RESUMO
The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located â¼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.
Assuntos
Bombyx/genética , Regulação da Expressão Gênica/fisiologia , Hormônios Juvenis/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Metamorfose Biológica/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Metamorfose Biológica/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
A juvenile hormone acid methyltransferase (JHAMT) was isolated as an abundant EST in a library of the corpora allata of the adult female mosquito Aedes aegypti. Its full length cDNA encodes a 278-aa protein that has 43% amino acid identity with BmJHAMT, a juvenile hormone acid methyltransferase previously cloned from Bombyx mori. Heterologous expression produced a recombinant protein that metabolizes farnesoic acid (FA) into methyl farnesoate, as well as juvenile hormone acid into juvenile hormone III (JH III) with exquisite stereo specificity. Real time PCR experiments showed that JHAMT mRNA levels are not an unequivocal indicator of JH III synthesis rates; the A. aegypti JHAMT gene, silent in female pupae, was transcriptionally activated just 4-6h before adult eclosion. Radiochemical methyltransferase assays using active and inactive corpora allata glands (CA) dissected from sugar and blood-fed females respectively, clearly indicated that significant levels of JHAMT enzymatic activity are present when the CA shows very low spontaneous rates of JH III synthesis. Having the last enzymes of the JH synthetic pathway readily available all the time might be critical for the adult female mosquito to sustain rapid dynamic changes in JH III synthesis in response to nutritional changes or peripheral influences, such as mating or feeding. These results suggest that this gene has different roles in the regulation of JH synthesis in pupal and adult female mosquitoes, and support the hypothesis that the rate-limiting steps in JH III synthesis in adult female mosquitoes are located before entrance of FA into the synthetic pathway.
Assuntos
Corpora Allata/enzimologia , Culicidae/enzimologia , Expressão Gênica , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Metiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Corpora Allata/química , Corpora Allata/crescimento & desenvolvimento , Culicidae/química , Culicidae/genética , Culicidae/crescimento & desenvolvimento , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/genética , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Juvenile hormone (JH) acid O-methyltransferase (JHAMT) is the enzyme that transfers a methyl group from S-adenosyl-l-methionine (SAM) to the carboxyl group of JH acids to produce active JHs in the corpora allata. While the JHAMT gene was originally identified and characterized in the silkworm Bombyx mori, no orthologs from other insects have been studied until now. Here we report on the functional characterization of the CG17330/DmJHAMT gene in the fruit fly Drosophila melanogaster. Recombinant DmJHAMT protein expressed in Escherichia coli catalyzes the conversion of farnesoic acid and JH III acid to their cognate methyl esters in the presence of SAM. DmJHAMT is predominantly expressed in corpora allata, and its developmental expression profile correlates with changes in the JH titer. While a transgenic RNA interference against DmJHAMT has no visible effect, overexpression of DmJHAMT results in a pharate adult lethal phenotype, similar to that obtained with application of JH analogs, suggesting that the temporal regulation of DmJHAMT is critical for Drosophila development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hormônios Juvenis/metabolismo , Metiltransferases/metabolismo , Animais , Corpora Allata/enzimologia , Corpora Allata/fisiologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/fisiologia , Feminino , Expressão Gênica , Marcação de Genes , Genitália Masculina/enzimologia , Genitália Masculina/fisiologia , Masculino , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Fenótipo , Interferência de RNA , S-Adenosilmetionina/metabolismo , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Juvenile hormone controls the timing of insect metamorphosis. As a final step of juvenile hormone biosynthesis, juvenile hormone acid O-methyltransferase (JHAMT) transfers the methyl group from S-adenosyl-l-methionine to the carboxyl group of farnesoic acid and juvenile hormone acid. The developmental expression profiles of JHAMT mRNA in the silkworm Bombyx mori and the fruitfly Drosophila melanogaster suggest that the suppression of JHAMT transcription is critical for the induction of larval-pupal metamorphosis, but genetic evidence for JHAMT function in vivo is missing. In this study, we identified three methyltransferase genes in the red flour beetle Tribolium castaneum (TcMT1, TcMT2 and TcMT3) that are homologous to JHAMT of Bombyx and Drosophila. Of these three methyltransferase genes, TcMT3 mRNA was present continuously from the embryonic stage to the final larval instar, became undetectable before pupation, and increased again in the adult stage. TcMT3 mRNA was localized in the larval corpora allata. Recombinant TcMT3 protein methylated farnesoic acid and juvenile hormone III acid, but TcMT1 and TcMT2 proteins did not. Furthermore, RNA interference-mediated knockdown of TcMT3 in the larval stage resulted in precocious larval-pupal metamorphosis, whereas knockdown of either TcMT1 or TcMT2 showed no visible effects on metamorphosis. Importantly, precocious metamorphosis caused by TcMT3 RNA interference was rescued by an application of a juvenile hormone mimic, methoprene. Together, these results demonstrate that TcMT3 encodes a functional JHAMT gene that is essential for juvenile hormone biosynthesis and for the maintenance of larval status.
