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1.
Insect Mol Biol ; 18(2): 171-81, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19320758

RESUMEN

In the present study, we characterized a 3320-bp genomic DNA fragment encoding two medfly (Ceratitis capitata) homologues of the Drosophila melanogaster heat shock protein 23 (hsp23) gene, named Cchsp23-alphaand -beta. The two medfly hsp23 genes are transcribed in opposite directions and encode two almost identical proteins. Furthermore, the two genes exhibit a very high degree of similarity in their 5' untranslated and proximal promoter regions. Phylogenetic analysis indicated that the CcHsp23 proteins are orthologous to Drosophila Hsp23 and Sarcophaga crassipalpis Hsp23. Structural analysis of the 5' flanking regions of the Cchsp23 genes revealed the presence of several putative heat shock elements. Both CcHsp23 genes are induced by heat in a similar manner. In addition to heat-induction, the Cchsp23 genes are expressed at several stages of normal development as well as in ovaries and testes. In general, the developmental expression patterns of the medfly genes are similar, suggesting that they are under similar regulatory mechanisms. However, the expression of the Cchsp23 genes differs significantly from the expression of the Drosophila hsp23 gene in certain embryonic and larval stages, suggesting differential regulation of the hsp23 genes in the two dipteran species. The expression of both Cchsp23 genes in adult flies is increased with age, especially in males.


Asunto(s)
Ceratitis capitata/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Drosophila , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Filogenia , Mapeo Restrictivo , Alineación de Secuencia
2.
Insect Mol Biol ; 17(6): 699-710, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19133079

RESUMEN

In the present study, a genomic DNA clone encoding the medfly homolog of Drosophila melanogaster hsp27 gene, named Cchsp27, was isolated. We sequenced a part of the clone containing the coding region, the 5' untranslated region and approximately 2.8 Kb of the 5' flanking region of the gene. Phylogenetic analysis of several insect small heat shock proteins, suggested that CcHsp27 is orthologous to Drosophila Hsp27 and Sarcophaga crassipalpis Hsp25. The Cchsp27 gene was mapped at the 81A division of the sixth chromosome which coincides with one of the major heat shock puffs of medfly. Structural analysis of the 5' flanking region of the Cchsp27 gene revealed the presence of five putative heat shock elements and one putative ecdysone response element. In addition to heat induction, the Cchsp27 gene was expressed at several stages of normal medfly development. In general, the developmental expression pattern of the Cchsp27 gene was similar to the respective pattern of Drosophila hsp27 gene. However, there were some important differences in certain developmental stages suggesting differential regulation of the hsp27 gene in the two dipterans species. Salivary gland culture experiments showed that the Cchsp27 gene is regulated by 20-hydroxyecdysone.


Asunto(s)
Ceratitis capitata/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Análisis por Conglomerados , Cartilla de ADN/genética , Componentes del Gen , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN
3.
Insect Mol Biol ; 15(3): 373-82, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16756556

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 , Transgenes
4.
Insect Mol Biol ; 15(6): 839-52, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17201776

RESUMEN

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.


Asunto(s)
Ceratitis capitata/embriología , Ceratitis capitata/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Proteínas de Choque Térmico/química , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo
5.
Insect Mol Biol ; 11(6): 553-65, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12421413

RESUMEN

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 Genética
6.
Genome ; 44(5): 752-62, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11681598

RESUMEN

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.


Asunto(s)
Cromosomas/fisiología , Dípteros/genética , Ecdisona/fisiología , Animales , Cromosomas/ultraestructura , Cicloheximida/farmacología , Regulación de la Expresión Génica/fisiología , Larva , Técnicas de Cultivo de Órganos , Pupa , Glándulas Salivales/fisiología , Glándulas Salivales/ultraestructura
7.
Insect Mol Biol ; 10(3): 249-54, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11437916

RESUMEN

The aim of development of a Mediterranean fruit fly Ceratitis capitata genetic sexing strain derives from the large scale SIT programmes being carried out to control this pest. Toward this direction, we present here the male-specific expression of the Drosophila melanogaster alcohol dehydrogenase (ADH) in medfly transgenic adults generated by Minos-mediated germ line transformation. This expression pattern is obtained by using a promoter fragment of the male-specific gene MSSP-alpha2 of the medfly. We show that the heterologous enzyme is functional in the medfly oxidizing both ethanol and 2-propanol. Although leading to an approximately twofold increase of total ADH activity in male compared to female transgenic adults, these expression levels are not enough for performing genetic sexing when high doses of environmental alcohol are applied. This could be achieved either by further enhancement of the transgene expression or by generating an Adh- line to host the Minos insertions.


Asunto(s)
Oxidorreductasas de Alcohol/genética , Dípteros , Proteínas de Drosophila , Drosophila melanogaster/enzimología , Alcohol Deshidrogenasa , Oxidorreductasas de Alcohol/fisiología , Alcoholes/farmacología , Animales , Animales Modificados Genéticamente , Dípteros/efectos de los fármacos , Dípteros/genética , Drosophila melanogaster/genética , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Masculino
8.
Insect Mol Biol ; 9(2): 185-95, 2000 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10762426

RESUMEN

A multigene family encoding male specific serum polypeptides (MSSPs) that show significant structural similarity to the family of insect odourant binding proteins, has been characterized in the medfly Ceratitis capitata. This family comprises seven members classified in three subgroups, MSSP-alpha, MSSP-beta and MSSP-gamma. The genes of subgroups alpha and beta are clustered in tandem in a 35-kb genomic region, and present an exceptionally high degree of similarity not only in their coding but also in the surrounding regions, while the genes of the gamma subgroup are drastically divergent. Although MSSPs are predominantly expressed in the male fat body, detailed expression studies suggest that individual members of this family are expressed in a distinct sex- and tissue-specific manner.


