RESUMO
A gene encoding the adult cuticular protein ACP-20 was isolated in Tenebrio. It consists of three exons interspersed by two introns, intron 1 interrupting the signal peptide. To understand the regulatory mechanisms of ACP-20 expression, ACP-20 promoter-luciferase reporter gene constructs were transfected into cultured pharate adult wing epidermis. Transfection assays needed the presence of 20-hydroxyecdysone, confirming that ACP-20 is up-regulated by ecdysteroids. Analysis of 5' deletion constructs revealed that three regions are necessary for high levels of transcription. Interaction experiments between intronic fragments and epidermal nuclear proteins confirmed the importance of intron 1 in ACP-20 transcriptional control, which results from the combined activity of regulatory cis-acting elements of the promoter and those of intron 1.
Assuntos
Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Metamorfose Biológica/fisiologia , Tenebrio/genética , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ecdisona/metabolismo , Ecdisona/farmacologia , Epiderme/metabolismo , Biblioteca Gênica , Genes Reporter/fisiologia , Íntrons/genética , Íntrons/fisiologia , Luciferases/metabolismo , Metamorfose Biológica/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Análise de Sequência de DNA , Tenebrio/fisiologia , Ativação Transcricional/genética , TransfecçãoRESUMO
In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.
Assuntos
Genes de Insetos/genética , Proteínas de Insetos/genética , Muda/genética , Tenebrio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/química , DNA/genética , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Genoma , Larva/química , Larva/genética , Larva/ultraestrutura , Dados de Sequência Molecular , Pupa/química , Pupa/genética , Pupa/ultraestrutura , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Tenebrio/química , Tenebrio/ultraestruturaRESUMO
The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.