RESUMO
Testis-specific tandemly repeated Stellate genes are part of the Ste-Su(Ste) genetic system required for male fertility in Drosophila melanogaster. Stellate genes encode a functional homolog of the ß-subunit of protein kinase CK2. Derepression of Stellate results in their over-expression, meiotic disturbances and male sterility. Stellate genes are represented by clustered copies in the X chromosome and carry promoters shared with another X-chromosome cluster, ßNACtes genes, encoding putative ß-subunits of the nascent polypeptide-associated complex. Using Electrophoretic Mobility Shift Assay, we revealed in the Stellate promoter three cis-acting elements, E-boxes, the loss of which greatly diminished the reporter gene expression in Drosophila testes. We identified that these E-boxes were recognized by helix-loop-helix protein, dUSF (Drosophila ortholog of mammalian USF) in testis nuclear extract. All three E-boxes were preserved in the promoters of both euchromatic and heterochromatic Stellate clusters. Two analogous E-boxes were detected in the promoters of 5'-copies of the duplicated ßNACtes gene pairs, whereas the 3'-copies lacked these sites but possessed a new binding site for a testis protein distinct from dUSF. Here we characterized a new type of testis-specific core promoter and identified dUSF as its interacting transcription factor.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Quinases/genética , Testículo/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica/genética , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , Fatores Estimuladores Upstream/metabolismoRESUMO
ECAR-LANS, the recombinant L-asparaginase from Erwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K (m) value was 98x10(-6) M for the main physiological substrate L-Asn and 3400x10(-6) M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.