Asunto(s)
Proteínas Sanguíneas/genética , Dípteros/genética , Genes de Insecto , Proteínas de Insectos/genética , Familia de Multigenes , Receptores Odorantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/clasificación , Proteínas de Insectos/clasificación , Masculino , Datos de Secuencia Molecular , Feromonas/metabolismo , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores Sexuales , Distribución Tisular
9.
Eur J Biochem ; 265(2): 798-808, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10504412

RESUMEN

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 Secuencia
10.
Insect Mol Biol ; 7(4): 345-53, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9723872

RESUMEN

Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.


Asunto(s)
Proteínas Sanguíneas/genética , Dípteros/genética , Proteínas de Insectos/genética , Receptores Odorantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Hemolinfa , Masculino , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
11.
Insect Mol Biol ; 7(3): 279-90, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9662478

RESUMEN

The cloning and the characterization of the heat shock 70 (hsp70) genes of the medfly C. capitata, a major agricultural pest, are presented. Six genomic clones were isolated by screening a medfly genomic library with an hsp70 genomic fragment of Drosophila melanogaster. They form two 30 kb contigs, both of which map cytogenetically in a single major heat shock puff (3L:24C) of the salivary gland polytene chromosomes. Restriction mapping and blot hybridization indicated the presence of six putative hsp70 genes in these two closely linked regions. The sequence of one of these genes suggests that it is a heat-inducible hsp70 gene. The 638-codon open reading frame shows 84% identity at the amino acid level (73.5% at the nucleotide level), relative to corresponding D. melanogaster sequences. The 5' untranslated leader sequence, approximately 200 bp long, is not interrupted by introns and is very rich (48%) in adenine residues, resembling Drosophila heat-inducible hsp70 genes. Furthermore, the promoter of this gene contains two characteristic heat shock elements close upstream from the TATA box. The levels of the hsp70 transcripts are very low at 25-30 degrees C, increase significantly at 33 degrees C and reach maximum at 39 degrees C.


Asunto(s)
Dípteros/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Insectos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN Complementario , Drosophila melanogaster/genética , Genes de Insecto , Proteínas HSP70 de Choque Térmico/química , Proteínas de Insectos/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
12.
Int J Biochem ; 25(3): 313-8, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8462721

RESUMEN

1. The lipopolysaccharide (LPS) and the extracellular products (slime) of a smooth, nonmucoid Pseudomonas aeruginosa strain (PAC IR) and its rough mutant (PAC 605) were subjected to a comparative biochemical analysis. 2. Chemical and electrophoretic analyses suggested that the slime preparation of both strains are composed mainly of similar carbohydrate components which are different from those of the respective lipopolysaccharides. 3. Chromatographic analysis of the two slime preparations on gel permeation HPLC columns revealed the presence of a major polysaccharide in both strains with an apparent molecular weight 29 kDa and a minor high molecular weight polysaccharide in the PAC IR strain.


Asunto(s)
Lipopolisacáridos/análisis , Pseudomonas aeruginosa/química , Carbohidratos/análisis , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Galactosa/análisis , Glucosa/análisis , Manosa/análisis , Peso Molecular , Mutación , Pseudomonas aeruginosa/genética , Ramnosa/análisis
13.
J Clin Microbiol ; 25(4): 656-9, 1987 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-3106409

RESUMEN

The slime material from a revertant nonmucoid variant, derived by serial passage of a heavily mucoid Pseudomonas aeruginosa strain isolated from a patient with bacteremia, was found to contain 16% uronic acids, 48.5% carbohydrates, 11% protein, and 2% lipids. Chromatographic analysis by ion exchange chromatography revealed that this extracellular material consisted of three fractions, one uronic acid fraction with properties similar to those of the alginate fraction of the parental strain and two other fractions consisting of neutral sugars and proteins in approximately a 5:1 ratio. In addition, the slime material from six other clinical macroscopic nonmucoid P. aeruginosa strains was found to contain alginate. These results demonstrate that alginate production in various amounts is a property shared by all P. aeruginosa strains.


Asunto(s)
Alginatos/biosíntesis , Pseudomonas aeruginosa/metabolismo , Alginatos/análisis , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Humanos
14.
Dev Biol ; 95(2): 492-6, 1983 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-6337889

RESUMEN

Four major hemolymph polypeptides (ceratitins) with molecular weights between 8.1 X 10(4) and 8.7 X 10(4) daltons have been identified in the fat body of late Ceratitis capitata larvae. Total fat body RNA from late larvae was translated in reticulocyte lysate, and the predominant in vitro translation products were shown to be the ceratitin precursors. The biosynthesis of these proteins during postembryonic development was studied in both tissue culture and cell-free system. Comparison of the biosynthetic patterns obtained in the two systems suggests a linear relationship between messenger concentration and protein synthesis. Three of these polypeptides show a coordinate pattern of synthesis and are immunologically related. After pupation, all four ceratitins are reabsorbed by the fat body where they accumulate.


Asunto(s)
Tejido Adiposo/metabolismo , Dípteros/crecimiento & desarrollo , Cuerpo Adiposo/metabolismo , Biosíntesis de Péptidos , Péptidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Sistema Libre de Células , Dípteros/metabolismo , Electroforesis en Gel de Poliacrilamida , Hemolinfa/metabolismo , Técnicas de Inmunoadsorción , Larva/metabolismo , Precursores de Proteínas/biosíntesis
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