RESUMO
This report presents the cDNA cloning, heat shock regulation and developmental expression of the hsp90 gene homologue of the Mediterranean fruit fly Ceratitis capitata (medfly). The isolated cDNA contained the coding region, the 3'UTR and most of the 5'UTR of the medfly hsp90 homologue, which was named Cchsp83. The deduced CcHSP83 polypeptide contained all the highly conserved amino acid segments that characterize the cytosolic members of the HSP90 family. Genomic analysis showed that the Cchsp83 gene is unique and was mapped at the 94C division of the sixth polytene chromosome. The size of the Cchsp83 mRNA was found to be approximately 2.7 kb. The predicted molecular mass of the CcHSP83 protein was 81.4 kDa, while the apparent molecular weight estimated by SDS-PAGE was approximately 90 kDa. Phylogenetic analysis based on 14 insect HSP90 amino acid sequences was consistent with the known phylogeny at low taxonomic level. The Cchsp83 gene is constitutively expressed in all stages of medfly development and is induced from a low level to several-fold by heat, depending on the developmental stage. Heat shock induction begins at 30 degrees C, reaching a maximum between 35 and 41 degrees C. Cchsp83 RNA expression is highly regulated during embryonic development; however, the temporal fluctuations in RNA levels during embryogenesis were not followed by similar fluctuations in the levels of the protein.
Assuntos
Ceratitis capitata/embriologia , Ceratitis capitata/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Proteínas de Choque Térmico/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por RestriçãoRESUMO
Clerocidin, a diterpenoid natural product, has been shown in vitro to inhibit DNA religation following cleavage by topoisomerase II. Herein, we characterize the efficacy and specificity of clerocidin in HeLa cells. Our results suggest that clerocidin recognizes topoisomerase II as its main intracellular target and binds to this enzyme prior to formation of the 'cleavable complex' with DNA. These pharmacological features attest to the promising chemotherapeutic potential of this natural product.
