Página 1 dos resultados de 20436 itens digitais encontrados em 0.018 segundos

Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant; Mutagênese em Petunia x hybrida Vilm. e isolamento de um novo mutante morfológico

BERENSCHOT, Amanda S.; ZUCCHI, Maria I.; TULMANN-NETO, Augusto; QUECINI, Vera
Fonte: Sociedade Brasileira de Fisiologia Vegetal Publicador: Sociedade Brasileira de Fisiologia Vegetal
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.34%
Traditionally, mutagenesis has been used to introduce novel genetic variability in ornamental crops. More recently, it has become a powerful tool in gene discovery and functional analyses in reverse genetics approaches. The present work aimed to compare the efficiency of physical and chemical agents in generating mutant populations of petunia. We have indirectly evaluated the genomic damage by analyzing developmental characteristics of the plantlets derived from treated seeds employing gamma radiation at 0, 20, 40, 60, 80 and 100 Gy and the alkylating agent ethyl-methanesulfonate (EMS) at 0, 0.05, 0.1, 0.15, 0.2 and 0.25% (v/v). Gamma rays and EMS caused developmental defects and decreased seedling viability in plants obtained from the mutagenized seeds. High mutagen doses reduced in approximately 44% the number of plants with primary leaves at 15 days after sowing (DAS) and decreased seedling survival rates to 55% (gamma) and 28% (EMS), in comparison to untreated controls. Seedling height decrease was proportional to increasing EMS dosage, whereas 40 and 60 Gy of gamma irradiation caused the most significant reduction in height. Moderate DNA damage allowing a high saturation of mutant alleles in the genome and the generation of viable plants for reverse genetics studies was correlated to the biological parameter LD50...

Mutagênese e tecnologia in vitro no melhoramento genético da pimenta-do-reino (Piper nigrum L.).; Mutagenesis and in vitro technology in the genetic improvement of black pepper (Piper nigrum L.).

Lemos, Oriel Filgueira de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 28/02/2003 Português
Relevância na Pesquisa
36.34%
O presente trabalho teve por objetivo desenvolver tecnologia in vitro e associá-la à mutagênese, e avaliar plantas V 5 e V6 quanto aos caracteres agronômicos de produção em área de ocorrência da doença fusariose, visando ao melhoramento genético da pimenta-do-reino para obtenção de plantas tolerantes e/ou resistentes à doença fusariose. A aplicação das técnicas in vitro iniciou-se através da obtenção de plantas doadoras de explantes, a partir de estacas em casa-de-vegetação e, de sementes e embriões zigóticos in vitro. O processo de micropropagação foi desenvolvido a partir de gemas de plantas obtidas in vitro através do estabelecimento de condições adequadas de cultivo em meios de cultura apropriados para multiplicação de gemas, enraizamento e obtenção de "plantlets", e de tipo de substrato para aclimatação e formação de mudas. Após a definição deste processo, gemas de plantas de casa-de-vegetação foram submetidas a diferentes tratamentos de assepsia e as sobreviventes micropropagadas. Seleção in vitro foi estabelecida ao cultivar isolados patogênicos do fungo Fusarium solani f. sp. piperis em meio Czapek-Dox e, através da curva de crescimento foi estabelecido o período de 28 dias de cultivo mais adequado para obtenção de filtrado da cultura do fungo. Diferentes concentrações de filtrado e formas de esterilização foram testadas em meio de cultura de multiplicação de gemas e determinou-se a concentração de 55% do filtrado do fungo (v/v) sob a esterilização por duas autoclavagens...

Estudo dos efeitos mutagênicos da poluição ambiental em trabalhadores de rua em São Paulo; Air pollution significantly influences mutagenesis in the oral mucosa: a study in inhabitants of Sao Paulo, Brazil

Negri, Ariadini
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 23/10/2009 Português
Relevância na Pesquisa
36.44%
A poluição atmosférica vem recebendo crescente atenção como problema de saúde pública, pois representa uma fonte de agentes que podem promover o stress oxidativo e danos ao DNA, levando a efeitos cancerígenos e mutagênicos. As vias aéreas superiores, incluindo a cavidade oral são as áreas mais expostas do sistema respiratório, a material particulado e gases, e podem ser facilmente acessadas para monitorar a exposição humana a estes agentes. Nosso objetivo foi analisar o impacto da poluição do ar sobre a incidência de mutagénese em habitantes de nossa cidade. Para o estudo da mutagênese, utilizamos o teste de micronucleous (Mn), em células do epitélio da mucosa oral. Uma média de 4 a 6 amostras foram coletadas de cada voluntário, que concordou em participar do estudo, em São Paulo (SP) (n = 39) e, em Peruibe (PER), uma pequena cidade à beira-mar (n = 24), utilizados como um grupo controle. Os níveis de material particulado (PM10) foram medidos pelo método gravimétrico, e o ozônio e NO2 foram medidos pelos amostradores passivos, bem como pelas medições realizadas pela CETESB no mesmo local da coleta. Os resultados expostos em relação à concentração de MN / células, mostraram uma diferença estatisticamente significativa comparando SP (0042 ± 0032) e Per (0023 ± 0019)...

Estudo da atividade inibitória da troponina I através de mutações sítio-dirigidas; Study of the inhibitory activity of troponin I by site-directed mutagenesis

Quaggio, Ronaldo Bento
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 06/10/1994 Português
Relevância na Pesquisa
36.34%
A troponina I (TnI) é a sub-unidade inibitória do complexo troponina, responsável pela regulação da contração do músculo esquelético. Foi demonstrado que sua ação inibitória sobre a Mg2+ATPase da actomiosina, deve-se principalmente à região entre os resíduos 96 e 116 (região do peptídeo inibitório). Para estudar o mecanismo de inibição a nível molecular, produzimos três mutantes na região do peptídeo inibitório através de mutações sítio-dirigidas. Substituímos os resíduos lisina 105 por ácido glutâmico (K105E), fenilalanina 106 por tirosina (F106Y) e arginina 113 por ácido glutâmico (R113E). As troponinas I mutantes foram expressas em E.coli, purificadas e ensaiadas em sua atividade inibitória, interações com os outros componentes do complexo regulatório e sua capacidade regulatória. Os resultados obtidos indicam que a mutação na posição 105 alterou a interação da proteína com a tropomiosina, diminuindo sua atividade inibitória e afinidade pela actina-tropomiosina. A substituição na posição 113 alterou a interação da proteína com a actina e com a actina-tropomiosina, também diminuindo a atividade inibitória na presença de tropomiosina e inviabilizando a inibição na ausência de tropomiosina. Já a substituição na posição 106 não produziu alteração detectável. Concluímos que o resíduo 105 faz parte do sítio de ligação da troponina I ao complexo actina-tropomiosina e que o resíduo 113 participa diretamente do mecanismo de inibição. Desta forma...

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

Portaro, Fernanda C. V.; Hayashi, Mirian A. F.; De Arauz, Luciana J.; Palma, Mario Sergio; Assakura, Marina T.; Silva, Célio L.; De Camargo, Antonio C. M.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 7400-7406
Português
Relevância na Pesquisa
36.51%
The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey

Viviani, Vadim R.; Arnoldi, Frederico G.C.; Ogawa, Florisbela T.; Brochetto-Braga, M.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 362-369
Português
Relevância na Pesquisa
36.44%
Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather...

Lentinula edodes (Shiitake) modulates chemically induced mutagenesis by enhancing pitting

Alves de Lima, Patrícia L.; Sugui, Marina M.; Petrício, Angela I.M.; Vilela, Lízia C.; Pinto, Andréa V.F.; Martins, Priscila R.; Kaneno, Ramon; Ribeiro, Daniel A.; Salvadori, Daisy Maria Favero; Ribeiro, Lúcia R.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 733-739
Português
Relevância na Pesquisa
36.34%
This study was undertaken to understand how Lentinula edodes modulates in vivo mutagenesis induced by alkylating agents in bone marrow and peripheral blood as described in our previous article. Male Swiss mice were pretreated for 15 consecutive days with aqueous extracts prepared from L. edodes, after which, the number of circulating blood cells, normal erythroid bone marrow cell cycling, and phagocytosis of micronucleated reticulocyte (MNRET) and activation of spleen macrophages were assessed. The results indicate that the antimutagenicity seen in bone marrow and peripheral blood is exerted by distinct compounds with different actions. The antimutagenic effect in bone marrow is exerted by compounds subject to degradation at deep-freeze storage temperature of -20 C. On the other hand, compounds responsible for antimutagenicity in peripheral blood are not subject to degradation at -20 C. The results also indicate that the antimutagenic action in peripheral blood leading to the reduction of circulating MNRET occurs in the spleen primarily through a phagocytic activity due to higher macrophage numbers and probably not due to the enhanced activation state of individual cells. © Mary Ann Liebert, Inc.

Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Quinta; Wiebe, Marilyn G.; Penttilä, Merja; Domingues, Lucília
Fonte: Landes Bioscience; Landes Bioscience Publicador: Landes Bioscience; Landes Bioscience
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
Relevância na Pesquisa
36.34%
To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth...

Separation of levan-formation and sucrose-hydrolysis catalyzed by levansucrase of Zymomonas mobilis using in vitro mutagenesis

Sangiliyandi,G.; Kannan,T.R.; Raj,K. Chandra; Gunasekaran,P.
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1999 Português
Relevância na Pesquisa
36.34%
A levansucrase (SacB) of Zymomonas mobilis capable of sucrose hydrolysis but not levan formation was isolated through invitro mutagenesis of cloned sacB gene. When the sacB mutant gene was expressed in Escherichiacoli strains, only 50% of the sucrose-hydrolysing activity (2.0 U/mg) was produced, compared to the wild type levansucrase (4.0 U/mg). Sequencing of the sacB mutant gene revealed changes of two amino acid residues (Phe-102 to Leu and Trp-261 to Lys in the levansucrase). The absence of mutation at the site of Cys of SacB is contradictory to the inhibition kinetics that demonstrated the involvement of Cys in conferring the levan-forming activity to the SacB. The present finding is useful in understanding the mechanism of selective modulation of levan-forming (polymerase) activity of levansucrase.

Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant

Berenschot,Amanda S.; Zucchi,Maria I.; Tulmann-Neto,Augusto; Quecini,Vera
Fonte: Brazilian Journal of Plant Physiology Publicador: Brazilian Journal of Plant Physiology
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2008 Português
Relevância na Pesquisa
36.34%
Traditionally, mutagenesis has been used to introduce novel genetic variability in ornamental crops. More recently, it has become a powerful tool in gene discovery and functional analyses in reverse genetics approaches. The present work aimed to compare the efficiency of physical and chemical agents in generating mutant populations of petunia. We have indirectly evaluated the genomic damage by analyzing developmental characteristics of the plantlets derived from treated seeds employing gamma radiation at 0, 20, 40, 60, 80 and 100 Gy and the alkylating agent ethyl-methanesulfonate (EMS) at 0, 0.05, 0.1, 0.15, 0.2 and 0.25% (v/v). Gamma rays and EMS caused developmental defects and decreased seedling viability in plants obtained from the mutagenized seeds. High mutagen doses reduced in approximately 44% the number of plants with primary leaves at 15 days after sowing (DAS) and decreased seedling survival rates to 55% (gamma) and 28% (EMS), in comparison to untreated controls. Seedling height decrease was proportional to increasing EMS dosage, whereas 40 and 60 Gy of gamma irradiation caused the most significant reduction in height. Moderate DNA damage allowing a high saturation of mutant alleles in the genome and the generation of viable plants for reverse genetics studies was correlated to the biological parameter LD50...

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Martinez, Sarah A.; Khayter, Cyd; Moore, Finola Elizabeth; Reyon, Deepak; Sander, Jeffry D.; Blackburn, Jessica S.; Ramirez, Cherie Lynn; Joung, Jae Keith; Langenau, David M.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.34%
Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together...

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Jiang, Lin
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
36.44%
Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide...

Lymphomagenesis in SCID-X1 Mice Following Lentivirus-mediated Phenotype Correction Independent of Insertional Mutagenesis and gamma c Overexpression

Ginn, S.; Liao, S.; Dane, A.; Hu, M.; Hyman, J.; Finnie, J.; Zheng, M.; Cavazzana-Calvo, M.; Alexander, S.; Trasher, A.; Alexander, I.
Fonte: Academic Press Inc Elsevier Science Publicador: Academic Press Inc Elsevier Science
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
Relevância na Pesquisa
36.44%
The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in γc-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing γc from the phosphoglycerate kinase (PGK) and Wiscott–Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-α (EF1α) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or γc overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety...

Mutagenesis as a Diversity Enhancer and Preserver in Evolution Strategies

Guerrero Madrid, José Luis; Gómez-Jordana, Alfonso; Berlanga, Antonio; Molina, José M.
Fonte: Springer Publicador: Springer
Tipo: info:eu-repo/semantics/acceptedVersion; info:eu-repo/semantics/conferenceObject; info:eu-repo/semantics/bookPart
Publicado em //2012 Português
Relevância na Pesquisa
36.44%
Mutagenesis is a process which forces the coverage of certain zones of the search space during the generations of an evolution strategy, by keeping track of the covered ranges for the different variables in the so called gene matrix. Originally introduced as an artifact to control the automated stopping criterion in a memetic algorithm, ESLAT, it also improved the exploration capabilities of the algorithm, even though this was considered a secondary matter and not properly analyzed or tested. This work focuses on this diversity enhancement, redefining mutagenesis to increase this characteristic, measuring this improvement over a set of twenty-seven unconstrained optimization functions to provide statistically significant results.; This work was supported in part by Projects CICYT TIN2008-06742-C02- 02/TSI, CICYT TEC2008-06732-C02-02/TEC, CAM CONTEXTS (S2009/TIC-1485) and DPS2008-07029-C02-02.; Proceedings of: 9th International Symposium on Distributed Computing and Artificial Intelligence (DCAI 2012). Salamanca, March 28-30, 2012

Melhoramento genético da levedura oleaginosa Lipomyces starkeyi por mutagênese aleatória, visando a produção de biocombustíveis de segunda geração; Genetic improvement of oleaginous yeast Lipomyces starkeyi through random mutagenesis, order to produce of second generation of biofuels

Eulalia Vargas Tapia
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 31/07/2012 Português
Relevância na Pesquisa
36.51%
Neste trabalho foram desenvolvidos estudos visando o melhoramento genético da linhagem de levedura Lipomyces starkeyi DSM 70296, visando sua utilização na biossíntese de precursores de biocombustíveis a partir de fontes renováveis. Inicialmente foram verificados os parâmetros de mutagênese. O melhoramento genético foi conduzido por mutagênese aleatória de DNA por irradiação ultravioleta. O tempo de exposição foi ajustado de forma a assegurar uma taxa de sobrevivência celular não superior a 5%, para obter indivíduos contendo elevado acúmulo de mutações no DNA. Os mutantes foram selecionados com o uso da cerulenina agente interferente ao metabolismo de interesse, de forma que fossem identificados os mutantes cujas alterações genéticas pudessem estar promovendo efeitos sobre este metabolismo. Os mutantes que demonstraram crescimento normal em meio de cultura suplementado com cerulenina foram considerados bons candidatos para estudos aprofundados. Nesta etapa foram selecionados 90 mutantes, dos quais foram selecionados os oito melhores candidatos para estudo através de fermentação em frascos agitados. A avaliação de desempenho fermentativo foi conduzida a partir da avaliação dos índices de crescimento e produtividade de lipídeo utilizando meio de cultura contendo xilose como única fonte de carbono. A fermentação da cepa padrão foi conduzida nas mesmas condições para permitir uma análise comparativa. Os resultados obtidos mostraram que um dos mutantes (identificado como A1) apresentou aumento significativo nos índices de produtividade de biomassa e lipídeo em relação à cepa padrão (teste de Tukey com 95% de significância). Este mutante foi então selecionado para estudo aprofundado através da fermentação em biorreator utilizando a mesma composição de açúcares observada em bagaço de cana-de-açúcar (30% glicose: 70% xilose)...

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

Aftab,Muhammad Nauman; Haq,Ikram-ul; Baig,Shahjahan
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2012 Português
Relevância na Pesquisa
36.44%
The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables...

A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

Monson, Rita; Smith, Deborah S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P.
Fonte: Frontiers Publicador: Frontiers
Tipo: Article; published version
Português
Relevância na Pesquisa
36.56%
This is the final version of the article. It was first available from Frontiers via http://dx.doi.org/10.3389/fmicb.2015.01442; Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a ?-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal...

Studies of Spontaneous Oxidative and Frameshift Mutagenesis in Saccharomyces cerevisiae

Mudrak, Sarah Victoria
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 6087624 bytes; application/pdf
Publicado em //2010 Português
Relevância na Pesquisa
36.67%

Preserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast Saccharomyces cerevisiae. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work...

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

Portaro, Fernanda C. V.; Hayashi, Mirian A. F.; De Arauz, Luciana J.; Palma, Mario Sergio; Assakura, Marina T.; Silva, Célio L.; De Camargo, Antonio C. M.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 7400-7406
Português
Relevância na Pesquisa
36.51%
The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

La lipofección incrementa la eficiencia de mutagénesis dirigida en células troncoembrionarias de ratón E14 TG2a

López-Heydeck,Sandra M.; Cajero-Juárez,Marcos; Alonso-Morales,Rogelio A.; Martínez-Castañeda,José S.; Robles-González,José F.; Barbabosa-Pliego,Alberto; Vázquez-Chagoyán,Juan C.
Fonte: Facultad de Medicina Veterinaria y Zootecnia, UNAM Publicador: Facultad de Medicina Veterinaria y Zootecnia, UNAM
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2009 Português
Relevância na Pesquisa
36.6%
Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo...