Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.
We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha 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 alpha 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 NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha 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 alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens; D006801:Humans; D046911:Macromolecular Substances; D008285:Major Histocompatibility Complex; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D006684:HLA-DR Antigens; D046911:Macromolecular Substances; D009841:Oligonucleotides; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA