Stuttgart, Universiẗat, Diss., 1999.

The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 95-107). / Microfiche. / x, 107 leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

No description available.

La bactériose vasculaire du riz (BLB) et à stries foliaires (BLS) causées respectivement par Xanthomonas oryzae pv. oryzae (Xoo) et X. oryzae pv. oryzicola (Xoc) sont deux maladies émergentes en Afrique de l'Ouest, suite à l'expansion de la culture du riz et à l'introduction de variétés à haut rendement au cours de ces dernières décennies. Trois nouvelles races de Xoo ont été caractérisées en Afrique dont on a montré, sur la base d'une analyse génétique, leur spécificité africaine. En revanche, une étude réalisée sur une dizaine de souches de Xoc isolées au Mali en 2003, démontre qu'elles sont apparentées à des souches de Xoc asiatiques. En Asie, plusieurs gènes de résistance à Xoo ont été identifiés et déployés dans les programmes de lutte contre BLB. Cependant aucun gène de résistance à Xoc n'a été encore identifié chez le riz. Les objectifs de notre étude étaient (i): d'implémenter les collections de souches de Xoo et Xoc Africaines disponibles mais incomplètes, à l'aide de nouvelles campagnes d'échantillonage réalisées de 2009 à 2012 dans différentes zones agroécologiques du Burkina Faso et du Mali, (ii) de déterminer la diversité génétique de ces souches, (iii) d'identifier et caractériser de nouvelles sources de résistance contre BLB et BLS au sein d'accessions de riz cultivées au Burkina Faso. Nos résultats ont montré que les souches africaines de Xoc sont hautement variables tant d'un point de vue génétique que du pouvoir pathogène. L'analyse par PCR de deux effecteurs de types III conservés (xopAJ et xopW) permet de différencier les souches de Xoc en deux groupes, xopAJ étant absent dans la majorité des souches et une insertion de 1050 bp étant détectée dans la séquence codante de xopW de certaines souches. Néanmoins, il apparait que la forte diversité génétique des Xoc n'est pas corrélée à leur origine géographique, ni à la période de collecte, ou à la nature de l'hôte. Les souches de Xoo caractérisées appartiennent toutes à la race A1 qui n'avait pas encore été signalée au Mali. Au regard de la diversité des souches et de leur évolution, il est important d'envisager un plan de surveillance épidémiologique à plus large échelle des populations de Xo dans les régions concernées en Afrique de l'Ouest. Enfin, nous avons montré que certaines variétés de riz cultivées au Burkina Faso présentent un phénotype de résistance spécifique des souches africaines de Xoo et ce, à tous les stades de développement de la plante. Ces données originales contrastent par rapport au phénotype des lignées de riz résistantes de référence (Xa4, xa5 et Xa7 efficaces uniquement au stade de tallage maximum). Eu égard à l'absence de gènes de résistance dans le riz efficaces contre Xoc, ces variétés qui constituent également une source de résistance efficaces contre la diversité des souches de Xoc africaines, offrent potentiellement un nouveau moyen pour assurer le contrôle du BLS au Burkina Faso et éventuellement dans d'autres pays Africains. / Bacterial Leaf Blight (BLB) and Bacterial Leaf Streak (BLS) diseases respectively caused by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae. pv oryzicola (Xoc) are two emerging diseases of rice in West Africa, due to the recent expansion of rice cultivation and introduction of improved rice varieties over the last decade. Three news Xoo races were characterized based on genetic analysis, demonstrating their african specificity. In contrast, a study achevied on about ten Xoc strains isolated in Mali in 2003, show that they are related to asian Xoc strains. In Asia, several R genes against Xoo have been identified and deployed in breeding program to control BLB. In contrast, no R gene against Xoc has been identified in rice. The objectives of this PhD thesis are to (i) complete the Xo collections of African isolates upon annual sampling operated from 2009 to 2012 in various agroecological areas of Burkina Faso and Mali, (ii) determine the genetic diversity of these strains , (iii) identify and characterize news sources of resistance genes to BLB and BLS within rice accessions cultivated in Burkina Faso.Our results showed that african Xoc are highly diverse genetically and phenotypically. PCR-based analyse of two conserved type III effector gene (xopAJ and xopW) differentiated two groups of Xoc strains, with xopAJ not detected in a majority of African Xoc strains and 1050 bp insertion detected in xopW gene for few strains. However, the high genetic diversity observed among the Xoc strains is not correlated to geographical origin, sampling data or host plant species. Xoo strains characterized belong all to race A1 previously reported by Gonzalez et al. (2007) in Burkina Faso. Given the diversity of X. oryzae strains and their evolution, it is essential to establish a large scale epidemiological monitoring of Xo populations in concerned regions in west Africa.At last, some accessions cultivated in Burkina Faso showed specific resistance to african Xoo strains at all plant development stages. These original data contrast with rice lines carring Xa4, xa5 and Xa7 resistance genes against BLB, which are only effective at maximun tillering stage.Given no sources of effective resistance genes against BLS is available in rice, these accessions which were also efficient against a set of Xoc strains representative of the diversity in Africa, represent a huge potential source for the control of BLS in Burkina Faso, and eventually in others african countries.

