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We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

Polymorphic restriction endonuclease sites within the HLA-DR alpha gene have been defined, localized, and used as genetic markers in the analysis of susceptibility to insulin-dependent diabetes mellitus (IDDM). Hybridization of Bgl II-digested human genomic DNA with a cDNA clone for the HLA-DR alpha chain (pDR alpha-1) has revealed three allelic restriction fragment lengths: 3.8 kilobase pairs (kb), 4.2 kb, and 4.5 kb. Hybridization of EcoRV-digested human genomic DNA with the same probe has revealed two allelic polymorphic restriction fragment lengths: 9.2 kb and 13.0 kb. By analysis of double digests of genomic DNA from individuals homozygous for each of the allelic variants, the polymorphic restriction sites were found to be clustered near the 3' end of the HLA-DR alpha gene. The observed correlations of DR alpha Bgl II restriction site variants with serologically determined DR specificities suggest linkage disequilibrium between the DR alpha and DR beta loci. The 3.8-kb fragment is correlated with the DR1 type (Pc = 4.4 X 10(-4)); and the 4.2-kb fragment, with a subset (B8,DR3) of the DR3 type (Pc = 5.1 X 10(-4)) and with the DR6 type. The segregation pattern of HLA-DR alpha polymorphic Bgl II restriction fragments was analyzed in six IDDM families. The observed association of IDDM with the Bgl II 4.2-kb DR alpha restriction variant is higher than with existing serological markers and supports the utility of this approach in elucidating IDDM inheritance.

The human class II gene, HLA-DR alpha, contains an octanucleotide sequence ATTTGCAT located approximately 40 base pairs upstream of the transcription initiation site. We have investigated the transcriptional function of the DR alpha octamer in human B-lymphoblastoid cells and non-B cells. Deletion and substitution mutagenesis of the octamer sequence greatly reduced the activity of the DR alpha promoter in both in vivo and in vitro cell-free transcription systems of B-cell origin. Conversely, these mutations did not affect promoter activity in several non-B-cell lines that express the DR alpha gene. Removal of octamer-binding proteins by in vivo titration with an octamer-containing competitor plasmid reduced DR alpha promoter activity in B-lymphoblastoid cells. These results suggest that a protein-octamer interaction, most likely involving the B-cell-specific octamer binding protein (OTF-2), is required for DR alpha promoter function in B-lymphoblastoid cells but not in non-B cells.

Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.

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.