Drosophila melanogaster mutant tan

Drosophila melanogaster gene tan was originally discovered in the early 20th century as a mutant strain lacking the dark pi gment pattern of wild-type (wt) f lies and, therefore, showing a light yellowish brown color (McEwen, 1918). Flies lack ing Tan function also exhibited abnormalities in vision (Benzer, 1967; Inoue et al. , 1988; True et al. , 2005), and tan males displayed an abnormal courtship behavior (Cook, 1980; Tomkins et al. , 1982). tan 1 ( t 1 ) and tan 3 ( t 3 ) alleles were found as spontaneous mutations, t 3 mutant being apparently lighter than t 1 (Brehme, 1941). tan is the structural gene for N- β -alanyldopamine hydrolase (NBAD-hydrolase or Tan protein), the enzyme that generates dopamine (DA) from NBAD (Wright, 1987; True et al. , 2005). Tan is expressed as a precursor protein of 43.7 kDa. Th is precursor is clea ved into two subunits of 29.9 and 13.8 kDa that apparently conform together a he terodimeric active protein (Wagner et al. , 2007). The enzyme that generates NBAD from DA, th e opposite reaction to the one catalyzed by Tan, is the NBAD-synthase or E bony protein (Wright, 1987; Pérez et al ., 1997), which is codified by the gene ebony . Since both Tan and Ebony ar e involved in cuticle tanni ng, carcinine re gulation, and NBAD metabolism in nervous tissue (Wright, 1987; Pérez et al. , 1997, 2004; Hovemann et al. , 1998; Borycz et al. , 2002; True et al. , 2005), it has been suggested that they function together in a system regulating the levels of dopamine during cuticle sclerotization a nd histamine in the visual metabolism (Borycz et al. , 2002; Pérez et al. , 2010). During the last few years, several publicati ons appeared regarding NBAD-synthase (Wappner et al ., 1996a, b; Pérez et al ., 1997, 2002, 2004, 2010; Hovemann et al. , 1998; Borycz et al. , 2002; Wittkopp et al., 2002; Schachter et al ., 2007), but very little is known about tan (True et al. , 2005; Wagner et al. , 2007). Thus, it was important to furthe r characterize the NBAD-hydrolase in D. melanogaster wt and in mutants t 1 and t 3.Fil: Badaracco, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Quesada Allue, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pérez, Martín Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages’ strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.EEA Cerro AzulFil: Miño, Samuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Cerro Azul; Argentina.Fil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Louge Uriarte, Enrique Leopoldo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Ciarlet, Max. Icosavax. Clinical Development; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentin

Phylogenetic analyses of typical bovine rotavirus genotypes G6, G10, P[5] and P[11] circulating in Argentinean beef and dairy herds

Group A rotavirus (RVA) is one of the main causes of neonatal calf diarrhea worldwide. RVA strains affecting Argentinean cattle mainly possess combinations of the G6, G10, P[5] and P[11] genotypes. To determine RVA diversity among Argentinean cattle, representative bovine RVA strains detected in diarrheic calves were selected from a survey conducted during 1997–2009. The survey covered the main livestock regions of the country from dairy and beef herds. Different phylogenetic approaches were used to investigate the genetic evolution of RVA strains belonging to the prevalent genotypes. The nucleotide phylogenetic tree showed that all genotypes studied could be divided into several lineages. Argentinean bovine RVA strains were distributed across multiple lineages and most of them were distinct from the lineage containing the vaccine strains. Only the aminoacid phylogenetic tree of G6 RVA strains maintained the same lineages as observed at the nucleotide level, whereas a different clustering pattern was observed for the aminoacid phylogenetic trees of G10, P[5] and P[11] suggesting that the strains are more closely related at the aminoacid level than G6 strains. Association between P[5] and G6(IV), prevalent in beef herd, and between P[11] and G6(III) or G10 (VI and V), prevalent in dairy herds, were found. In addition, Argentinean G6(III), G10, P[5] and P[11] bovine RVA strains grouped together with human strains, highlighting their potential for zoonotic transmission. Phylogenetic studies of RVA circulating in animals raised for consumption and in close contact with humans, such as cattle, contribute to a better understanding of the epidemiology of the RVA infection and evolution.Fil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Matthijnssens, J.. University of Leuven. Rega Institute for Medical Research; BélgicaFil: Louge Uriarte, Enrique Leopoldo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Bilbao, Gladys Noemí. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Fernandez, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parra, G. I.. National Institutes of Health; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

