Project: Research project

Project Details


The hsp70 heat shock gene in Drosophila is associated with a collection of
nonhistone chromosomal proteins that establish and maintain the
transcriptional potential of the gene before induction by heat shock or
other stress. RNA polymerase II and TFIID are among this collection of
proteins. the RNA polymerase II appears to have initiated transcription
but arrested elongation approximately 25 nucleotides downstream of the
transcription start. The long-term objective of this proposal is to
understand the pathway of interactions that is responsible for this
potentiated state. The types of interactions that are involved might play
a key role in establishing the patterns of gene expression during early
stages in development and for maintaining the transcriptional potential of
a gene until induction occurs at an appropriate stage. The project has
four specific aims: 1) Determine the molecular architecture of a normal
hsp70 promoter that has been transformed into Drosophila. 2) Identify
sequences that are essential for the potentiated state by determining the
molecular architecture of mutant hsp70 promoters that have been transformed
into flies. 3) Characterize protein-DNA and protein-protein interactions
that occur during reconstitution of protein-DNA complexes on the hsp70
promoter. 4) In vitro transcription analysis of normal and mutant hsp70

P element-mediated transformation will be used to introduce single copies
of normal and mutant hsp70 promoters into Drosophila. Genomic footprinting
and protein-DNA crosslinking will be used to identify protein-DNA
interactions and a nuclear run-on assay will be used to determine if RNA
polymerase II is transcriptionally engaged on the promoters. Biochemical
studies implicate 4 regions of the promoter that may be involved. These
are the TATA element, the start site, a conserved sequence element centered
at +25 and GAGA elements that are interdigitated with the heat shock
regulatory elements. The molecular genetic analysis will be complemented
with biochemical studies of highly purified protein fractions isolated from
the nuclear extract of uninduced Drosophila embryos. In vitro and in vivo
analysis with normal and mutant promoters should provide a comprehensive
understanding of the potentiated state.
Effective start/end date5/1/924/30/93


  • National Institute of General Medical Sciences

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