Isolation of cDNA clones encoding HLA-DR alpha chains.

Abstract

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 a 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 ofcDNA, 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 a 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 ofHLA-DR a chains. The translation product ofHLA-DR a chain mRNA purified by hybridization-selection gives a single a chain spot on two-dimensional gels, whereas the a chain released from the a//3 HLA-DR complex gives about seven distinct spots. Finally, the results ofanalysis ofgenomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic a chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.

A defect in the regulation of major histocompatibility complex class II gene expression in human HLA-DR negative lymphocytes from patients with combined immunodeficiency syndrome.

“…RNA was prepared from PHA-induced blasts of two patients. These RNAs were analysed by Northern blot hybridization for the presence of mRNA specific of HLA-DR, 3-chains, or Ii-chains, using our specific cDNA probes (21,22,23). The same blot containing 6 and 1 gg of control RNA and 13 jig of RNA from lymphocytes of patient R.A.…”

Molecular organization of the HLA-SB region of the human major histocompatibility complex and evidence for two SB beta-chain genes.

“…The cloning of class II p-chain cDNA clones from a DR4,w6 cell line has been described (9 (9), HLA-DR a chain (16), and the SB p chain. Occasionally a fragment from a cosmid was subcloned to obtain a more detailed map.…”

Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain.

“…Preparation of RNA and screening of the cDNA library Total RNA (de Preval and Mach, 1983) and poly(A)+ RNA were prepared as described. The cDNA clones were constructed from an enriched mRNA fraction as reported by Wake et al, (1982). Screening of the cDNA library and preparation of plasmid DNA have already been described (Long et al, 1983).…”