The major objective is to elucidate fundamental mechanistic aspects of transcription, as well as transcriptional regulatory mechanisms, for genes whose activities are related to particular states of cell growth, proliferation, and differentiation. The genes to be analyzed include various human and amphibian (Xenopus) genes encoding 5S and tRNAs (class III), and histone proteins (class II). Having previously demonstrated accurate transcription initiation for a variety of genes in systems reconstituted with crude extracts or with RNA polymerases and partially purified factors, we now propose for the genes of interest: (1) to complete the delineation and purification of the respective transcription factors, including both general and regulatory (e.g., tissue- or cell-stage specific) factors; (2) to investigate in detail the sites and mechanisms of action of these factors (e.g., interactions with specific DNA sequences and possible functions via site-specific chromatin structural perturbations); (3) to monitor alterations in the activities or levels of these factors during critical growth and developmental transitions (to correlate with gene activity); (4) to use a recently isolated cDNA encoding a 5S gene-specific regulatory factor to study the structure, function, and regulation of the corresponding gene and to further study structure-function relationships in the factor itself; (5) as part of the above, to use site-directed mutagenesis and in vivo gene transfer systems to analyze the expression and regulation of the various genes in vivo; and (6) in conjunction with these studies, to continue the isolation and structural and functional analysis of both Xenopus and human histone genes, especially those which are cell-cycle regulated versus those which are expressed constituitively or only at certain developmental steps (e.g., during oogenesis or during the cessation of cell proliferation). These studies should provide fundamental information regarding transcriptional mechanisms in eukaryotes as well as insights into transcriptional controls which are operative during normal growth and development and which are altered in abnormal states such as neoplasia. (M)
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