The overall aim of the proposed research is to determine the mechanism by which a steroid hormone, ecdysone, stimulates transcription of a set of related genes, the yolk protein genes of Drosophila melanogaster. We have already cloned these genes, sequenced them, identified the sequences complementary to the mRNAs, shown that their transcription rate is likely to be controlled by the hormone concentration and transcribed the cloned genes with a partially purified Drosophila RNA polymerase II. We now propose to use in vitro transcription to investigate transcription initiation and control of that initiation by ecdysone and ancillary factors. Our experiments have demonstrated that in vivo and in vitro transcription initiates upstream of the CAP site. By pulse-chase and in vitro mutagenic experiments we will determine whether or not these upstream initiation events lead to mature mRNA by way of processing events. The same methodology will be used to determine whether or not transcription and processing are coupled vectoral processes and to identify and purify the presumed processing activities. The objective of this processing study is to determine where the hormonally controlled initiation occurs. Similar methods will be used to identify nucleotides and proteins required for the observed specific in vitro initiation of transcription. Finally, I propose to investigate the relation between in vitro initiation and in vivo, hormonal control of transcription. In order to accomplish this I plan to use two assay systems (addition of cellular extract and fractions thereof to the pol II transcription reaction and DNA binding assays for cellular proteins with specific affinity for the genes) to detect other factors, presumably proteins, that are necessary for hormonal control but are missing from the in vitro pol II transcription system. Current knowledge suggests that the apparent general mechanism of steroid hormone action (receptor mediated interaction with the chromatin or DNA) is similar in Drosophila and mammals. I believe that our current analysis of the yolk protein gene system is sufficiently advanced that further analysis is likely to make a substantial contribution to understanding the mechanism by which a hormone-receptor complex controls transcription rate. This is certainly a fundamental process that must work well in a healthy human.
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