The overall objective is to elucidate the nature and mechanism of action of those host and viral-encoded factors which regulate the transcription of both viral and host genes in cells productively or abortively infected by, or transformed by, adenovirus 2. Using purified DNA templates we have demonstrated accurate in vitro transcription of host and adenoviral (early, intermediate, land late) class II genes and host and viral (VA RNA) class III genes in systems reconstituted with crude extracts or purified RNA polymerases and partially purified host factors. We now propose to (1) complete the resolution and purification to homogeneity of the various host transcription factors, using a battery of conventional and more sophisticated techniques, (2) to determine the nature, specificity, and site and mechanism of action of each, as well as quantitative or qualitative variations in each during infection/transformation, (3) to identify, by complementation of purified systems with known viral proteins or with partially purified components from infected cells, viral regulatory factors and to similarly characterize these, (4) as part of the above, to analyze the structure and function of various natural and reconstituted nucleoprotein templates/transcription complexes and their role in transcriptional regulation (particularly by viral proteins), (5) as part of the above, to use site-directed mutagenesis to determine specific DNA sequences important for expression and regulation in vitro, and (6) to complement (and verify) the in vitro analyses with corresponding determinations of those cis-acting DNA sequences and transacting viral genes (products) required for expression and regulation in vivo (using various viral infection and viral gene transfection/transformation protocols). While adenovrirus genes will be emphasized initially, a long-range goal is to include similar studies of virus-modulated host genes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034223-04
Application #
3171969
Study Section
Virology Study Section (VR)
Project Start
1983-04-01
Project End
1987-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Krensky, A M (1991) Molecular basis of transplant rejection and acceptance. Pediatr Nephrol 5:422-7
Huang, D H; Roeder, R G (1988) Delineation of DNA sequences that are important for in vitro transcription from the adenovirus EIIa late promoter. Mol Cell Biol 8:1906-14
Hough, P V; Mastrangelo, I A; Wall, J S et al. (1987) The gene-specific initiation factor USF (upstream stimulatory factor) bound at the adenovirus type 2 major late promoter: mass and three-dimensional structure. Proc Natl Acad Sci U S A 84:4826-30
Reinberg, D; Roeder, R G (1987) Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE. J Biol Chem 262:3310-21
Reinberg, D; Horikoshi, M; Roeder, R G (1987) Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. J Biol Chem 262:3322-30
Pieler, T; Hamm, J; Roeder, R G (1987) The 5S gene internal control region is composed of three distinct sequence elements, organized as two functional domains with variable spacing. Cell 48:91-100
Van Dyke, M W; Roeder, R G (1987) Two forms of transcription factor TFIIIC in extracts from HeLa cells. Nucleic Acids Res 15:5031-9
Reinberg, D; Roeder, R G (1987) Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains. J Biol Chem 262:3331-7
Van Dyke, M W; Roeder, R G (1987) Novobiocin interferes with the binding of transcription factors TFIIIA and TFIIIC to the promoters of class III genes. Nucleic Acids Res 15:4365-74
Hawley, D K; Roeder, R G (1987) Functional steps in transcription initiation and reinitiation from the major late promoter in a HeLa nuclear extract. J Biol Chem 262:3452-61

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