Title

Identification, purification and functional analysis of ecdysterone receptor from Drosophila melanogaster

Date of Award

1990

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Richard W. Voellmy, Committee Chair

Abstract

The ecdysterone receptor regulates the expression of an array of genes during Drosophila puparium formation, and therefore, plays a key role in development. Studies have been conducted to identify and purify the ecdysterone receptor from a hormone-responsive Drosophila cell line S3. Specifically, based on the previous findings that showed that hormonal regulation of hsp23 and 27 genes was mediated by ecdysterone-responsive elements (EREs, which were distinct from heat shock element, HSE), a cellular factor which was presumably identical with ecdysterone receptor was identified by various approaches: in vivo competition assays, exoIII protection assays and gel retardation assays. The factor was then purified by virtue of its selective binding to ERE sequences using specific DNA affinity chromatography, and as expected, was determined to be ecdysterone receptor. The protein has a subunit MW and 130kD. It can bind to a variety of distantly similar sequences.Functional analysis showed that purified ecdysterone receptor could stimulate ERE-containing, hormone-inducible promoters in a cell-free transcription system, and that, in vivo, EREs from hsp27 and 23 gene promoters could confer hormone inducibility on an otherwise uninducible hsp70 gene core promoter.Ecdysterone receptor (ERE binding protein) could be detected early in Drosophila development, as shown by binding studies involving probe containing hsp27 gene ERE sequences and staged embryonic nuclear extracts. Future studies to unravel aspects of ecdysterone receptor function in different stages of Drosophila development promises to be of interest.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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