This proposal focuses on the transcriptional regulation of an adenovirus (Ad) early promoter (E2-early) which transcribes a region of the viral genome that encodes a 72 kD single stranded DNA binding protein, an 80 kD precursor terminal protein and the 140 kD DNA polymerase all of which are vital for virus growth. Transcriptional regulation of the E2-early promoter involves protein-protein interactions between multiple cellular transcription factors and several virus-encoded transactivators. During the previous grant period, we have defined the cis-acting sequence elements of this promoter within the context of the viral chromosome, identified several cellular transcription factors that bind to these sequence elements and the role of the virus-encoded transactivators that regulate transcription of this promoter. We propose to continue these studies and use genetic and biochemical approaches to elucidate the mechanisms by which this promoter is regulated by the cellular transcription factors and the virus-encoded transactivators. We have the following specific aims: Recent results show that in addition to ElA, the E2 early promoter is transactivated by one of the Ad early region 4 polypeptides (E4 6/7) and the single stranded DNA binding protein. We will use genetic and biochemical approaches to determine the mechanism of transactivation of the E2-early promoter by these gene products. We will attempt to clone the cDNA that encodes the cellular transcription factor E2F which plays a critical role in E2-early promoter transcription. The E2F also plays an important role in cell cycle regulation and binds to retinoblastoma susceptibility gene (RB) product, an RB related protein pl07, cycline A and cdk2 kinase. A cDNA clone of E2F is a valuable reagent for a number of studies which include studies of structure-functional relationship of E2F, and its role in cell cycle regulation. We will dissect the in vivo DNA-protein interactions of the E2-early promoter in basal and virus induced transcription with the ligation mediated PCR amplification approach. In this study, we will use a series of Ad mutants that we previously constructed, which contain mutations in different transcription factor binding sites of the E2-early promoter.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020156-13
Application #
2003276
Study Section
Virology Study Section (VR)
Project Start
1983-08-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1998-12-31
Support Year
13
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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Porras, A; Bennett, J; Howe, A et al. (1996) A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays. J Virol 70:6902-8
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Malhotra, P; Manohar, C F; Swaminathan, S et al. (1993) E2F site activates transcription in fission yeast Schizosaccharomyces pombe and binds to a 30-kDa transcription factor. J Biol Chem 268:20392-401
Swaminathan, S; Malhotra, P; Manohar, C F et al. (1993) Activation of a dual adenovirus promoter containing nonconsensus TATA motifs in Schizosaccharomyces pombe: role of TATA sequences in the efficiency of transcription. Nucleic Acids Res 21:2737-46

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