Pioneering studies on the gene order, DNA sequence and evolution of the MHC class I region in the new world primate Callthrix jacchus
Pionieruntersuchungen der Gen-Reihenfolge, DNA-Sequenz und Evolution der MHC-Klasse-I-Region in Neue-Welt-Primaten Callithrix jacchus
by Natalia Regina Mesa Herrera
Date of Examination:2007-07-05
Date of issue:2007-08-02
Advisor:Prof. Dr. Johannes Heinrich Matthaei
Referee:Prof. Dr. Jürgen Wienands
Referee:Prof. Dr. Ernts A. Wiemmer
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EnglishAnalysis of MHC class I region in common marmoset (Callithrix jacchus) was made with 42 BAC clones. These clones containing class I region genes were identified for the purpose of both restriction mapping and PCR sequencing. Contigs 1, 3 and 4 of BAC clones were obtained for genomic analysis by comparison of EcoRI fragments characterized by Southern blot fingerprint hybridization with framework and class I gene probes. A gene map for the interval BAT1 - PUO5F1 was constructed by comparison of the Caja EcoRI fragments so characterized with the computer-split EcoRI fragments of the Hs class I region having known gene assignments. Ancillary evidence needed for identification of a fragment obtained from six BAC clones was obtained by analysis of sections of the Hs DNA sequence near POU5F1 for class I pseudogenes using a collection of all Hs class I pseudogene sequences for comparison of their alignment by the neighbour-joining program. Seven new pseudogenes were thereby detected within only 50 kb from POU5F1, each one classified by its greatest similarity with a known pseudogene. Adequately refined, this method might be useful for finding and classifying all of the many class I pseudogenes. A genetic typification of 42 BAC clone inserts by PCR with various primers was performed. This knowledge was used for pilot sequencing in the class I region of Caja. The resulting 55 sequences were localized and potentially gene-assigned by comparative alignment, generally with DNA of Hs, in many cases also of other primates and mammals. These sequence localizations and assignments were defined by their first and last bp numbers on the scales of Shiina and also of the Venter genome. For survey, they were both tabulated and entered into a to-bp-scale map drawn for the Hs class I region. TS1, one of the two Caja sequences established by Prof. Takashi Shiina using a BAC clone selected by the author, was shown to contain within its 2994 bp four different pseudogenes of peculiar interest: on the right hand strand, two class I pseudogenes (ps1 and ps2); on the left hand strand, reading anti-parallel, a rather young ferritine heavy chain pseudogene (psfth1) apparently unique to Caja; and next to it exist a Vorspann v, potentially an ancient 5 FR (flanking region) which in its reverse-complementary, the right hand strand turns out to be the known pseudogene HCGIV-02, as a very highly identical, almost completely coincident sequence. The two new pseudogenes, ps1 and ps2, were recognized as being useful indicators for the duplicated DNA as found in the two duplicons B and C of Hs and Patr that contain the ps1, ps2 and v homologues near the classical HLA genes B and C, respectively. Alignment of ps1, ps2 and v could be observed as well in all of the 14 Mamu B duplicons of the rhesus monkey Macacca mulatta. High identity alignment of ps1, ps2 and v is being searched for in the vicinities of other classical HLA genes being conditions for duplicons. Physical mapping and sequence analysis of the MHC class I region of Callithrix jacchus is to supply new genomic basic data, which further analyses of the MHC region for evolution of MHC, disease susceptibility, immune reactivity and transplantation repulsion of the MHC to make possible. Additionally the map offers the basis for following Sequencing of the BAC clones of the individual contigs.
Keywords: MHC; class I region; Callithrix jacchus; framework gene; evolution
Der Weissbüschelaffe Callithrix jacchus dient als Tiermodell zur Erforschung von Infektionen, Arzneimittelwirksamkeiten, der Parkinson-Krankheit sowie Organtransplantationen. Ferner wurde an ihm die Durchführbarkeit von Zell- und Gentherapie bei immunologischen und hämatologischen Störungen getestet. Dieser Neuweltaffe ist von besonderer Bedeutung wegen seiner Ähnlichkeit zum Menschen. Ziel dieser Arbeit war es die vier Klasse-I-Intervalle (Contig 1-4), einschließlich der flankierenden Framework-Gene, der MHC-Klasse-I-Region des Weissbüschelaffen Callithrix jacchus auf genomischer Ebene zu analysieren. Hierzu wurde eine BAC-Bank (CHORI 259) von Callithrix jacchus mit Sonden von zwei MHC-Klasse-I-Genen und mehreren Framework-Genen gescreent. Southern-Blot- und Sequenzanalyse der MHC-Klasse-I-positiven BAC-Klone ermöglichte die Erstellung einer Karte über die Anordnung der MHC-Klasse-I-Gene im Weissbüschelaffen. Die physikalische Kartierung der MHC-Klasse-I-Region von Callithrix jacchus liefert neue Grundlagen zur weiteren Analyse der MHC Evolution und zur Erforschung von verschiedenen Krankheiten, die mit MHC assoziert sind. Außerdem dient die Karte als Grundlage für die komplette Sequenzierung der MHC Region.
Schlagwörter: MHC; Klasse-I-Region; Callithrix jacchus; Framework-Gen; Evolution