The long-term goal of this proposed research is to understand how gene expression is regulated during development. The model system understudy is the Drosophila alcohol dehydrogenase (Adh) gene, a single-copy gene that is transcribed from two promoters into two different ADH mRNAs, the larval and the adult during development. It is proposed that the cis- and trans-acting elements that regulate the developmental expression of Adh be identified and their interactions studied at the molecular level. The cis-acting transcription signals will be identified by comparing the expression of the cloned wild-type Adh to that of specific deletion mutants in the promoter regions, and that of naturally-occurring variant Adh genes cloned from other Drosophila species using an in vivo transient assay in homologous Drosophila cultured cells. The changes in chromatin structure that accompany specific transcription from the larval and the adult promoters will be studied using several nucleases as probes. The transcribing Adh templates will be the transfected exogenous Adh wild-type and specific promoter mutant genes, and the endogenous, in situ, Adh genes of ADH-positive cell lines. Cultured Drosophila cells that express ADH will be analyzed for the presence of factors that bind specifically to the larval and adult promoter regions. Both in vivo assays by transfection and biochemical studies will be used to identify and characterize these putative regulatory factors. Use of the multiple promoters for a single gene is quite common in nature. The knowledge obtained from this study should be of central interest and relevant to other developmental systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034850-01
Application #
3286574
Study Section
Molecular Biology Study Section (MBY)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
Schools of Arts and Sciences
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Benyajati, C; Mueller, L; Xu, N et al. (1997) Multiple isoforms of GAGA factor, a critical component of chromatin structure. Nucleic Acids Res 25:3345-53
Jackson, J R; Benyajati, C (1993) DNA-histone interactions are sufficient to position a single nucleosome juxtaposing Drosophila Adh adult enhancer and distal promoter. Nucleic Acids Res 21:957-67
Ayer, S; Benyajati, C (1992) The binding site of a steroid hormone receptor-like protein within the Drosophila Adh adult enhancer is required for high levels of tissue-specific alcohol dehydrogenase expression. Mol Cell Biol 12:661-73
Benyajati, C; Ewel, A; McKeon, J et al. (1992) Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila. Nucleic Acids Res 20:4481-9
Ewel, A; Jackson, J R; Benyajati, C (1990) Alternative DNA-protein interactions in variable-length internucleosomal regions associated with Drosophila Adh distal promoter expression. Nucleic Acids Res 18:1771-81
Ayer, S; Benyajati, C (1990) Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines. Mol Cell Biol 10:3512-23
Le, L; Ayer, S; Place, A R et al. (1990) Analysis of formaldehyde-induced Adh mutations in Drosophila by RNA structure mapping and direct sequencing of PCR-amplified genomic DNA. Biochem Genet 28:367-87
Benyajati, C; Ayer, S; McKeon, J et al. (1987) Roles of cis-acting elements and chromatin structure in Drosophila alcohol dehydrogenase gene expression. Nucleic Acids Res 15:7903-20