RESUMEN
The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos-mediated germ-line transformation. The activity of each promoter was evaluated in 15 transformed lines by beta-galactosidase quantitative assays. The heat-inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The -263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the -263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo.
Asunto(s)
Ceratitis capitata/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Animales , Ceratitis capitata/genética , Ceratitis capitata/crecimiento & desarrollo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Estadios del Ciclo de Vida , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transformación Genética , TransgenesAsunto(s)
Ceratitis capitata/genética , Cromosomas/genética , Animales , Ceratitis capitata/anatomía & histología , Ceratitis capitata/crecimiento & desarrollo , Bandeo Cromosómico , Mapeo Cromosómico , Cromosomas/ultraestructura , Intercambio Genético/genética , Drosophila melanogaster/genética , Femenino , Genes de Insecto , Marcadores Genéticos , Masculino , Mecanorreceptores/ultraestructura , Especificidad de Órganos , Glándulas Salivales/ultraestructura , Cromosomas Sexuales/genética , Cromosomas Sexuales/ultraestructura , Especificidad de la EspecieRESUMEN
Using 5' RACE with specific primers for the ecdysone receptor B1 isoform of the Mediterranean fruit fly (medfly), Ceratitis capitata, we isolated a cDNA clone encoding the specific region of the medfly ecdysone receptor A isoform (CcEcR-A). The CcEcR-A-specific region was very similar to the EcR-A-specific region of Drosophila melanogaster and less similar to the EcR-A-specific regions of Lepidoptera. The developmental expression of both CcEcR-A and CcEcR-B1 mRNAs was studied in whole animals, salivary glands and ovaries by RT-PCR, using isoform-specific primers. Both CcEcR mRNAs are present in very early embryos, decrease to very low levels during the first hours of embryogenesis and are highly expressed in all consequent embryonic stages. During metamorphosis both isoforms are present showing two peaks; the first at the larval-prepupal transition and the second during the second half of prepupal development. These peaks are correlated with the two puffing cycles and the two major 20-hydroxyecdysone (20E) increases that occur during medfly metamorphosis. CcEcR-B1 mRNA was directly induced in larval salivary glands in vitro by 20E, even at very low concentrations of the hormone, while CcEcR-A mRNA was slightly induced only by high 20E concentrations and in the absence of a protein synthesis inhibitor. During oogenesis, the CcEcR mRNAs were expressed synchronously, peaking at the beginning of both previtellogenic and vitellogenic phases.
Asunto(s)
Ceratitis capitata/crecimiento & desarrollo , Ceratitis capitata/genética , Regulación de la Expresión Génica , Filogenia , ARN Mensajero/genética , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ceratitis capitata/clasificación , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Glándulas Salivales/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The construction of the first balancer chromosome, FiM1, for the medfly Ceratitis capitata is described. This chromosome has three overlapping pericentric inversions and is marked with dominant and recessive mutations. The inversion breakpoints of FiM1 suppress recombination throughout the length of the fifth chromosome, allowing lethal mutations to be recovered and maintained. This chromosome will provide a powerful tool for the manipulation of laboratory stocks, in particular, the recovery of new mutant and transgenic strains. We demonstrate the use of FiM1 for the recovery and maintenance of chromosomes carrying lethal mutations.
Asunto(s)
Cromosomas , Dípteros/genética , Animales , Aberraciones Cromosómicas , Inversión Cromosómica , Femenino , Fertilidad/genética , Genes Letales , Genes Recesivos , Marcadores Genéticos , Técnicas Genéticas , Masculino , Mutación , Recombinación GenéticaRESUMEN
In this paper, we report the chromosomal localization of ceratotoxins, a gene family encoding antibacterial female-specific peptides from the mediterranean fruit fly Ceratitis capitata. The analysis of both polytene and mitotic chromosomes by in situ hybridization shows that ceratotoxins are the first case of female-specific X-linked genes from the medfly C. capitata. Southern blot analysis reveals that the ceratotoxin gene family is not specifically amplified in the female reproductive accessory glands of C. capitata.
Asunto(s)
Dípteros/genética , Ligamiento Genético , Proteínas de Insectos/genética , Cromosoma X/genética , Animales , Southern Blotting , Mapeo Cromosómico , Cromosomas/metabolismo , Femenino , Hibridación in Situ , Hibridación Fluorescente in Situ , Proteínas de Insectos/metabolismo , MitosisRESUMEN
In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5'-untranslated region and the complete 3'-untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.
Asunto(s)
Dípteros/metabolismo , Proteínas de Insectos/genética , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Ecdisterona/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación Fluorescente in Situ , Proteínas de Insectos/química , Larva/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/metabolismo , Receptores de Esteroides/química , Alineación de SecuenciaRESUMEN
The results of two screens for mutations and chromosomal aberrations in Ceratitis capitata are presented. Three dominant mutations were recovered, including Sb, which is associated with a homozygous lethal translocation between the third and fifth chromosomes, T(3;5)Sb, with the fifth chromosome breakpoint adjacent to y. The T(3;5)Sb chromosome is maintained by selecting for Sb in a T(3;5)Sb, w2 Sb y2 wp/w2 y2 wp stock and can be used to distinguish between other chromosomes carrying differential combinations of the recessive markers w2 y2 wp. The ability to isolate particular marked chromosomes is essential in order to recover an inversion-based balancer chromosome. In addition to the recovery of dominant mutations, gamma-ray induced somatic mosaics of w2 and y2 and zygotic w mosaics were found. The generation of zygotic mosaics following mutagenesis can give mutants with a mosaic germ line that fail to breed true in the first generation. A screen of 22,830 irradiated chromosomes failed to recover variegating alleles of w, although such alleles might be recovered in a larger screen. The high frequency of dominant mutations and the instability at the w locus in our stocks implies a background level of dysgenic activity. These results have implications for the construction and long-term maintenance of genetically modified strains.