34 4.1 Preparation of materials………………………………………………. 34 4.2 Filters of carrying bacterial artificial chromosome (BAC) clones for screening………………………………………………………………. 35 4.2.1 Source and origin of BAC bank CHORI of common marmoset (Callithrix jacchus)…...………………………………………………… 35 4.2.2 Organization of BAC bank CHORI 259 from of the new world monkey common marmoset (Callithrix jacchus) on microtiter plates and on nylon filters…………………………………………….. 35 4.2.3 Procedure for hybridization on high density filters…………………. 35 4.3 Preparation of a BAC clone cultures………………………………… 37 4.4 Isolating plasmid DNA (Miniprep)……………………………………. 38 4.5 Quantification of DNA…………………………………………………. 39 4.5.1 UV-absorption spectroscopy method……………………………….. 39 4.5.2 Minigel method………………………………………………………… 40 4.6 Restriction digestion of plasmid DNA ………………………………. 41 4.7 Separation of large DNA fragments by agarose gel electrophoresis………………………………………………………… 42 4.8 Extraction of DNA fragments from agarose gels…………………… 43 4.9 Random primer method………………………………………………. 46 4.10 Analysis of plasmid DNA by blotting and hybridization……………. 46 4.10.1 Southern blotting………………………………………………………. 46 4.10.2 Hybridization analysis of DNA………………………………………... 48 4.11 Autoradiography……………………………………………………….. 49 4.12 Polymerase chain reaction (PCR)…………………………………… 49 4.13 Inverse PCR……………………………………………………………. 51 4.14 DNA sequencing ……………………………………………………… 52 5 Results………………………………………………………………….. 54 Table of contents III 5.1 Identification of BAC clones from Callithrix jacchus containing MHC class-I region genes …………………………………………… 55 5.1.1 Contig 1, the interval BAT1 - TCF19………………………………… 58 5.1.2 Contig 3, the intervals TRIM39 - TRIM26…………………………… 59 5.1.3 Contig 4, interval TCTEX4 – MOG…………………………………… 61 5.2 Genomic analysis by Southern blot hybridization …………………. 62 5.2.1 Contig 1, the interval BAT1 – TCF19………………………………… 62 5.2.1.1 Southern blot analysis of contig 1…………………………………… 62 5.2.1.2 Evaluation of hybridization results (contig 1)……………………….. 77 5.2.1.3 Construction of a contig 1 map………………………………………. 81 5.2.2 Contig 3, the interval TRIM26 – TRIM39……..………….………..... 84 5.2.2.1 Southern blot analysis of contig 3……………..…………………….. 84 5.2.2.2 Evaluation of hybridization results (contig 3)…..…………………… 87 5.2.2.3 Contribution to a contig 3 map..……………………………………… 87 5.2.3 Contig 4, the intervall TCTEX4 - MOG …..…………………………. 89 5.2.3.1 Southern blot analysis of contig 4…………………………………… 89 5.2.3.2 Evaluation of hybridization results (contig 4)……………………….. 91 5.2.3.3 Contribution to a contig 4 map..……………………………………… 91 5.3 PCR and sequence analysis of BAC clone inserts….……………... 92 5.3.1 Polymerase chain reaction (PCR) analysis………………………… 92 5.3.2 Approximative alignment of sequences to homologoues DNA of Hs and other mammals by BLAST………….……………………….. 96 5.3.3 Sequences of contig 1………………………………………………… 99 5.3.4 Sequences of contig 3………………………………………………… 103 5.3.5 Sequences of contig 4……...…………………………………………. 106 5.4 Identification of the Callithrix jacchus sequences by comparative alignment……………………………………………………………….. 106 5.4.1 Programs used for identification of sequences by localization and gene assignment……………………………………………………..... 106 5.4.2 Collection and presentation of sequence data……………………… 113 5.4.3 Demonstration of individual sequences identified by localization and possibly gene assignment in the Hs genome or proteome sequence K19161C12-TS2…………………………………………… 114 Table of contents IV 5.4.4 K28-347D1-TCF19, nucleotide sequence…………………………... 116 5.4.5 MOG sequences……………………………………………………….. 118 5.5 Evolution of class-I sequences and framework genes found in Callithrix jacchus compared to their homologous in other primates and mammals………………………………………………………….. 120 5.5.1 Gene trees class-I of genes…………………………..………………. 120 5.5.2 Gene trees of framework genes……………………………………… 122 5.6 TS1: teachings and challenges………………………………………. 124 5.6.1 A gene map of TS1 and its homologous genomic vicinities in the duplicons B and C of Hs.…………………….……………………….. 124 5.6.2 The two new class I pseudogenes present in the HLA-B and -C duplicons of Hs, Patr and other mammals, also in the 14 Mamu-B duplicons………………………………………………………………... 127 5.6.3 A MHC ferritin heavy chain pseudogene in Callithrix jacchus…….. 134 5.6.4 Characterization and possible interpretations of “Vorspann” v, the sequence next to which a ferritin transposon has been inserted…. 141 6 Discussion……………………………………………………………… 142 6.1 Physical map of the intervals from MHC class-I……………………. 144 6.1.1 Physical map of the BAT1 – TCF19 interval (contig 1)……………. 144 6.1.2 Physical map of the intervals CAT56 – TRIM39 and TRIM 39 – TRIM26 (contig 2 and contig 3 respectively)………………………... 145 6.1.3 Physical map of interval TCTEX4 and MOG (contig 4)…………… 145 6.2 Sequence analysis of BAC clones…………………………………… 146 6.2.1 BAT1 gene in Callithrix jacchus……………………………………… 146 6.2.2 TCF19 gene in Callithrix jacchus…………………………………… 146 6.2.3 MOG gene in Callithrix jacchus……………………………………… 146 6.3 On alignment of sequences in general and in particular those of the Callithrix jacchus………………….………………………………. 147 6.3 On pseudogenes in the MHC class I region………………………… 147 6.4 On duplicons in the MHC class I region…………..………………… 148 6.5 On transposon especially the pseudo ferritin found in MHC Caja transposon in the evolution of MHC……………………..…………... 148 6.6 On potential value of sequence TS1 for molecular immunogenetics………………………………………………………... 148 Table of contents V 7 Summary………………………………………………………………... 150 8 References..…………………………………………………...…......... 152 9 Annex: The 55 Callithrix jacchus sequences……………………….. 175 Acknowledgements Lebenslauf Aims and Scopes 1 ……If you cannot study function, study structure! Francis Crick 1. Aims and scopes The object of these pilot studies is to contribute to the structural analysis of the MHC class I region in a lower primate, the new world monkey Callithrix jacchus (Caja). The aims are: • Selection of BAC clones containing inserts apt to genomic and sequence analysis of the MHC class I region; • Establishing the contigs belonging MHC class I in Callithrix jacchus of inserts for obtaining a restriction map and hybridization with class I probe and framework gene, this framework gene is flanking the MHC class I region. • Typifying BAC clone inserts selecting various primers by PCR. This knowledge should be useful for a complete walking chromosome sequencing of the entire class I region in Caja having been started recently by Prof. T. Shiina; • Identification and localization of the 55 sequences obtained by alignment in the Hs genome and other species. • Using the special opportunity of studying one sequence, TS1 containing apparently three different kinds of pseudogenes, interesting by an arrangement on both DNA strands, much more however, as they seem to concern three unsolved basic problems in immunogenetics, i.e. gene clustering by duplication, control of gene expression by promotor regions (5’FR), and transposition of (pseudo)genes. The scope of these pilot investigations in some parts is
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