The long term objectives of this proposal are to elucidate the molecular mechanisms of steroid hormone-regulated transcription at a biochemical level. The proposed experiments will focus on the steroid-inducible transcription of the proviral DNA of mouse mammary tumor virus (MMTV) with the goals of assessing the role of trans-acting factors in establishing the basal rate of MMTV transcription and eventually determining how this basal rate is regulated.
The specific aims are 1) to identify the functional elements of the MMTV promoter by mutagenesis techniques, including linker scanning and oligonucleotide-directed, site-specific mutagenesis, 2) to identify nuclear proteins that recognize these elements, 3) to purify proteins that mediate MMTV transcription, and 4) to analyze basal MMTV transcription in vitro. Activity of mutant MMTV promoters will be assessed with a transient transfection assay that has been well characterized; promoter activity will be monitored by expression of a linked reporter gene encoding chloramphenicol acetyltransferase, as well as by direct analysis of MMTV transcripts. Proteins that recognize MMTV promoter elements will be identified using a gel electrophoresis mobility shift assay, and binding will be further characterized by DNAse I and exonuclease III footprinting and by methylation protection and interference. Purification of transcription factors will be accomplished by sequence-specific DNA affinity chromatography, as well as by other chromatographic means. In vitro transcription assays will be performed with nuclear extracts derived from tissue culture cells or bovine liver. In vitro transcription will allow functional assays for purified transcription factors to be established and will provide a means to begin mechanistic studies on promoters function based on recently described methods to dissect functional steps in transcription initiation by differential sensitivity to Sarkosyl. The basic processes by which genes are controlled have broad implications for many health-related issues, including an understanding of the mechanisms of aberrant gene expression and of cellular transformation.
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| Huang, M; Lee, J W; Peterson, D O (1993) Functional redundancy of octamer elements in the mouse mammary tumor virus promoter. Nucleic Acids Res 21:5235-41 |
| Pazzagli, M; Devine, J H; Peterson, D O et al. (1992) Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells. Anal Biochem 204:315-23 |
| Lee, J W; Moffitt, P G; Morley, K L et al. (1991) Multipartite structure of a negative regulatory element associated with a steroid hormone-inducible promoter. J Biol Chem 266:24101-8 |
| Toohey, M G; Lee, J W; Huang, M et al. (1990) Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus. J Virol 64:4477-88 |
