Isolation and developmental expression of the ecdysteroid-induced GHR3 gene of the wax moth Galleria mellonella.
Three degenerate primers were designed to match the most conserved regions within the DNA-binding domains of several selected members of the steroid hormone receptor family. Use of these primers in the polymerase chain reaction with cDNA from Galleria mellonella prepupae detected a 177 bp fragment that had 87% identity to the Manduca sexta gene MHR3 and 75% to the Drosophila melanogaster DHR3 gene, and therefore was named "GHR3". Screening of a Galleria penultimate instar cDNA library with this fragment yielded a cDNA clone that contained a 557 codon open reading frame, predicting a 62.3 kDa protein. The deduced amino acid sequence of GHR3 showed 92% overall identity with the MHR3 protein and 97 and 70% identity with DHR3 in the putative DNA- and ligand-binding domains, respectively. Hybridization of whole body RNA revealed high GHR3 mRNA levels during both the larval and pupal molts, coincident with the molt-inducing ecdysteroid pulses, and low or undetectable levels during the first half of the last instar. During the larval-pupal transformation, no GHR3 mRNA was found at the beginning of the stemmatal pigment retraction at the onset of the ecdysteroid rise; maximal levels were observed 4 h later, coincident with the peak ecdysteroid titer (over 2.3 micrograms 20E equivalents/ml hemolymph). Two mRNAs (4.6 and 3.6 kb) were detected when the ecdysteroid titer was high. Injection of 2 micrograms/gm 20E into isolated final instar larval abdomens induced the appearance of the 4.6 kb mRNA within 1.5 h; the mRNA level then reached maximum by 3 h and declined by 6 h. No 3.6 kb mRNA was detectable during that time. A 10-fold lower 20E dose caused only trace induction by 3 h.
Jindra
M
M
Institute of Entomology, Czech Academy of Sciences, Ceské Budĕjovice.
Sehnal
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F
Riddiford
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics*; D026461:Ecdysteroids; D004441:Ecdysterone / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D017344:Genes, Insect; D007301:Insect Hormones / Q000502:physiology*; D019476:Insect Proteins; D007447:Invertebrate Hormones / Q000502:physiology*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011679:Pupa; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear; D013256:Steroids / Q000502:physiology*; D016335:Zinc Fingers / Q000235:genetics
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D026461:Ecdysteroids; D007301:Insect Hormones; D019476:Insect Proteins; D007447:Invertebrate Hormones; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013256:Steroids; D004441:Ecdysterone