The major long range objective of the proposed research is to determine the mechanisms involved in the regulation of transcription and RNA polymerases during growth and developmental transitions. The major emphasis will be placed on transitions where quiescent tissues are induced to proliferate by environmental or hormonal signals. Specific objectives include: (1) To determine the subunit structural relationships of nuclear RNA polymerases among eukaryotic organisms (i.e., animal, plant, and fungal) by using subunit-specific antibodies raised against RNA polymerases as probes. RNA polymerase subunit protein blotting will identify the structural relationships of these enzymes at the subunit level and provide information on the conservation of the structures through evolution. (2) To determine whether the alteration in RNA polymerase subunit structures plays a role in the regulation of transcription during growth and development. This will be evaluated by analysis of RNA polymerase structures during development using subunit-specific antibodies as probes. (3) To develop soluble in vitro transcription systems that are capable of accurate transcription from sources that would allow the purification of transcription factors to homogeneity. (4) To use the available in vitro transcription systems for analyzing the function of individual RNA polymerase subunits. Subunit-specific antibodies to the RNA polymerases will facilitate these studies by their ability to bind to an block specific sites on the RNA polymerases, and thus inhibit a specific process in transcription. (5) To initiate experiments on the isolation of RNA polymerase subunit genes and cDNA clones to subunit mRNAs. The RNA polymerase subunit-specific antibodies will provide probes for immunological screening of cDNA libraries. The isolation of these genes and cDNA clones will provide probes for studying the regulation of RNA polymerase synthesis during growth and developmental transitions. These studies on RNA polymerase architecture, subunit function, and regulation of synthesis should provide insight into transcriptional regulation in eukaryotes especially in cases where cells are induced to abnormal levels of proliferation.
