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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021626-13
Application #
3270607
Study Section
Genetics Study Section (GEN)
Project Start
1977-12-01
Project End
1988-02-29
Budget Start
1986-12-01
Budget End
1988-02-29
Support Year
13
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Brandeis University
Department
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Jacoby, D B; Wensink, P C (1996) DNA binding specificities of YPF1, a Drosophila homolog to the DNA binding subunit of human DNA-dependent protein kinase, Ku. J Biol Chem 271:16827-32
An, W; Wensink, P C (1995) Integrating sex- and tissue-specific regulation within a single Drosophila enhancer. Genes Dev 9:256-66
An, W; Wensink, P C (1995) Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes. EMBO J 14:1221-30
Lossky, M; Wensink, P C (1995) Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor. Mol Cell Biol 15:6943-52
O'Donnell, K H; Chen, C T; Wensink, P C (1994) Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene. Mol Cell Biol 14:6398-408
Jacoby, D B; Wensink, P C (1994) Yolk protein factor 1 is a Drosophila homolog of Ku, the DNA-binding subunit of a DNA-dependent protein kinase from humans. J Biol Chem 269:11484-91
O'Donnell, K H; Wensink, P C (1994) GAGA factor and TBF1 bind DNA elements that direct ubiquitous transcription of the Drosophila alpha 1-tubulin gene. Nucleic Acids Res 22:4712-8
Coschigano, K T; Wensink, P C (1993) Sex-specific transcriptional regulation by the male and female doublesex proteins of Drosophila. Genes Dev 7:42-54
Logan, S K; Wensink, P C (1990) Ovarian follicle cell enhancers from the Drosophila yolk protein genes: different segments of one enhancer have different cell-type specificities that interact to give normal expression. Genes Dev 4:613-23
Mitsis, P G; Wensink, P C (1989) Identification of yolk protein factor 1, a sequence-specific DNA-binding protein from Drosophila melanogaster. J Biol Chem 264:5188-94

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