Cloning and characterization of CcEcR

Abstract

In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5 H -untranslated region and the complete 3 H -untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.

Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

“…DmEcR, CfEcR, BaEcR, and BmEcR are underlined as their proteins contain a Tyr at the critical ligand specificity site. Data bank search yielded EcR protein sequences from 16 species: 6 Diptera species, the Mediterranean fruit fly Ceratitis capitata (CcEcR [67]), the sheep blowfly Lucilia cuprina (LcEcR [23]), the yellow fever mosquitoes A. aegypti (AaEcR, 12) and A. albopictus (not shown, as its EcR I box is 100% identical to AaEcR [28]), the midge Chironomus tentans (CtEcR [27]), and D. melanogaster (DmEcR [34]); 6 Lepidoptera species, the spruce budworm C. fumiferana (CfEcR [35]), squinting bush brown Bicyclus anynana (BaEcR [R. K. Reinhardt, P. Weber, and P. B. Koch, submitted to GenBank, accession no. CAB63236]), the silkworm B. mori (BmEcR [32,60]), the tobacco budworm Heliothis virescens (HvEcR [41]), the tobacco hornworm M. sexta (MsEcR [18]), and the buckeye Junonia coenia (JcEcR [R. K. Reinhardt, P. Weber, and P. B. Koch submitted to GenBank, accession no.…”

EcR-AExpression in the Brain and Ovary of the Honeybee (Apis mellifera L.)

“…Amino-acid sequences Identity(%) (Verras et al, 1999), 1-MMKRRWSNNGG-11 Lucilia cuprina (Hannan et al, 1997), 1-MMKRRWSNNGG-11 Aedes albopictus (Jayachandran and Fallon, 2000) 1-MMKRRWSNNGG-11 Aedes aegypti (Cho et al, 1995) 1-MR-RRWSNNGG-10 Choristoneura fumiferana (Perera et al, 1999) …”

“…insect origins. The N-terminal 10 amino-acid residues "M(M/ R)(K/-)RRWSNNGG" are well conserved among 12 insect species [Ceratitis capitata (Verras et al, 1999), Lucilia cuprina (Hannan et al, 1997), Plodia interpunctella (Siaussat, et al, 2004), Aedes albopictus (Jayachandran et al, 2000), Aedes aegypti (Cho et al, 1995), Choristoneura fumiferana (Perera et al, 1999), Chilo suppressalis (Minakuchi, et al, 2002), Manduca sexta, (Fujiwara et al, 1995), Bombyx mori (Hossain et al, 2006), Leptinotarsa decemlineata (Ogura et al, 2005), Spodoptera frugiperda (Chen et al, 2002) and Drosophila melanogaster (Koelle et al, 1991)], all of the insect species from which EcR-B1 isoforms have been identified (Table 2). We then examined whether this sequence was encoded between exons 2 and 3 of AmEcR, where the exon for the B1 isoform, if any, was expected to exist.…”

Biochemical mode of action and differential activity of new ecdysone agonists against mosquitoes and moths

“…These data (Kothapalli et al, 1995)], Cs [Chilo suppressalis (Minakuchi et al, 2002)], Pi (Plodia interpunctata, Accession no. AAR84611), Cc [Ceratitis capitata (Verras et al, 1999)], Dm [Drosophila melanogaster (Koelle et al, 1991)], Lc [Lucilia cuprina (Hannan and Hill, 1997)], Ae [Aedes aegypti (Cho et al, 1995)], Ct [Chironomus tentans (Imhof et al, 1993)], Hv [Heliothis virescens (Martinez et al, 1999)], Ms [Manduca sexta (Fujiwara et al, 1995)], Bm [Bombyx mori (Kamimura et al, 1996;Swevers et al, 1995)], Lm [Locusta migratoria EcR (Saleh et al, 1998)], Tm [Tenebrio molitor EcR (Mouillet et al, 1997)], Mp [Myzus persicae (Hill, 1999)], Up [fiddler crab Uca pugilator (Chung et al, 1998)], Ama [ixodid tick Amblyomma americanum (Guo et al, 1997)]. …”