We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the α chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A) + mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR α chain, were used to prime a cDNA synthesis reaction on various poly(A) + mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH 2-terminal amino acids of the HLA-DR α chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR α mRNA was confirmed by primer extension and blot analysis. The CDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A) + mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of CDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR α chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR α chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.
|Number of pages
|Proceedings of the National Academy of Sciences of the United States of America
|Published - 1982