Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

Minakuchi, Chieka; Nakagawa, Yoshiaki; Kiuchi, Makoto; Seino, Atsushi; Tomita, Shuichiro; Kamimura, Manabu

doi:10.1016/s0965-1748(02)00165-0

Cited by 22 publications

(25 citation statements)

References 53 publications

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“…These results clearly show that EcR and USP expressed with PRO-TEIOS bind to hsp27 EcRE as a heterodimer, as observed for EcR and USP of several insects prepared with rabbit reticulocyte lysate. 1,3,11,19,[35][36][37] Significant specific binding of PonA to EcR, but not to USP, was detected (Fig. 4).…”

Section: Resultsmentioning

confidence: 90%

“…The entire coding region of C. suppressalis EcR (B1 isoform, encoding a 547-aa protein) 30) or USP 19) was subcloned into the vector pEU3-NII (Toyobo Co., Ltd.) and then the mRNA was synthesized from the construct using ThermoT7 RNA polymerase (Toyobo Co., Ltd.) according to the manufacturer's instructions. After each mRNA had been purified by gel filtration with a MicroSpin G-25 Column (Amersham Biosciences, Buckinghamshire, England), a translation reaction was performed at 26°C for 20 hr using wheat germ extract.…”

Section: Preparation Of Proteinmentioning

confidence: 99%

“…19,30,32) Briefly, 0.5 ml of EcR and/or USP was incubated in a 20 mM modified HEPES buffer (pH 7.5) on ice for 30 min, then 1 ng of [a-…”

Section: Gel Mobility Shift Assaymentioning

confidence: 99%

“…Insect EcR and USP have also been expressed with an E. coli system, 10,[38][39][40][41][42][43][44][45] but in most cases, the expression of the entire coding region of these proteins is difficult because of low yield and poor function. In contrast, EcR and USP have been synthesized successfully and quickly using in vitro transcription/translation systems such as rabbit reticulocyte lysate [1][2][3][11][12][13][14][15][16][17][18][19][20] and wheat germ extract (PROTEIOS) (this study).…”

Section: )mentioning

confidence: 99%

“…To date, successful in vitro transcription/translation with rabbit reticulocyte lysate has been reported for EcR and USP from various insects. [1][2][3][11][12][13][14][15][16][17][18][19][20] Recently, a highly efficient cell-free protein synthesis system using wheat embryos was developed to synthesize large amounts of functional proteins. To date, a number of proteins such as glucocorticoid receptor, 21,22) dihydrofolate reductase, 23) luciferase, 23) green fluorescent protein 23) and tobacco mosaic virus (TMV) replicase 23) have been successfully expressed with wheat germ cell-free extract.…”

Section: Introductionmentioning

confidence: 99%

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Preparation of Functional Ecdysteroid Receptor Proteins (EcR and USP) Using a Wheat Germ Cell-Free Protein Synthesis System

Minakuchi

Nakagawa

Soya³

et al. 2004

J. Pestic. Sci.

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Ecdysone receptor (EcR) and ultraspiracle (USP) of the lepidopteran Chilo suppressalis were expressed using a wheat germ cell-free protein synthesis system, and the functions of the expressed proteins were analyzed. In a gel mobility shift assay, the EcR/USP heterodimer bound to the Drosophila melanogaster hsp27 ecdysone response element. In ligand-receptor binding assays, a potent ecdysteroid, ponasterone A, bound to the EcR/USP complex with a high affinity (K D =2.1 nM). For a series of ecdysteroids and N-tert-butyl-N,NЈ-dibenzoylhydrazines, the binding activity toward the in vitro expressed EcR/USP complex using a wheat germ system was linearly correlated with that to the endogenous receptor proteins in the cell-free preparation of C. suppressalis integument. These results indicate that functional ecdysteroid receptor proteins were successfully expressed in a wheat germ protein synthesis system.

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Section: Resultsmentioning

confidence: 90%

Section: Preparation Of Proteinmentioning

confidence: 99%

“…19,30,32) Briefly, 0.5 ml of EcR and/or USP was incubated in a 20 mM modified HEPES buffer (pH 7.5) on ice for 30 min, then 1 ng of [a-…”

Section: Gel Mobility Shift Assaymentioning

confidence: 99%

Section: )mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Preparation of Functional Ecdysteroid Receptor Proteins (EcR and USP) Using a Wheat Germ Cell-Free Protein Synthesis System

Minakuchi

Nakagawa

Soya³

et al. 2004

J. Pestic. Sci.

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New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects

et al. 2017

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Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators ( IGR s), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle ( USP ) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR– USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay ( RGA ) systems to detect molting hormone (ecdysone) activity by introducing EcR– USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose‐dependent and ecdysteroid‐specific manner. Furthermore, the insect order‐selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR– USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.

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Evolution of Nuclear Hormone Receptors in Insects

Laudet

Bonneton

2005

Comprehensive Molecular Insect Science

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Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

Cited by 22 publications

References 53 publications

Preparation of Functional Ecdysteroid Receptor Proteins (EcR and USP) Using a Wheat Germ Cell-Free Protein Synthesis System

Preparation of Functional Ecdysteroid Receptor Proteins (EcR and USP) Using a Wheat Germ Cell-Free Protein Synthesis System

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects

Evolution of Nuclear Hormone Receptors in Insects

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