# INACTIVATION OF GENE EXPRESSION BY DNA AGENTS

> **NIH NIH R01** · UNIVERSITY OF GEORGIA · 1988 · $105,418

## Abstract

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.

## Key facts

- **NIH application ID:** 3171804
- **Project number:** 5R01CA034066-05
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **SAM.gov UEI:** NMJHD63STRC5
- **Principal Investigator:** Robert Ivarie
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1988
- **Award amount:** $105,418
- **Award type:** 5
- **Project period:** 1983-07-01 → 1990-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3171804

## Citation

> US National Institutes of Health, RePORTER application 3171804, INACTIVATION OF GENE EXPRESSION BY DNA AGENTS (5R01CA034066-05). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3171804. Licensed CC0.

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