Master of Science / Department of Plant Pathology / Erick D. DeWolf / Wheat blast, caused by Magnaporthe oryzae pathotype triticum, has emerged as a serious problem for wheat production in South America and recently emerged as a threat to wheat production in Bangladesh. To prepare for the possible introduction of wheat blast in to the United States, it would be helpful to identify areas of the country most at risk for blast epidemics. Because wheat blast occurs primarily in tropical and subtropical regions of the world, cold winter temperatures may restrict the establishment of the blast pathogen in the United States. Therefore, the first objective of this research was to quantify the freeze-thaw tolerance of the wheat blast pathogen in naturally infected wheat rachises from Bolivia and to measure the viability of the conidia after exposure to various treatments. The results indicate that exposing the fungus in moist residue to multiple freeze-thaw cycles is more damaging than exposing the fungus in moist residue to longer, single freezes. When in dry residue, the fungus was not harmed by the freeze-thaw cycles. Freezing and thawing of the wheat blast fungus in moist residue significantly affected its ability to produce viable conidia. The second objective of this research was to identify environmental conditions that could be conducive for wheat blast epidemics by examining historical epidemics of rice blast, caused by Magnaporthe oryzae pathotype oryza. The dataset used in this analysis consisted of 60 site-years of historical observations of rice blast levels from Arkansas, Louisiana, and Texas. These observations were coupled with monthly and weekly summaries of hourly weather variables based on temperature, relative humidity, precipitation, and regional moisture indices. Classification trees and logistic regression were used to identify variables associated with rice blast epidemics. The results indicate that rice blast epidemics are favored by cooler April temperatures and higher levels of precipitation in June. Preliminary models for rice blast based on these variables were able to correctly classify epidemic years with >75% accuracy. In the future, the results of this project will be used as part of a risk assessment for a wheat blast introduction and establishment in the United States.

The ascomycete fungus Magnaporthe oryzae, causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and M. oryzae is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made M. oryzae an ideal model system for understanding general plant-pathogen interactions as well. The objective of this dissertation was to characterize the M. oryzae homologs of Histoplasma capsulatum RYP (Required for Yeast Phase) genes that are required for transition to the parasitic phase. H. capsulatum is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37°C, the host body temperature. Four H. capsulatum RYP genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. RYP1 is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. RYP2 and RYP3 are homologs of vosA and velB, members of the Velvet family, best characterized in Aspergillus nidulans, where they coordinate morphological differentiation with secondary metabolism. RYP4 is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the M. oryzae RYP1 homolog, RIG1 (Required for Infectious Growth), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. Δrig1 was blocked in the transition to infectious hyphal growth, similar to H. capsulatum ryp1, which could not transition to the yeast phase. Deletion mutants of M. oryzae RYP2, RYP3, and RYP4 homologs were similar to the wild type in somatic growth and pathogenicity indicating that although RIG1 is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the RYP pathogenicity genes identified in H. capsulatum. Δrig1 is the first M. oryzae mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Entomology, Waite Agricultural Research Institute, 1978.

