This is a proposal to study mechanisms of gene regulation during the mammalian cell cycle using the histone and the cellular thymidine kinase genes as model systems. The major goal of this proposal is to carry out an in-depth analysis of the cis-acting DNA sequence and the trans-acting factors required for the cell cycle regulated gene expression in vivo. Recently, it has been demonstrated that sequences flanking the mammalian histone genes can confer transcriptional control on heterologous fusion genes, resulting in cell cycle regulation of their mRNA levels. In this proposal, the sequences around the histone gene will be subjected to in vitro mutagenesis. A series of recombinants will be transfected into recipient mammalian cells and the effect of deletions on the cell cyclic transcriptional control will be examined. This approach has been used successfully to define domains of DNA critical for regulatory function. Similar analysis of the cellular thymidine kinase gene will be performed with techniques already established for the histone gene system. Our focus is to dissect the transcriptional regulatory component of the thymidine kinase gene system. From our deletion analysis, we will determine if a consensus sequence for cell cycle regulatory sequence can be identified. In addition, we will test if cell cycle regulatory element has enhancer-like properties. For the characterization of the cellular factors involved in the cell cycle regulation, deletion mutant constructs will be transfected into mammalian recipient cells to probe if the factors act in a positive or negative manner. Competition experiments will be performed to confirm the regulatory nature of the factors, and to determine their relative binding affinities, concentrations during the cell cycle and their specificity toward other cell cycle regulated genes. Binding assays of the DNA and protein fractions extracted from dividing and G1 arrested cells will further identify the DNA regions capable of interacting with the protein factors and provide for assay systems towards their purification. This research will contribute to our knowledge of co-ordinate gene control during the mammalian cell cycle. Consequently, it may prove useful in understanding diseases, such as cancer, that may result from aberrations of gene expression during the cell cycle.
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