MOLECULAR GENETIC BASIC FOR HLA POLYMOPHISM

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The objective for this proposal is to investigate the molecular genetic basis for the extensive polymorphism which exists for the HLA Class II histocompatibility antigens. Since these gene products are expressed on Epstein-Barr virus transformed B-lymphoblastoid cell lines (B-LCL), the approach will be to determine the nucleotide sequence of cloned cDNAs constructed from RNA of B-LCL. Because of the extensive polymorphism of the HLA-Class II determinants observed in the population with at least 13 serologically detectable HLA-DR haplotypes, it will be necessary to sequence several genes for each supertypic DR specificity (e.g. several DR1 haplotypes, several DR2 haplotypes, etc.). B-LCL from individuals who are homozygous for HLA as a result of being derived from offspring of first cousin marriages will be obtained from collaborating laboratories throughout the world as part of the ongoing preparations for the Xth International Histocompatibility Workshop. cDNA libraries from 20 representative B-LCL will be prepared in Lambdagt 10. The libraries will be screened with HLA Class II DNA probes and appropriate cloned genes will be prepared for nucleotide sequencing. The clones will be distributed to participating laboratories as inserts in the plasmid vector, pUC12. The clones will be made available during late Fall of 1986. The sequencing of the clones will be performed by the investigators who participate in this component of the Xth International Histocompatibility Workshop. Each laboratory will be responsible for subcloning the insert fragment into M13. It is anticipated that this collective effort on cDNA nucleotide sequencing of HLA class II genes will contribute substantially to the identification of gene and allele specific nucleotide sequences.