Isolation of cDNA clones encoding HLA-DR alpha chains.
HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.
D000818:Animals; D001402:B-Lymphocytes; D002051:Burkitt Lymphoma; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification; D006801:Humans; D046911:Macromolecular Substances; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014981:Xenopus
D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA