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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034066-05
Application #
3171804
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1983-07-01
Project End
1990-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Arnold, T E; Farrance, I K; Morris, J et al. (1991) Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter. DNA Cell Biol 10:105-12
Clark, T G; Morris, J; Akamatsu, M et al. (1990) A bovine homolog to the human myogenic determination factor myf-5: sequence conservation and 3' processing of transcripts. Nucleic Acids Res 18:3147-53
Farrance, I K; Ivarie, R (1989) Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide. Anal Biochem 179:60-5
Farrance, I K; Eadie, J S; Ivarie, R (1989) Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer. Nucleic Acids Res 17:1231-45
Davis, R B; Morris, J; Ivarie, R (1987) The polypeptide P16 is a carboxy-terminal cleavage product of rat growth hormone in anterior pituitary and GH3 pituitary tumor cells. Mol Endocrinol 1:102-8
Phillips, G J; Arnold, J; Ivarie, R (1987) The effect of codon usage on the oligonucleotide composition of the E. coli genome and identification of over- and underrepresented sequences by Markov chain analysis. Nucleic Acids Res 15:2627-38
Phillips, G J; Arnold, J; Ivarie, R (1987) Mono- through hexanucleotide composition of the Escherichia coli genome: a Markov chain analysis. Nucleic Acids Res 15:2611-26
Ivarie, R (1987) Thymine methyls and DNA-protein interactions. Nucleic Acids Res 15:9975-83
Morris, J; Kushner, S R; Ivarie, R (1986) The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans. Mol Biol Evol 3:343-55
Ivarie, R; Morris, J A (1986) Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation. Mol Cell Biol 6:97-104

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