The rat is an important model for studying organ graft rejection and susceptibility to certain complex diseases. The MHC, the RT1 complex, plays a decisive role in controlling these traits. We have cloned the telomeric class I region of the RT1 complex, RT1-C/E/M, of the BN inbred rat strain in a contig of overlapping P1-derived artificial chromosome clones encompassing ∼2 Mb, and present a physical map of this MHC region. Forty-five class I exon 4-hybridizing BamHI fragments were detected, including the previously known rat class I genes RT1-E, RT-BM1, RT1-N, RT1-M2, RT1-M3, and RT1-M4. Twenty-six non-class I genes known to map to the corresponding part of the human and mouse MHC were tested and could be fine mapped in the RT1-C/E/M region at orthologous position. Four previously known microsatellite markers were fine mapped in the RT1-C/E/M region and found to occur in multiple copies. In addition, a new, single-copy polymorphic microsatellite has been defined. The expression profiles of several class I genes and the 26 non-class I genes were determined in 13 different tissues and exhibited restricted patterns in most cases. The data provide further molecular information on the MHC for analyzing disease susceptibility and underline the usefulness of the rat model.
Inhaltsverzeichnis __________________________________________________________________________________________ II 3.2.5 Kleinansatz zur Präparation von PAC-DNA 3.2.6 Kleinansatz zur Plasmidisolierung (lange Methode) 3.2.7 Minipräparation von Plasmid-DNA 3.3 Konzentrations-und Reinheitsbestimmung von DNA 3.4 Spaltung von DNA mit Restriktionsenzymen 3.4.1 Partieller Verdau genomischer DNA für Bank-Konstruktion 3.5 Gelelektrophorese von Nukleinsäuren 3.6 Transfer von DNA aus Agarosegelen (Southernblot) 3.7 Radioaktive DNA-Markierung mittels random-priming Reaktion 3.8 Radioaktive Endmarkierung synthetischer Oligonukleotide durch γ-[ 32 P]-dATP 3.9 Hybridisierung der Southernblots 3.10 Autoradiographie 3.11 Bakterien-Kolonie-Screening erstellter Subbanken 3.12 Erstellung von DNA dot blots 3.13 Isolierung von DNA-Fragmenten aus Gelen 3.13.1 Isolierung von DNA-Fragmenten aus Agarosegelen mittels JETQUICK spin columns 3.13.2 Isolierung von DNA-Fragmenten aus Agarosegelen durch Elektroelution 3.14 Dephosphorylierung freier 5'-Enden 3.15 Ligation von DNA-Enden 3.16 Nukleotidsequenzanalyse doppelsträngiger DNA 3.17 Endsequenzierung der PAC-Klone durch inverse PCR 3.18 Reverse Transkription 3.19 Polymerasekettenreaktion (PCR) 3.20 3'RACE (rapid amplification of cDNA ends) 3.21 Long range PCR 3.22 Klonierung von PCR-Produkten 3.23 Screening einer PAC-Bank 3.24 Herstellung elektrokompetenter Bakterienzellen 3.25 Elektroporation 4 Ergebnisse 4.1 Konstruktion einer genomischen Bank aus der LEW.1W-Ratte (RT1 u -Haplotyp) 4.2 Etablierung von Hybridisierungsproben 4.2.1 Sonden aus klassischen MHC-Klasse-I-Genen 4.2.2 Herstellung von Hybridisierungsproben aus MHC-Klasse-Ib-Genen 4.2.3 Herstellung von Hybridisierungssonden aus verschiedenen "Ankergenen" 4.2.4 Herstellung von STS-Markern bei der Ratte und Maus 4.3 Klonierung und physikalische Kartierung der RT1-C/M Region anhand einer genomischen Bank aus der BN Ratte (RT1 n -Haplotyp) 4.3.1 Contig 1 4.3.1.1 Sequenzierung der Enden einiger PAC-Klone aus dem Contig 1 4.3.1.2 11/3R-homologe Gene im Contig 1 Inhaltsverzeichnis __________________________________________________________________________________________ III 4.3.1.3 Subklonierung und Teil-Sequenzierung einiger Klasse-I-Gene aus dem Contig 1 4.
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