| Summary: | (a) The class III region of the human MHC is approximately 1.1 Mbp in size. Almost 50% has been cloned in two overlapping cosmid clusters by chromosome walking from the complement/ 210H gene cluster and from the TNF a locus. The complement/ 210H cosmid cluster consists of 45 independent recombinants, spaning 390 kb. The TNFα and TNF β cosmid cluster consists of 16 independent recombinants, spanning 150 kb. Each genomic insert has been characterised by restriction enzyme digestion and Southern blot analysis to produce a detailed restriction map of the cloned region. (b) Single copy DNA sequences isolated from the cosmid clones have been hybridised to Southern blots of genomic DNA digests separated by pulsed-field gel electrophoresis (PFGE). This analysis has established the orientation and precise position of the complement/ 210H gene cluster within the class III region relative to the class I and class II genes. Furthermore, probes from the TNF cosmid cluster have been used to show that the TNF genes lie ~250 kb centromeric to the HLA-B locus. The gene order from the centromere is HLA-DR-210HB-C2-TNF α-TNF β-HLA-B, with HLA-OR separated from 210HB by 300-350 kb, and C2 separated from HLA-B by 650 kb. (c) Genomic probes have been used in conjunction with PFGE to define the positions of RTF islands within the cloned portion of the class III region. Single copy sequences associated with these islands have been hybridised to Southern blots of DNA from a variety of animal species to look for cross-hybridisation indicative of phylogenetic conservation of coding sequences. These probes have been used to screen Northern blots and define island related transcripts. The products of 15 genes have been mapped within the cloned region. Of these, two have been identified as duplicated members of the HSP 70 family. The remaining loci appear to be single copy sequences. One (Gll) has been cloned and characterised.
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