Transforming growth factor alpha is a 50 amino acid mitogenic peptide that shares structural and functional homology with EGF. It is produced by many transformed cells, as well as developing and normal mammalian tissues, indicating a broad role in nature. Very little is known, however, about the regulatory mechanisms involved in TGF-alpha mRNA expression. The existence of cloned neoplastic cell lines derived by chemical transformation of a rat liver epithelial cell have provided a model in which to explore these mechanisms. Using certain cell lines from this lineage, it is the long range goal of this proposal to elucidate the mechanisms regulating the TGF-alpha gene and to identify how TGF-alpha mRNA expression is altered in its various contexts. Accordingly, we will: (1) perform nuclear run-on assays, and methylation and DNase I sensitivity assays to ascertain if regulatory control occurs at the level of transcription and is influenced by structural changes in the gene; (2) further define those sequences representing positive and negative regulatory elements of the TGF-alpha promoter by CAT assay using wild-type and mutagenized constructs containing the putative regulatory sequences; (3) recognize activities from cell extracts which bind to identified regulatory sequences by performing gel shift and DNA footprinting assays; (4) examine the role of 3' untranslated sequences in TGF-alpha mRNA stability, and hence TGF-alpha mRNA accumulation. This will be accomplished by preparing chimeric constructs which contain varying amounts of 3' untranslated sequences from the TGF-alpha gene and an easily identified reporter gene. These will be used in a transfection experiments and the stability of the resultant transcripts evaluated by S1 nuclease digestion analysis subsequent to treatment of the transfectants with actinomycin D.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Physician Scientist Award (K11)
Project #
5K11DE000300-03
Application #
3086200
Study Section
NIDCR Special Grants Review Committee (DSR)
Project Start
1991-04-01
Project End
1995-07-31
Budget Start
1993-04-01
Budget End
1994-07-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Dentistry
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599