cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.
A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002268:Carboxypeptidases / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D011132:Polyribosomes / Q000276:immunology; D012333:RNA, Messenger / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D012333:RNA, Messenger; D004247:DNA; D002268:Carboxypeptidases