The proposed experiments are directed toward understanding the molecular basis by which the DNA alkylating agent and chemical carcinogen, ethly methanesulfonate (EMS), enhances methylation of cytosines in CpG sites and inactivates the expression of the rat prolactin (rPRL) gene in an GH3 rat pituitary tumor cells. Toward this end, the transcriptional activity of the gene will be assayed in wildtype, deficient and revertant GH3 cells by measuring the number of RNA polymerase II molecules engaged in rPRL gene transcription by nuclear runoff transcription. Primary transcripts of the rPRL gene will also be assayed by Northern blotting. The 5' and 3' ends of the rPRL mRNA will be mapped by primer extension and S1 nuclease digestion to determine whether the same transcription start and termination sites are used in variant and revertant lines. Cytoplasmic turnover rate of the mature rPRL message will also be measured to determine whether stability of the mRNA is affected in the deficient cells. To determine whether rPRLdeficient cells have lost the expression of a transacting factor, transient expression vectors containing the promotors and 5' flanking regions of the rPRL and rat growth hormone (rGH) gene will be used to transfect wildtype and deficient lines. Regions of flanking DNA containing cisacting enhancer sites will be assayed for binding potential regulatory factors extracted from wildtype and variant cell nuclei. Potential EMS target sequences in the rat rPRL gene which when modified by EMS stimulate enzymatic methylation and inactivates rPRL expression will be sought. CpG sites in the 5' flanking region will be methyulated and assayed for their ability to alter rPRL promotor function and genomic sequencing will be used to locate CpG sites whose methylation correlates with the expression pattern of the gene in wildtype and deficient lines. Sites hypersensitive to DNAse I digestion will also be analyzed to correlate with expression of the gene in wild-type and variant liens. The activity of mammalian DNA methylase on CpG sites containing N7ethylguanine 5' and 3' to cytosine will be measured to determine whether the N7ethyl adduct is responsible for in vitro stimulation of DNA methylase activity on EMS-treated poly(dCdG).poly(dC-dG). An in vivo assay using a nested set of restriction enzyme sites overlapping at a GCG site will be used to measure whether N7ethyl adducts at either G site stimulates enzymatic methylation of cytosine in the site.