Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis.
cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.
Minakuchi
Chieka
C
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, 606-8502, Kyoto, Japan.
Nakagawa
Yoshiaki
Y
Kiuchi
Makoto
M
Seino
Atsushi
A
Tomita
Shuichiro
S
Kamimura
Manabu
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D007915:Lepidoptera / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008024:Ligands; D012333:RNA, Messenger; D014157:Transcription Factors; C066030:USP protein, Drosophila