FREITAS, A. C. Produção de proteases por Aspergillus em fermentação semi-sólida utilizando torta de canola. 83 f. 2009. Dissertação (Mestrado em Engenharia Química) – Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2009. / Submitted by Marlene Sousa (mmarlene@ufc.br) on 2016-03-21T19:12:22Z No. of bitstreams: 1 2009_dis_acfreitas.pdf: 1152918 bytes, checksum: 4a33865f89d978715f9c8c42e00d1485 (MD5) / Approved for entry into archive by Marlene Sousa(mmarlene@ufc.br) on 2016-03-28T17:44:50Z (GMT) No. of bitstreams: 1 2009_dis_acfreitas.pdf: 1152918 bytes, checksum: 4a33865f89d978715f9c8c42e00d1485 (MD5) / Made available in DSpace on 2016-03-28T17:44:50Z (GMT). No. of bitstreams: 1 2009_dis_acfreitas.pdf: 1152918 bytes, checksum: 4a33865f89d978715f9c8c42e00d1485 (MD5) Previous issue date: 2009-02-19 / The proteases belong to the class of enzymes with the capacity to hydrolyze peptidic connections into protein and protein fragments, modifying the substrate by great selectivity and specification. The semi-solid fermentation is a fermentation process in which the growth of microorganisms and the formation of the products occur on the surface of the solid substrate next to the absence of free water, usually using natural raw materials as a source of carbon and energy. On this study, it was done the semi solid cultivation of filament fungi, in order to produce proteases using as a substrate a cake of canola. It was evaluated, in a first study, the potential of different strains of Aspergillus for the production of proteases. The strains studied were Aspergillus niger (CNPAT 001, IOC 207, IOC 4222, IOC 4220 and IOC 3883) and Aspergillus oryzae IV. The best strain obtained for the production of protease was a strain of Aspergillus oryzae IV. Later on, it was studied the influence of the different quantities of water and coke canola added; the best production was obtained in proportion with 40 mL of water to 100 g of cake. After determination to medium of moisture, was evaluated the temperature of incubation, the best results were obtained in media incubated at 20° C. Further, studies were done to determine the best concentration of inoculum, the concentrations tested were 1 × 104, 1 × 105, 1 × 106 and 1 × 107 spores per gram of medium, being 1 × 107 to better concentration analyzed. The influence of supplementation of fermentation medium with sources of phosphorus, carbon and nitrogen was evalueted. Supplementation of the cake of canola, with 1% (w/w) monobasic sodium phosphate did not influence positively the production of proteases. Supplementation of medium with different sources of nitrogen were studied in the proportion of 1.0% (w/w) in the weight of the substrate, being the yeast extract of the best source evaluated, showing increased production of proteases. Then, it was tested the incubation period of the medium without supplementation of nutrients, between 0 and 240 hours of fermentation with samples being removed every 24 h of fermentation process. The highest production occurred at 96 h with production of 336 U.g-1 protease. After this stage, was evaluated the supplementation of medium with different carbon sources at different concentrations, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10.0, 12, 5 15.0 % (w/w) in the weight of the substrate; the increased production of proteases occurred in the medium with the addition of glucose at a concentration of 7.5 % obtaining 452 Ug-1. In the last stage, was performed the characterization of the medium during the fermentation process by of a chemical and physicochemical analytical determinations. The increased production of proteases obtained was 452 U.g-1 in 96 hours of fermentation process, in media supplemented with 7.5 % glucose, at the fermentation conditions: temperature of incubation of the medium at 20° C, damp cake with 40 mL water per 100 g of substrate and concentration of inoculum of 1 × 107 spores.g-1. / As proteases fazem parte da classe de enzimas com a capacidade de hidrolisar ligações peptídicas em proteínas e fragmentos de proteínas, modificando os substratos com grande seletividade e especificidade. A fermentação semi-sólida é um processo fermentativo no qual o crescimento do microrganismo e a formação dos produtos ocorrem na superfície do substrato sólido próximo à ausência de água livre, geralmente utilizando matéria-prima natural como fonte de carbono e energia. Realizou-se neste estudo o cultivo semi-sólido de fungos filamentosos, visando à produção de proteases utilizando como substrato a torta de canola. Avaliando-se, em um primeiro estudo, o potencial de diferentes linhagens de Aspergillus para a produção de proteases. As linhagens estudadas foram Aspergillus niger (CNPAT 001, IOC 207, IOC 4222, IOC 4220 e IOC 3883) e Aspergillus oryzae IV. A melhor linhagem obtida para a produção de protease foi a linhagem de Aspergillus oryzae IV. Em seguida, estudou-se a influência da adição de diferentes volumes de água a torta, onde a melhor produção foi obtida nos meios com proporção 40 mL de água para 100 g de torta. Determinada a melhor umidade do meio, avaliou-se a temperatura de incubação, os melhores resultados obtidos foram em meios incubados a 20°C. Na sequência, foram feitos estudos para determinação da melhor concentração de inóculo, as concentrações testas foram 1×104, 1×105, 1×106 e 1×107 esporos por grama de meio, sendo 1×107 a melhor concentração analisada. Avaliou-se a influência da suplementação do meio fermentativo com fontes de fósforo, carbono e nitrogênio. A suplementação da torta de canola, com 1% (m/m) de fosfato de sódio monobásico não influenciou positivamente na produção de proteases. A suplementação do meio com diferentes fontes de nitrogênio foram estudadas na proporção de 1,0 % (m/m) em relação ao peso do substrato, sendo o extrato de levedura a melhor fonte avaliada, apresentando maior produção de proteases. Em seguida, testou-se o período de incubação do meio sem suplementação de nutrientes, entre 0 e 240 horas de fermentação com amostras sendo retiradas a cada 24 h de processo fermentativo. A maior produção ocorreu em 96 h com produção de 336 U.g-1 de proteases. Após esta etapa, avaliou-se a suplementação do meio com diferentes fontes de carbono em diferentes concentrações, 1,0; 2,0; 3,0; 4,0; 5,0; 7,5; 10,0; 12,5 15,0 % (m/m) em relação ao peso do substrato, a maior produção de proteases ocorreu no meio com adição de glicose na concentração de 7,5 % obtendo 452 U.g-1. Na última etapa, fez-se a caracterização do meio ao longo do processo fermentativo, através de determinações analíticas de ordem químicas e físico-químicas. A maior produção de proteases obtida foi de 452 U.g-1 em 96 horas de processo fermentativo, nos meios suplementados com 7,5 % de glicose, nas seguintes condições fermentativas: temperatura de incubação do meio a 20°C, torta umedecida com 40 mL de água por 100 g de substrato e concentração de inóculo de 1×107 esporos.g-1.