Cloning and characterization of LcEcR: a functional ecdysone receptor from the sheep blowfly Lucilia cuprina.
Degenerate oligonucleotides were designed on the basis of conserved amino acid sequences in the DNA binding domains of the ecdysone receptors from Drosophila melanogaster (DmEcR) and Chironomus tentans (CtEcR). Using these oligonucleotides a fragment encoding part of the DNA binding domain of the Lucilia cuprina ecdysone receptor (LcEcR) was amplified by polymerase chain reaction (PCR) from genomic DNA and cloned. This cloned fragment was used to screen a cDNA library which was prepared from Lucilia larvae at the late third instar. A full-length LcEcR gene was isolated within a 3336 bp cDNA clone. The conceptually translated amino acid sequence of this open reading frame (757 amino acids) contained all five domains typical of a steroid hormone receptor. Alignment comparisons and phylogenetic analyses indicated that LcEcR most closely resembled the B1 isoform of DmEcR relative to other known insect steroid receptors, including six insect EcRs. An antisense RNA probe specific for the 3' end of LcEcR was used in ribonuclease protection assays to detect significant levels of LcEcR mRNA in embryos, late third instar larvae, pupae and adult females during Lucilia development. This pattern parallels the pattern of expression observed for DmEcR mRNAs during Drosophila development. The LcEcR gene was engineered for expression in mammalian cells, and we now report that the cloned LcEcR is functional and can act as an ecdysteroid-dependent transcription factor in mammalian cells.
Hannan
G N
GN
C.S.I.R.O. Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, NSW, Australia.
Hill
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004175:Diptera / Q000737:chemistry / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D008969:Molecular Sequence Data; D012333:RNA, Messenger; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D012756:Sheep / Q000469:parasitology
D018076:DNA, Complementary; D012333:RNA, Messenger; D011987:Receptors, Steroid; C055469:ecdysone receptor