CLONING AND CHARACTERIZATION OF LCECR - A FUNCTIONAL ECDYSONE RECEPTOR FROM THE SHEEP BLOWFLY LUCILIA-CUPRINA

Degenerate oligonucleotides were designed on the basis of conserved am ino acid sequences in the DNA binding domains of the ecdysone receptor s from Drosophila melanogaster (DmEcR) and Chironomus tentans (CtEcR), Using these oligonucleotides a fragment encoding part of the DNA bind ing domain of the Lucilia cuprina ecdysone receptor (LcEcR) was amplif ied by polymerase chain reaction (PCR) from genomic DNA and cloned, Th is cloned fragment was used to screen a cDNA library which was prepare d from Lucilia larvae at the late third instar, A full-length LcEcR ge ne was isolated within a 3336 bp cDNA clone, The conceptually translat ed amino acid sequence of this open reading frame (757 amino acids)con tained all five domains typical of a steroid hormone receptor, Alignme nt comparisons and phylogenetic analyses indicated that LcEcR most clo sely resembled the B1 isoform of DmEcR relative to other known insect steroid receptors, including six insect EcRs, An antisense RNA probe s pecific for the 3' end of LcEcR was used in ribonuclease protection as says to detect significant levels of LcEcR mRNA in embryos, late third instar larvae, pupae and adult females during Lucilia development, Th is pattern parallels the pattern of expression observed for DmEcR engi neered for expression in mammalian cells, and we now report that the c loned LcEcR is functional and can act as an ecdysteroid-dependent tran scription factor in mammalian cells, (C) 1997 Elsevier Science Ltd.