Isolation of a human DNA repair gene by selection in Chinese hamster ovary cells
Alkylation of DNA at the O/sup 6/-position of guanine represents a potent mutagenic and carcinogenic lesion. O/sup 6/-Methylguanine DNA methyltransferase is the repair system responsible for catalyzing the transfer of the methyl group to a cysteine of the protein in a suicide reaction. The gene controlling its expression in mammalian systems is designated mex. Resistance to chloroethylnitrosourea (CNU) is also mediated by this protein; this was used to select cells into which the max gene has been introduced. DNA purified from human liver has been transfected into mex/sup -/ CHO cells by the CaPO/sub 4/ method. pSV2gpt, containing a marker gene, gpt, was cotransfected. The transformed cells were initially selected for the expression of gpt (mycophenolic acid resistance) and reselected in CNU for mex/sup +/. Several clones were resistant to both demonstrating the linkage of these genes. A cosmid library was made from a mex/sup +/gpt/sup +/ clone and grown in a gpt/sup -/ strain of E. coli. gpt/sup +/ colonies were selected and the cosmid DNA rescued. One of the tested cosmid DNA's produced CNU resistance upon introduction into CHO cells. This cosmid was subcloned, restriction endonuclease-treated and a 5.3 kb fragment showed mex activity. This fragment is being further characterized and the DNA sequenced.
- Research Organization:
- Univ. of Nebraska Medical Center, Omaha
- OSTI ID:
- 6365839
- Report Number(s):
- CONF-870644-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA
ALKYLATION
DNA REPAIR
BIOCHEMISTRY
GENES
CHEMICAL COMPOSITION
DNA SEQUENCING
NITROSOUREAS
TOXICITY
TRANSFERASES
ENZYME ACTIVITY
BIOLOGICAL MARKERS
CHO CELLS
ESCHERICHIA COLI
MAN
ANIMAL CELLS
ANIMALS
BACTERIA
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CHEMICAL REACTIONS
CHEMISTRY
ENZYMES
MAMMALS
MICROORGANISMS
NITROSO COMPOUNDS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PRIMATES
RECOVERY
REPAIR
STRUCTURAL CHEMICAL ANALYSIS
VERTEBRATES
560300* - Chemicals Metabolism & Toxicology