INACTIVATION OF GENE EXPRESSION BY DNA ALKYLATING AGENTS

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The proposed experiments will determine the molecular basis by which the DNA alkylating agent and chemical carcinogen, ethyl methanesulfonate (EMS), stably and heritably inactivates prolactin (rPRL) gene expression at high frequency by increasing the methylation of cytosine residues in the genome of GH3 rat pituitary tumor cells. Biochemical and molecular techniques will be utilized to test the hypothesis that alkylation of DNA by EMS promotes "maintenance" methylation of the modified DNA. Increases in the 5-methylceytosine content in DNA from GH3 cells treated with EMS will be assayed by metabolic labelling techniques and by in vitro DNA methylase assays. Partially purified preparations of mammalian DNA methylase will be used to measure the effect of various alkyl adducts on the methyl-accepting capacity of DNA. Alkylated DNAs will also be introduced into cultured cells by gene transfer techniques and subsequent methylation of the introduced DNA will be assayed by restriction enzyme digestions and Southern genomic blotting techniques. The structural and functional consequences of rPRL gene inactivation will also be assayed in the rPRL-deficient, revertant and wild type cells. The extent of methylation of CpG sequences in the rPRL gene and its flanking regions will be assayed by restiction enzyme digestions and genomic Southern blotting techniques. It will also be determined whether inactivation results in (1) a reduced sensitivity of the rPRL gene to digestion by DNAse I; (2) an acquisition of hypersensitive sites in 5 feet flanking sequences, and (3) a decrease in the level of nuclear precursor RNAs to cytoplasmic pre-rPRL messenger RNA. A HeLa cell mutant with a missense mutation in the gene for hypoxanthine phosphoribosyl transferase (HPRT) will be used to develop a model genetic system for monitoring the frequency of activation and inactivation of a silent HPRT gene in response to a variety of chemical carcinogens.