The overall goal of the proposed research is to determine how the transcriptional enhancer of the Moloney murine leukemia virus regulates the expression of the virus in lymphoid and myeloid hematopoietic cells, and contributes to the T cell specificity of transformation. In vitro characterization of the Moloney enhancer has identified a complex array of nuclear factor binding sites. Our recent genetic analysis has enabled us to identify two of these sites, the LVb site and adjacent core element as important determinants of the thymic disease specificity of the Moloney virus. We now plan to use molecular genetics and biochemistry to further characterize the nuclear proteins that associate with the LVb/core region of the Moloney enhancer. We also plan to identify Moloney enhancer sequences that regulate viral gene expression in myeloid cells.
Our specific aims are: 1. To identify and characterize nuclear proteins that bind to the LVb/core element in the Moloney enhancer. Microaffinity DNA precipitation assays will be used to identify and distinguish between ubiquitous, tissue specific, and TPA inducible nuclear factors that bind to the LVb/core region of the Moloney enhancer. 2. To obtain and characterize T cell specific cDNA clones that encode the core-binding protein. cDNA libraries prepared from T cells will be screened for core binding factors by one of the methods listed in 2.1 - 2.3 isolated cDNA clones will be characterized for DNA binding specificity, sequenced, and analyzed for tissue-specific expression. 3. To identify enhancer sequences that mediate transcription from the Moloney enhancer in myeloid cells. A panel of Moloney enhancer mutants, each containing a point mutation in one of the nuclear factor binding sites on the enhancer, will be analyzed for transcriptional activity in myeloid leukemia cell lines representing progressive stages of differentiation, by transient CAT assays.
