LcUSP, an ultraspiracle gene from the sheep blowfly, Lucilia cuprina: cDNA cloning, developmental expression of RNA and confirmation of function.
A DNA sequence corresponding to most of the DNA-binding domain of a Lucilia cuprina ultraspiracle protein (LcUSP) was amplified by PCR from genomic DNA and cloned. This cloned fragment was used to screen a L. cuprina cDNA library and to isolate a full-length LcUSP encoding sequence within a 3800-bp cDNA clone. The conceptually translated amino acid sequence of this open reading frame (467 amino acids) was used in alignment comparisons and phylogenetic analyses to reveal that LcUSP most closely resembles DmUSP relative to other known insect nuclear hormone receptors. An antisense RNA probe specific for the 5' end of Lcusp was used in ribonuclease protection assays to detect significant levels of Lcusp RNA throughout L. cuprina development. Highest levels were detected in embryos, late third instar larvae, pupae and adult females. This pattern parallels the pattern of expression observed for Dmusp RNAs during Drosophila melanogaster development. Finally, the LcUSP sequence was engineered for expression in mammalian cells and we now report that the cloned LcUSP is functional in vivo and can act as a partner for a chimeric L. cuprina ecdysone receptor to form an ecdysteroid-dependent transcription factor in mammalian cells.
Hannan
G N
GN
CSIRO Molecular Science, Sydney Laboratory, PO Box 184, North Ryde, NSW 2113, Australia. garry.hannan@molsci.csiro.au
Hill
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D004175:Diptera / Q000235:genetics*; D029721:Drosophila Proteins; D005260:Female; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D006801:Humans; D019476:Insect Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors; D017386:Sequence Homology, Amino Acid; D012756:Sheep / Q000469:parasitology; D014157:Transcription Factors / Q000145:classification / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; C432946:ultraspiracle protein, Lucilia cuprina