06-Methylguanine DNA methyltransferase (OMMT) is an important protein that functions in the repair of 06-methylguanine lesions in DNA that may be of some importance in carcinogenesis, as well as in cancer chemotherapy. The long-term objective of the research is to understand the nature of the OMMT gene in mammalian systems, the details of its molecular biology, and the manner of its regulation.
The specific aims of this proposal are to: a) isolate the human OMMT gene from 2 cosmids (102 and A-36) that upon transfection are able to confer resistance to chloroethylnitrosourea (CNU) in CHO-D422 (mex-) cells, determine the DNA sequence of this gene, and examine its conservation in the human, rat, mouse and hamster; b) characterize the human OMMT mRNA, determine its rate of transcription in the livers of gamma- irradiated rats by using northern hybridization analysis, and ascertain if methylated OMMT can act as a trans-activator: c) determine the amino acid sequence of OMMT from protein that has been separated by SOS-PAGE, prepare antibodies to OMMT by using synthesized oligopeptides, and ascertain the stability of OMMT in the livers of the irradiated rats; and d) determine the specificity of OMMT for ras sequences that have been methylated at various guanines within and in the region of codon 12 by using synthetic 15 mer double-stranded oligodeoxyribonucleotides; and e) characterize the DNA repair capabilities of Cosmid 102-transformed excision repair-deficient CHO mutant cells, uV 5, UV 20 and UV 41, in regard to nitrosoureas, MNNG, and cis-platinum.
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