ß-lactamasas producidas por enterobacterias resistentes a amoxicilina-ácido clavulánico aisladas en Buenos Aires, Argentina: un nuevo gen blaTEM

Resistance to β-lactam/β-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second- and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a (1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV β-lactamases (K. pneumoniae and P. mirabilis). A new blaTEM variant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8 μg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrum β-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Badaracco, Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ayala, Juan. Centro de Biología Molecular "Severo Ochoa"; EspañaFil: Rodriguez, Cynthia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Famiglietti, Angela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Avances sobre la caracterización del “mal de la tela” en plantas de yerba mate Ilex paraguariensis a. St. Hil.

La yerba mate (Ilex paraguariensis A. St. -Hil.) es un cultivo primario y de gran importancia para el noreste de Argentina. En los últimos años, la enfermedad conocida como “mal de la tela”, cuyo agente causal es Ceratobasidium niltonsouzanum, está ocasionando importantes daños en la producción; y debido a la poca información existente su manejo resulta muy difícil. Al inicio de la enfermedad los signos incluyen cordones de micelios blancos visibles sobre las ramas y hojas infectadas. Las hojas se necrosan, se secan y quedan colgando de las ramas por la hifa del hongo. A partir de hojas y ramas sintomáticas, muestreadas de distintas partes de la provincia de Misiones, fue aislado el agente causal para su posterior caracterización morfológica y molecular. Las colonias fueron aisladas y crecieron rápidamente sobre medios de APG (agar papa glucosado), presentando un color blanquecino al inicio, tornándose luego de un color castaño. Con la caracterización morfológica se pudo determinar que el hongo se encuentra distribuido en distintas partes de la provincia de Misiones, afectando las plantaciones con distintos niveles de incidencia. Pruebas de patogenicidad, conducidas en laboratorio, permitieron conocer el progreso de la enfermedad bajo condiciones ambientales favorables. El objetivo del presente trabajo fue contribuir al conocimiento de la enfermedad, con la descripción de síntomas y signos, condiciones predisponentes y caracteres morfológicos y moleculares del agente causal del “mal de la tela”.Yerba mate (Ilex paraguariensis A. St. Hil.) is a primary and important crop in northeastern Argentina. In past years, a disease called white thread blight caused by Ceratobasidium niltonzousanum is having a very important impact in the production and has not been well characterized. Thus, the objective of this work was to complement the knowledge with symptom descriptions, morphological and molecular characterization of the causal agent, and pathogenicity tests. Signs of the disease include white mycelial cords visible on the infected branches and leaves with twig dieback, diseased leaves dry up but remain attached to the plant hanging by the fungal hyphae. Symptomatic leaves and branches were sampled from different parts of the province of Misiones. The causal agent was isolated for its subsequent morphological and molecular characterization. The colonies grow rapidly on PDA (potato dextrose agar) and are white at the beginning but later become light brown. With the morphological characterization it was possible to determine that the fungus is distributed in different parts of the province of Misiones, affecting the plantations at different levels of incidence. Pathogenicity tests conducted in the laboratory allowed to know the progress of the disease under favorable environmental conditions. This work constitutes a contribution to the knowledge of this disease that affects yerba mate plantations.EEA MontecarloFil: Dummel, Delia Marlene. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina.Fil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Badaracco, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (CCT) Nordeste; ArgentinaFil: Kramer, Rodrigo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Rohatsch, Pablo Heriberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Agostini, Juan Pedro. Universidad Nacional de Misiones. Facultad de Ciencias Forestales. Cátedra de Protección Vegetal; Argentin

An Unwanted Association: The Threat to Papaya Crops by a Novel Potexvirus in Northwest Argentina

An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.Instituto de Patología VegetalFil: Cabrera Mederos, Dariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Cabrera Mederos, Dariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Torres, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones en Bacteriología y Virología Molecular (IBaViM); ArgentinaFil: Portal, Orelvis. Universidad Central “Marta Abreu” de Las Villas. Facultad de Ciencias Agropecuarias. Departamento de Biología; CubaFil: Portal, Orelvis. Universidad Central “Marta Abreu” de Las Villas. Facultad de Ciencias Agropecuarias. Centro de Investigaciones Agropecuarias; CubaFil: Jaramillo Zapata, Margarita. Universidad de San Pablo-T; ArgentinaFil: Trucco, Veronica Milagros. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Trucco, Veronica Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Flores, Ceferino Rene. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; ArgentinaFil: Ortiz, Claudio Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; ArgentinaFil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Acuña, Luis Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Nome Docampo, Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Nome Docampo, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Quito-Avila, Diego. Centro de Investigaciones Biotecnológicas del Ecuador. Escuela Superior Politécnica del Litoral; EcuadorFil: Bejerman, Nicolas Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Bejerman, Nicolas Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Castellanos Collazo, Onias. Fondo para la Investigación Científica y Tecnológica (FONCYT); ArgentinaFil: Castellanos Collazo, Onias. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Sánchez-Rodríguez, Aminael. Universidad Técnica Particular de Loja. Departamento de Ciencias Biológicas; EcuadorFil: Giolitti, Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Giolitti, Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentin

Actualización sobre los virus que infectan papaya en Argentina

PosterLa papaya Carica papaya se cultiva en las regiones tropicales y subtropicales, y se establece como una alternativa sustentable en el norte de Argentina Con el objetivo de generar conocimientos para aportar al manejo de las virosis que afectan papaya en Argentina, se realizaron evaluaciones en las principales áreas productorasInstituto de Patología VegetalFil: Cabrera Mederos, Dariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Cabrera Mederos, Dariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Portal, O. Universidad Central “Marta Abreu” de Las Villas. Facultad de Ciencias Agropecuarias. Santa Clara; CubaFil: Acuña, Luis Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Badaracco, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (CCT) Nordeste; ArgentinaFil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; ArgentinaFil: Rodríguez, E. Instituto de Enseñanza Agropecuaria (IEA) N9; ArgentinaFil: Nickel, A. Instituto de Enseñanza Agropecuaria (IEA) N9; ArgentinaFil: Sáez, S. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; ArgentinaFil: Nome Docampo, Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Nome Docampo, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Torres, C. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jaramillo, M. Universidad de San Pablo T; ArgentinaFil: Trucco, Veronica Milagros. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Trucco, Veronica Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Ortiz, Claudio Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; ArgentinaFil: Flores, Ceferino Rene. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; ArgentinaFil: Castellanos Collazo, Onias. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Giolitti, Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Giolitti, Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentin

Aspects of chromatin structure and induced transcription activity on salivary glands of Rhynchosciara americana (Diptera: Sciaridae)

Os cromossomos politênicos da glândula salivar dos dípteros proporcionam condições excepcionalmente favoráveis à observação, em microscopia óptica, da estrutura da cromatina bem como da atividade gênica. Neste trabalho, a distribuição de modificações epigenéticas e de marcadores associados à atividade de transcrição foi estudada em cromossomos politênicos de larvas Rhynchosciara americana em condições normais de crescimento e também submetidas a tratamentos que levam ao estresse, um dos quais (anestesia por éter dietílico) realizado pela primeira vez nesta espécie. Na recuperação do estresse por anestesia, o locus do gene hsp70 apareceu transcricionalmente ativo embora, frequentemente, a marcação de RNA polimerase II não ocupava todo o volume do pufe como ocorre usualmente na resposta ao estresse por choque térmico, sugerindo descondensação parcial do locus. A localização cromossômica da proteína Sex-lethal, postulada como um fator de splicing genérico em espécies da família Sciaridae, foi estudada pela primeira vez em larvas submetidas ao estresse. A ausência da mesma em loci cromossômicos que estão ativos em transcrição em condições estressantes sugere que Sex-lethal não seja um fator de splicing genérico. Além disto, a localização desta proteína em regiões transcricionalmente inativas durante o estresse sugere que Sex-lethal cumpra funções ainda não descritas. Entre os dados de localização de histona H3 metilada em lisina 4, marcador epigenético usualmente associado à transcrição, destacou-se a forte marcação de uma região particular dentro da secção cromossômica A-9. Esta região, entretanto, apresenta sinais muito fracos de anti-híbrido DNA/RNA e de RNA polimerase II, sugerindo atividade transcricional muito baixa ou ausente. Observou-se também que o marcador epigenético conservado de heterocromatina, H3me3K9 (histona H3 trimetilada em lisina 9), não está presente na mesma; mas, por outro lado, detectou-se na região sinal significativo da proteína da heterocromatina de Sciara coprophila. Com o objetivo de conhecer sequências de DNA presentes na região, foi produzida uma biblioteca plasmidial produzida a partir de microdissecção cromossômica do sub-setor de A-9 seguida de DOP-PCR. O produto de amplificação foi usado como sonda em hibridação in situ e marcou, além da região microdissecada, extremidades cromossômicas não centroméricas, indicando que estas regiões compartilham sequências repetitivas. Algumas puderam ser identificadas após o seqüenciamento, tais como repetições em tandem e elementos genéticos móveis enquanto que outras não apresentaram similaridade significativa com sequências disponíveis em bancos de dados. Os dados obtidos sugerem que o sub-setor de A-9 analisado assemelha-se à heterocromatina, porém composta de uma combinação aparentemente não usual de marcadores epigenéticosPolytene chromosomes of dipteran salivary glands provide optimal visualization of chromatin structure as well as gene activity at the cytological level. In this work, the distribution of epigenetic and transcription-associated markers was studied in polytene chromosomes of Rhynchosciara Americana larvae, either under during normal growth or stressful treatments, one of which (anaesthesia by ether) has been observed for the first time in this species. During the recovery from the anesthesia, the hsp70 gene locus was transcriptionally active although RNA polymerase II detection did not occupied the entire volume of the puff -as usually happens during the heat shock response-, suggesting partial puff decondensation. The chromosomal localization of the sciarid Sex-lethal protein, which was supposed to be a general splicing factor, was studied for the first time in stressed larvae and was not detected in transcriptionally active loci. Hence, the Sex-lethal protein may not be a general splicing factor. Moreover, Sex-lethal localization in transcriptionally inactive regions during stress situations suggests it plays still unknown roles. Antibodies to histone H3 methylated at lysine 4 -an epigenetic marker of transcription- labeled strongly at a particular region within chromosome section A-9. This region, in contrast, displays very low levels of both DNA/RNA hybrid and RNA polymerase II, suggesting, if any, poor transcriptional activity. Interestingly, the conserved epigenetic heterochromatin marker H3me3K9 (histone H3 trimethylated at lysine 9) is not significantly present in this region, even though the Sciara coprophila heterochromatin protein was clearly detected. In order to sample DNA sequences contained in this region, a plasmid library was produced by performing a microdissection of A-9 sub-section followed by DOP-PCR. The total microdissection product was used as a probe, and hybridized, in addition to the microdissected region, to non-pericentric chromosome ends, indicating that repetitive sequences are shared by these regions. Some sequences could be identify, after sequencing, such as tandem repeats and mobile elements, but others failed to display significant similarity to sequences deposited in databases. The results suggest that the sub-section analyzed resembles heterochromatin but being composed of an apparently unusual combination of epigenetic marker