Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts.
Antisera were raised in rabbits to three peptides which correspond to sequences of amino acids present in human O6-alkylguanine-DNA alkyltransferase (residues 1-11, 8-20 and 197-207). These antisera recognized the alkyltransferase protein on Western blots of proteins from a number of Mer+ human tumor cell lines but this protein was found to be absent from Mer- tumor cell lines. The alkyltransferase protein was also detected by these antisera in some extracts from human liver samples but other human liver extracts having a high alkyltransferase activity failed to react with antibodies directed towards the carboxyl terminus of the protein, suggesting that part of this region can be removed by protease action without loss of activity or that there is genetic variability in this region. This result indicates that studies with a number of antisera or with an antibody known to be directed towards an essential, invariant region of the alkyltransferase will be needed for unequivocal detection of the alkyltransferase by antibody screening. The immunoreactive human alkyltransferase protein was absent from CHO cell lines that had previously been selected for alkyltransferase expression after transfection with human DNA. It is therefore probable that the hamster gene has been reactivated in these cells.
Pegg
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AE
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey 17033.
Wiest
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L
Mummert
C
C
Dolan
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ME
D000818:Animals; D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000276:immunology* / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010455:Peptides / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010455:Peptides; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase