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1
Martin, Stephen Lewis. "Novel methods for mutagenesis." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302789.
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Ennis, Don Gregory. "Genetics of SOS mutagenesis." Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184602.
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Previous genetic evidence suggested that RecA was required in SOS mutagenesis for its regulatory role and perhaps some other nonregulatory role (Mount, 1977; Blanco et al., 1982). I undertook a genetic study which confirmed the above studies and provided further evidence that RecA protein appeared to have a dual "role in mutagenesis; first, the cleavage of LexA repressor for the derepression of specific SOS genes and second, one or more additional role(s). For these studies a new phage mutagenesis assay was developed which allows rapid scoring of SOS mutagenesis in a large number of host mutants. I next conducted a genetic analysis to determine if the newly defined RecA mutagenesis function was separable by mutation from the numerous other phenotypes which are known to be influenced by RecA protein. From the study of recA mutants it appears that the RecA mutagenesis function(s) is genetically separable from the following RecA phenotypes: LexA cleavage, lambda cI repressor cleavage, UV resistance and homologous recombination. In addition, I discovered that the LexA cleavage function and lambda cI cleavage function is also separable. I also studied in some detail the novel genetic properties that I uncovered for recA432 mutant strains. recA432 was defined as a mutagenesis defective allele (Kato and Shinoura, 1977). LexA cleavage in recA432 cells was more easily induced that in recA⁺ cells, causing lethal filamentation of these mutant cells even at very low UV doses. I concluded that the basis for the Mut⁻ phenotype was this strain's propensity to lethally filament, which complicated the detection of mutant cells. In another set of experiments, I examined the regulatory requirements for SOS mutagenesis and Weigle phage-reactivation; I wanted to determine which SOS operons must be derepressed for this process. lexA(Ind⁻) mutant cells are defective in mutagenesis because they cannot derepress specific SOS genes required in this process. I found that the selective derepression of umuDC was sufficient to restore mutagenesis to these lexA(Ind⁻) mutants; however, derepression of umuDC and recA was required for phage reactivation.
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Porto, Marília de Paula [UNESP]. "Ação moduladora do citral e eugenol em eventos genéticos em magrófagos murinos in vitro." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/92460.
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porto_mp_me_botib.pdf: 2132236 bytes, checksum: b1dd297e7ebc3769eafbc4dbcaa5705a (MD5)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Devido a propriedades terapêuticas, várias plantas e seus constituintes químicos vêm sendo muitas vezes utilizados como o primeiro recurso para o tratamento de diversas doenças. Nesse contexto, compostos isolados de plantas têm sido alvos de inúmeros estudos que avaliam, além da atividade, seus possíveis mecanismos de ação. Dentre os compostos com potencial quimioprotetor, o citral e o eugenol merecem atenção devido suas estruturas químicas de monoterpeno e de composto fenólico, respectivamente, e por seus potenciais anti-inflamatório, antiparasitário e antioxidante. Considerando que mutação no DNA pode ser a primeira etapa de várias doenças, e que lesões induzidas nessa molécula podem ser prevenidas ou moduladas por compostos naturais, este estudo objetivou avaliar, pelo teste do cometa, o potencial genotóxico do citral (25, 50 e 100 Tg/mL) e do eugenol (0,31, 0,62, 1,24 e 2,48 Tg/mL), após diferentes tempos de tratamento (6, 10, 24 e 30 h) e, também, seus possíveis efeitos moduladores sobre danos induzidos no DNA pela doxorrubicina (DOX), em diferentes protocolos de tratamento (pré, pós e simultaneamente à DOX) e momentos de análise (12, 24 e 36 h), em macrófagos peritoneais de camundongos. Além disso, foi avaliado o potencial toxicogenômico do citral e do eugenol por meio da modulação da expressão dos genes NF-kB1, COX-2 e TNF-α (relacionados a processos inflamatórios) em macrófagos ativados ou não por lipopolissacarideo de bactéria (LPS). Os resultados mostraram que ambos os compostos apresentaram potencial genotóxico. No caso do citral, a genotoxicidade foi observada para as duas maiores concentrações, mas apenas no tempo de 6h; para o eugenol, o aumento de danos no DNA foi detectado para todas as concentrações, em pelo menos um momento de análise. Com relação ao potencial...
Because of the therapeutic properties, several plants and their chemical constituents have been used for treatment of various diseases. Therefore, isolated compounds from plants have been targets of numerous studies that evaluate their activity and mechanisms of action. Among compounds with chemopreventive potential, citral and eugenol have gain attention because of their chemical structures, monoterpene and phenol,respectively, and for their anti-inflammatory, antioxidant and antiparasitic potentials. Since DNA mutation is the first step for some diseases, and since the lesions induced in this molecule can be prevented or modulated by natural compounds, aim of the present study was first to evaluate the genotoxic potential of citral (25, 50 and 100 Tg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 Tg/mL) at different times after exposure (6, 10, 24 and 30 h), and then, their ability to modulate DNA damage induced by doxorubicin (DOX) at different treatment protocols (pre, post and simultaneous with DOX) and times (12, 24 and 36 h) in mice peritoneal macrophages. In addition, the toxicogenomic potential of citral and eugenol by modulating the expression of NF-KB1, COX-2 and TNF-α (related to inflammatory processes) genes in macrophages activated or not by bacterial lipopolysaccharide (LPS) was also investigated. The results showed that both compounds have genotoxic potential. In the case of citral, genotoxicity was observed for the two major concentrations, but only 6h after the exposure. For eugenol, increased DNA damage was detected for all concentrations, in at least one moment of analysis. Related to the antigenotoxicity, both citral and eugenol presented protective effects at different concentrations and treatment protocols, and the more effective activities were detected at simultaneous and pre-treatment... (Complete abstract click electronic access below)
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Porto, Marília de Paula. "Ação moduladora do citral e eugenol em eventos genéticos em magrófagos murinos in vitro /." Botucatu : [s.n.], 2012. http://hdl.handle.net/11449/92460.
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Orientador: Daisy Maria Fávero SalvadoriCoorientador: Glenda Nicioli da Silva
Banca: Luís Fernando Barbisan
Banca: Denise Crispim Tavares
Resumo: Devido a propriedades terapêuticas, várias plantas e seus constituintes químicos vêm sendo muitas vezes utilizados como o primeiro recurso para o tratamento de diversas doenças. Nesse contexto, compostos isolados de plantas têm sido alvos de inúmeros estudos que avaliam, além da atividade, seus possíveis mecanismos de ação. Dentre os compostos com potencial quimioprotetor, o citral e o eugenol merecem atenção devido suas estruturas químicas de monoterpeno e de composto fenólico, respectivamente, e por seus potenciais anti-inflamatório, antiparasitário e antioxidante. Considerando que mutação no DNA pode ser a primeira etapa de várias doenças, e que lesões induzidas nessa molécula podem ser prevenidas ou moduladas por compostos naturais, este estudo objetivou avaliar, pelo teste do cometa, o potencial genotóxico do citral (25, 50 e 100 Tg/mL) e do eugenol (0,31, 0,62, 1,24 e 2,48 Tg/mL), após diferentes tempos de tratamento (6, 10, 24 e 30 h) e, também, seus possíveis efeitos moduladores sobre danos induzidos no DNA pela doxorrubicina (DOX), em diferentes protocolos de tratamento (pré, pós e simultaneamente à DOX) e momentos de análise (12, 24 e 36 h), em macrófagos peritoneais de camundongos. Além disso, foi avaliado o potencial toxicogenômico do citral e do eugenol por meio da modulação da expressão dos genes NF-kB1, COX-2 e TNF-α (relacionados a processos inflamatórios) em macrófagos ativados ou não por lipopolissacarideo de bactéria (LPS). Os resultados mostraram que ambos os compostos apresentaram potencial genotóxico. No caso do citral, a genotoxicidade foi observada para as duas maiores concentrações, mas apenas no tempo de 6h; para o eugenol, o aumento de danos no DNA foi detectado para todas as concentrações, em pelo menos um momento de análise. Com relação ao potencial... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Because of the therapeutic properties, several plants and their chemical constituents have been used for treatment of various diseases. Therefore, isolated compounds from plants have been targets of numerous studies that evaluate their activity and mechanisms of action. Among compounds with chemopreventive potential, citral and eugenol have gain attention because of their chemical structures, monoterpene and phenol,respectively, and for their anti-inflammatory, antioxidant and antiparasitic potentials. Since DNA mutation is the first step for some diseases, and since the lesions induced in this molecule can be prevented or modulated by natural compounds, aim of the present study was first to evaluate the genotoxic potential of citral (25, 50 and 100 Tg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 Tg/mL) at different times after exposure (6, 10, 24 and 30 h), and then, their ability to modulate DNA damage induced by doxorubicin (DOX) at different treatment protocols (pre, post and simultaneous with DOX) and times (12, 24 and 36 h) in mice peritoneal macrophages. In addition, the toxicogenomic potential of citral and eugenol by modulating the expression of NF-KB1, COX-2 and TNF-α (related to inflammatory processes) genes in macrophages activated or not by bacterial lipopolysaccharide (LPS) was also investigated. The results showed that both compounds have genotoxic potential. In the case of citral, genotoxicity was observed for the two major concentrations, but only 6h after the exposure. For eugenol, increased DNA damage was detected for all concentrations, in at least one moment of analysis. Related to the antigenotoxicity, both citral and eugenol presented protective effects at different concentrations and treatment protocols, and the more effective activities were detected at simultaneous and pre-treatment... (Complete abstract click electronic access below)
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Silva, Ana Carolina Buzinari da [UNESP]. "Análise de uma biblioteca de mutantes de Xanthomonas citri subsp. citri quanto à patogenicidade." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/121954.
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000817146.pdf: 479870 bytes, checksum: d3427b349dc719a504332ddf82064866 (MD5)O estudo da interação planta-patógeno é de grande importância para o entendimento do cancro cítrico, justificando assim a busca por genes que estejam ligados a patogenicidade e virulência em Xanthomonas citri subsp. citri (Xac), agente causal dessa doença. Neste estudo, foi realizada mutagênese aleatória por inserção do transposon EZ-Tn5 in vitro no genoma da Xac. Obteve-se 8000 mutantes onde 292 foram conduzidos em ensaio experimental in planta. Cinco mutantes expressaram sintomatologia alterada, dois com ausência total de sintomas e três com leve hiperplasia. A análise da sequência dos genes onde se inseriu o transposon indicam mutações nos genes purF, yapH, oar, um gene que codifica uma proteína hipotética (XAC 0196), e na região entre os genes pobB (XAC0362) e glpR (XAC0361). As análises de curvas de crescimento bacteriano in planta demonstraram que, exceto o gene purF, todos os demais podem ser genes envolvidos na patogenicidade de Xac. Dois destes, yapH e oar são descritos como relacionados à adesividade bacteriana, evidenciando que a interferência nesse processo exerce influência direta no sucesso da infecção de Xac. Destaca-se também a importância da identificação de uma proteína hipotética, já que essa apresentou sintomatologia atenuada quando ocorreu a inserção do transposon
The study of plant-pathogen interaction is very important to citrus canker understanding, justifying the search for virulence and pathogenicity related genes in Xanthomonas citri subsp. citri (Xac), causal agent of this disease. In this study, a random mutagenesis by Tn5 transposon insertion into Xac’s genome was performed. Eight thousand mutants were produced and 292 mutants were tested in planta. From those, five mutants expressed altered symptomatology, two showed complete absence of symptoms and three reduced hyperplasia. Gene sequences analysis where transposon was inserted, indicated mutations in purF, yapH and oar genes, in a region that codes for a hypothetical protein (XAC0196), and in a region between pobB (XAC0362) and glpR (XAC0361) genes. Analysis of bacteria growth curve in planta showed that, except for purF gene, all the others genes may be involved in Xac pathogenicity. Two of these genes, yapH and oar, are described as bacterial adhesion related genes, highlighting that interference in this process has direct influence in the Xac infection success. The importance of hypothetical protein identification is emphasized, since it presented attenuated symptomatology when mutated
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Terrazas, Peterson Menezes. "Estudo do potencial genotóxico da Gutiferona A em diferentes células de camundongos in vitro /." Botucatu, 2013. http://hdl.handle.net/11449/108542.
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Orientador: Edson Luis MaistroBanca: Maria Aparecida Marin Morales
Banca: Cláudia Aparecida Rainho
Resumo: Garcinia achachairu (GAC) é uma planta de origem boliviana que vem sendo utilizada na medicina popular para o tratamento de distúrbios gástricos, reumatismo, inflamações e como cicatrizante. A caracterização fitoquímica do extrato desta planta revelou que, uma benzofenona, a Gutiferona A (GA), é um dos seus compostos majoritários, que segundo estudos recentes, apresenta importante atividade antioxidante e antimicrobiana. Considerando o interesse em se aprofundar as análises do potencial farmacológico da GA e a inexistência de estudos que avaliem a sua toxicidade genética, o presente estudo foi elaborado visando investigar o potencial genotóxico e mutagênico da GA em diferentes células de camundongos in vivo, utilizando alguns dos testes tradicionais na área de mutagênese, como o Ensaio Cometa (EC) para a verificação da genotoxicidade e o Teste do Micronúcleo (TM) para a verificação da mutagenicidade. O experimento foi conduzido com camundongos Suíços albinos machos (Mus musculus) de 12 semanas, divididos em cinco grupos, constituídos cada um por seis animais. O grupo controle negativo recebeu, via gavagem, 0,3 mL de DMSO 1%. O grupo controle positivo, recebeu intraperitonealmente, 80 mg/kg de doxorrubicina. Os grupos tratados receberam, via gavagem, 0,3 mL da GA nas doses de 15, 30 e 60 mg/kg. Para a avaliação da genotoxicidade foi coletado sangue da veia caudal dos camundongos (4 e 24 horas após o tratamento), células do fígado, medula óssea, cérebro e testículos (coletadas 24 horas após o tratamento). Para a avaliação da mutagenicidade, foram coletadas células da medula óssea 24 horas após o tratamento. A citotoxicidade foi avaliada pela contagem de 200 eritrócitos policromáticos (PCE) e normocromáticos (NCE) e determinação de sua razão (PCE/NCE). Na amostra de sangue de 4h, analisadas pelo EC, os resultados obtidos mostraram que nas doses de 30 mg/kg e 60 mg/Kg. A análise ...
Abstract: Garcinia achachairu (GAC) is a native plant from Bolivia that has been used in folk medicine for the treatment of gastric disorders, rheumatism, inflammation and as a healing. The phytochemical characterization of this plant extract revealed that the benzophenone guttiferone A (GA) is one of its major compounds, which according to recent studies, has important antioxidant and antimicrobial activity. Considering the interest in deepening the analysis of the pharmacological potential of GA and the lack of studies assessing its genetic toxicity, the present study was designed in order to investigate the genotoxic and mutagenic effects of GA in different cells of mice in vivo, using some of the traditional tests in the mutagenesis area, the Comet Assay (CA) for genotoxicity evaluation and the Micronucleus Test (MT) for the mutagenicity assessment. The experiment was conducted in Swiss albino male mice (Mus musculus) with 12 weeks, divided into five groups with six animals each. The negative control group received, by oral gavage, 0.3 mL of 1% DMSO. The positive control group received, intraperitoneally, 80 mg/Kg of doxorubicin. The treated groups received 0.3 ml of GA at 15, 30 and 60 mg/kg, by gavage. For the genotoxicity evaluation, blood was collected from the tail vein of the mice (4 and 24 hours after treatment), and liver, bone marrow, brain and testicular cells were collected 24 hours after treatment. For the mutagenicity assessment, bone marrow cells were collected 24 hours after treatment. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes and their ratio (PCE/NCE) determined. For the 4 h blood sample, the results with GA at doses of 30 and 60 mg/kg showed that was a statistically significant increase in DNA damage in comparison to the negative control. For the 24 h blood sample, only 60 mg/kg dose showed significant genotoxicity. The analysis of ther ...
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Silva, Ana Carolina Buzinari da. "Análise de uma biblioteca de mutantes de Xanthomonas citri subsp. citri quanto à patogenicidade /." Jaboticabal, 2014. http://hdl.handle.net/11449/121954.
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Orientador: Maria Inês Tiraboschi FerroCoorientador: Jesus Aparecido Ferro
Banca: Flavia Maria de Souza Carvalho
Banca: Fabrício José Jaciani
Resumo: O estudo da interação planta-patógeno é de grande importância para o entendimento do cancro cítrico, justificando assim a busca por genes que estejam ligados a patogenicidade e virulência em Xanthomonas citri subsp. citri (Xac), agente causal dessa doença. Neste estudo, foi realizada mutagênese aleatória por inserção do transposon EZ-Tn5 in vitro no genoma da Xac. Obteve-se 8000 mutantes onde 292 foram conduzidos em ensaio experimental in planta. Cinco mutantes expressaram sintomatologia alterada, dois com ausência total de sintomas e três com leve hiperplasia. A análise da sequência dos genes onde se inseriu o transposon indicam mutações nos genes purF, yapH, oar, um gene que codifica uma proteína hipotética (XAC 0196), e na região entre os genes pobB (XAC0362) e glpR (XAC0361). As análises de curvas de crescimento bacteriano in planta demonstraram que, exceto o gene purF, todos os demais podem ser genes envolvidos na patogenicidade de Xac. Dois destes, yapH e oar são descritos como relacionados à adesividade bacteriana, evidenciando que a interferência nesse processo exerce influência direta no sucesso da infecção de Xac. Destaca-se também a importância da identificação de uma proteína hipotética, já que essa apresentou sintomatologia atenuada quando ocorreu a inserção do transposon
Abstract: The study of plant-pathogen interaction is very important to citrus canker understanding, justifying the search for virulence and pathogenicity related genes in Xanthomonas citri subsp. citri (Xac), causal agent of this disease. In this study, a random mutagenesis by Tn5 transposon insertion into Xac's genome was performed. Eight thousand mutants were produced and 292 mutants were tested in planta. From those, five mutants expressed altered symptomatology, two showed complete absence of symptoms and three reduced hyperplasia. Gene sequences analysis where transposon was inserted, indicated mutations in purF, yapH and oar genes, in a region that codes for a hypothetical protein (XAC0196), and in a region between pobB (XAC0362) and glpR (XAC0361) genes. Analysis of bacteria growth curve in planta showed that, except for purF gene, all the others genes may be involved in Xac pathogenicity. Two of these genes, yapH and oar, are described as bacterial adhesion related genes, highlighting that interference in this process has direct influence in the Xac infection success. The importance of hypothetical protein identification is emphasized, since it presented attenuated symptomatology when mutated
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Baeza, Centurión Pablo 1989. "Understanding alternative splicing using deep mutagenesis." Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668749.
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Alternative pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from the transcript and exons are joined together to form a mature mRNA. To study the effects of mutations on alternative splicing, we built a mutant library containing all combinations of 12 mutations that accumulated during the emergence of an alternatively-spliced human exon: FAS exon 6. This allowed us to study the effects of individual nucleotide substitutions in thousands of different closely-related genetic contexts. We show that the effect of the same mutation on exon inclusion depends non-monotonically on the inclusion levels of the exon before the mutation is made, with mutations having their strongest splice-altering effects in exons with intermediate levels of inclusion, and their smallest effects in exons that are always skipped or always included. After performing deep mutagenesis in two highly included (constitutive) exons, we find that, in agreement with our previous results, mutations have a very small effect on the inclusion of constitutive exons with the exception of mutations in exon-intron boundaries (splice sites). Since alternative splicing is frequently perturbed in human genetic diseases, we then put these results in a more practical context to address the question of the extent to which random mutations in the human genome are likely to have splice-altering effects. Since most human exons are highly included, we conclude that a random mutation is unlikely to have a splice altering effect, and that altered splicing should only be considered a likely proximal disease mechanism for mutations that affect alternatively spliced exons (included at intermediate levels) or disrupt splice sites.El empalme alternativo es un proceso de la expresión génica en eucariontes en el que los intrones del transcrito se eliminan, dejando únicamente exones para formar un RNAm maduro. Para estudiar los efectos de mutaciones en este proceso, diseñamos una librería de mutantes con las 12 mutaciones (y todas sus combinaciones) que surgieron a lo largo de la evolución enhumanos de un exón alternativo: el exón 6 de FAS. Esto nos permitió estudiar los efectos de cada mutación en miles de contextos genéticos distintos. Descubrimos que la misma mutación puede tener efectos muy diferentes en el empalme de un exón dependiendo de los niveles de inclusión del mismo. Los mayores efectos se observan en exones con niveles intermedios de inclusión, mientras que los menores efectos ocurren en exones con niveles de inclusión muy altos o muy bajos. Tras mutagenizar dos exones constitutivos, confirmamos que, con la excepción de mutaciones en los sitios de empalme, es poco probable que una mutación afecte la inclusión de dichos exones. Dado que el empalme alternativo es un proceso se encuentra alterado en muchas enfermedades genéticas humanas, pusimos nuestros resultados en un contexto más práctico al plantearnos qué probabilidad hay de que una mutación al azar sea capaz de alterar dicho proceso. Ya que la gran mayoría de exones humanos tienen altos niveles de inclusión, concluimos que es poco probable que una mutación escogida al azar sea capaz de alterar los niveles de inclusión de algún exón. De hecho, esto sólo es probable en el caso de mutaciones en los sitios de empalme o de aquellas que afecten la inclusión de un exón alternativo.
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Brown, Jeremy Stuart. "Signature tagged-mutagenesis of aspergillus fumigatus." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322287.
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Seaman, Jonathan. "Signature-tagged mutagenesis in Rhizobium leguminosarum." Thesis, University of Reading, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499374.
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Rhizobia are a diverse group of symbiotic alpha-proteobacterial diazotrophs which enter a relationship with specific leguminous plants, in which the plant supplies the bacteria with required compounds whilst the bacteria reduce atmospheric nitrogen into ammonia that the plant uses as a nitrogen source. Modification of rhizobial strains has produced mutants more effective at fixing nitrogen, which in turn results in an increase in biomass of host plants under laboratory conditions but these strains are frequently out competed by wild-type strains in field studies or lost in the intervening years of a crop rotation. This study aimed to establish a library of mutants and a system for screening these strains en masse to identify some of the genes involved in competitive rhizosphere colonization in Rhizobium leguminosam.
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Hart, Nicola Jayne. "Molecular outcomes of mutagenesis in wheat." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445180.
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Brown, R. B. "Ultraviolet light mutagenesis in Myxococcus xanthus." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373103.
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Herbert, Mark A. "Signature tagged mutagenesis of Haemophilus influenzae." Thesis, Open University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340734.
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Timms, Andrew Robert. "Spontaneous mutagenesis in stressed Escherichia coli." Thesis, Open University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244410.
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Basiran, Mohd Nazir. "DNA insertion mutagenesis in higher plants." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35436.
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Rúnarsdóttir, Saga. "Site-Directed Mutagenesis Studies of FucO." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235136.
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Al-Khatib, Haifa Yousef. "Site Directed Mutagenesis of Dienelactone Hydrolase." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc277940/.
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The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.
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Chen, Wei 1965. "Site Directed Mutagenesis Of Dienelactone Hydrolase." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc500900/.
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The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.
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Williams, Neal Kenneth. "Overexpression and mutagenesis of mammalian dihydroorotase." Thesis, The University of Sydney, 1993. https://hdl.handle.net/2123/26596.
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A central 2.1 kb region from a partial cDNA clone of the hamster CAD gene, pCAD142, has been sub-cloned and its nucleotide sequence determined. N-terminal and internal amino acid sequences obtained from the purified dihydroorotase domain isolated from elastase digests of trifunctional dihydroorotate synthetase have been aligned with the deduced amino acid sequence from the CAD cDNA. These results show conclusively that dihydroorotase is the central domain of trifunctional dihydroorotate synthetase.
A C-terminal sequence of two amino acid residues from the native dihydroorotase domain was also aligned with the cDNA sequence, and a single possible site for the two residues was located. On this basis, the dihydroorotase domain was predicted to consist of 344 amino acid residues, with a calculated molecular weight of 37 726 Da. This value for the molecular weight of the domain is considerably lower than 44 kDa observed by electrophoretic analysis of the native domain, bringing into question the reliability of the C-terminal sequencing data. The amino acid sequences of 8 dihydroorotases have been aligned. The dihydroorotase sequence has not been well conserved during evolution, and only 22 residues are invariant among the compared species. The dihydroorotase domain is separated from the aspartate transcarbamylase domain at the C-terminus of the trifunctional protein, by a large bridging sequence of 117 amino acids. The region contains 28 proline residues, and may consist of multiple 13-turns.
An Escherichia coli plasmid expression vector, pCW12, was constructed that incorporates a high copy number origin of replication, the strong hybrid tac promoter, and the leader sequence of the highly-expressed gene 10 of bacteriophage T7. The region of CAD cDNA corresponding to the dihydroorotase domain predicted by peptide sequencing was sub-cloned into pCW12, and the heterologous protein overproduced by the resulting pCW13 to -30% of total E. coli protein. This recombinant dihydroorotase domain was insoluble and had no enzymic activity, and attempts to refold the polypeptide into an active conformation were unsuccessful.
A series of larger cDNA fragments, varying in the length of their C-terminal extensions, was then sub-cloned into pCW12 for protein expression. The longest fragment included the entire aspartate transcarbamylase domain, and the shortest was only 6 codons longer than pCW13. All but the shortest of these plasmids led to the expression of dihydroorotase activity in the pyrC" E. coli strain S01263. The shortest active construct, pCW27, contained 12 additional C-terminal codons to the predicted native domain. The purified recombinant hamster dihydroorotase expressed from pCW27 is a dimer, with a calculated monomer molecular weight of 39.0 kDa, and catalytic properties similar to the isolated native domain.
The amino acid sequence of the hamster dihydroorotase domain has been compared with the zinc-binding regions of other enzymes. The most conserved region of the dihydroorotases, a nonapeptide, shows strong homology with a sequence from the carbonic anhydrases which includes 2 of the 3 histidine residues that bind the zinc at the active site of this enzyme. These His residues correspond to 2 invariant histidines of the dihydroorotases; His15 and His17 of the hamster domain. An Asp and 5 His residues have been replaced using site-directed mutagenesis. The mutated recombinant enzymes were expressed in E. coli, and tested for zinc-binding. The results support the assignment of His15 and His17 as zinc ligands. The conserved nonapeptide from hamster dihydroorotase homologous to the carbonic anhydrase zinc-binding site has been modelled from the crystal structure of human carbonic anhydrase II. It is proposed that Asp13 is hydrogen-bonded to His15, forming an "Asp-His-Zn2+ catalytic triad" which is commonly observed in zinc enzymes. It is further suggested that Arg19 may interact electrostatically with a carboxylate group of the substrate.
Site-directed mutagenesis of 9 conserved dihydroorotase residues has been used to examine their importance in substrate binding and catalysis. In all but 4 of the 13 dihydroorotase mutants constructed, enzymic activity was abolished as a result of the amino acid substitutions. The kinetic parameters of these 4 active mutant enzymes were determined, and the data found to be consistent with Lys239 forming a hydrogen bond with an uncharged group on the substrate, but not having a direct role in catalysis. The conserved amino acid Asp230, which has been implicated by these studies in affecting zinc binding, is found to be a crucial catalytic residue. The catalytic mechanisms of dihydroorotase, carbonic anhydrase and the zinc proteases are compared, and a common function for the zinc atom recognized. The results presented in this work have led to the proposal of a revised mechanism for the hydrolysis of dihydroorotate catalyzed by hamster dihydroorotase.
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Daggett, Kelly Anne. "Development and applications of codon scanning mutagenesis a novel mutagenesis method that facilitates in-frame codon mutations /." College Park, Md. : University of Maryland, 2009. http://hdl.handle.net/1903/9989.
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Thesis (Ph.D.) -- University of Maryland, College Park, 2009.Thesis research directed by: Dept. of Chemistry and Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Ozato, Kenjiro. "Mutagenesis, speciation, and genome analysis in medaka." Laboratory of Freshwater Fish Stocks Bioscience Center Nagoya University, 1994. http://hdl.handle.net/2237/13793.
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Dobrowsky, Adrian. "DNA polymerase-catalyzed expansion mutagenesis in vitro." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ40041.pdf.
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Fowler, kay. "Transposon mutagenesis of Strepromyces coelicolor A3(2)." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247105.
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Maughan, William Neil. "Pancreatic deoxyribonuclease I : a study using mutagenesis." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324794.
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Fontaine, Michael Christopher. "Allel-replacement mutagenesis of group A streptococci." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285338.
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Rogers, S. D. "DNA repair and mutagenesis in Penicillium chrysogenum." Thesis, University of Westminster, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233044.
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Scott, Henry Hepburne. "#alpha#B-crystallin expression, mutagenesis and immunoreactivity." Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284449.
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Hall, Neil. "Mutagenesis of nitrate reductase in Aspergillus nidulans." Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364155.
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Leggate, Daniel Richard. "Characterisation and mutagenesis of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621894.
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Smedley, Damian Paul. "Mutagenesis of a Bacillus thuringiensis entomocidal toxin." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627459.
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Vermulst, Marc. "Untangling mitochondrial mutagenesis and aging in mice /." Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/6321.
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Chang, Thomas Kyu-Young Barton Jacqueline K. Gray Harry B. Richards John. "Gene synthesis, expression, and mutagenesis of azurin /." Diss., Pasadena, Calif. : California Institute of Technology, 1991. http://resolver.caltech.edu/CaltechETD:etd-06142007-132218.
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SICOURI, LARA. "TUMOR SUPPRESSOR MUTAGENESIS DRIVEN BY DNA DEAMINASE." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/365729.
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Genomic instability is commonly associated with pathological disorders including cancer. The progressive accumulation of genetic abnormalities in cancer-associated genes can confer cellular autonomous proliferation, contributing to the oncogenic transformation. Although animal models have been instrumental to the understanding of the molecular mechanisms responsible for tumor progression, there are severe limitations for success. This thesis work aimed at the development of an innovative animal model that could recapitulate the ongoing lifelong accumulation of DNA lesions, leading to neoplastic transformation. To this end, the natural DNA mutating enzyme AID was fused to sequence specific DNA binding proteins. They were engineered to target tumor suppressor genes and to induce low-frequency mutagenesis in cell lines and in zebrafish. A TALE-Aid fusion protein was targeted to the p53 locus of mouse cell lines, and the Aid-dependent mutations were monitored by next-generation sequencing. The induced mutations occurred at a comparable frequency to those observed in Aid-induced non-immunoglobulin gene targets in B cells. Mutations were found mostly in the DNA binding domain of p53, possibly reflecting AID- hotspot residues in p53. Our approach also induced mutations that have not been characterized previously, and could provide further insight into p53 dependent oncogenesis. When TALE-AID were expressed and targeted to the p53 locus of zebrafish embryos, the activity induced developmental abnormalities with variable severity, leading to both an increased mortality, and impaired ovarian maturation and fertility. We have developed and initially characterized a novel tool for the in vitro / in vivo study of the accumulation of mutations in cancer-associated genes.
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Terrazas, Peterson Menezes [UNESP]. "Estudo do potencial genotóxico da Gutiferona A em diferentes células de camundongos in vitro." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/108542.
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000753046.pdf: 1011046 bytes, checksum: 0e27bed9fbd866a00fb519db604313c6 (MD5)Garcinia achachairu (GAC) é uma planta de origem boliviana que vem sendo utilizada na medicina popular para o tratamento de distúrbios gástricos, reumatismo, inflamações e como cicatrizante. A caracterização fitoquímica do extrato desta planta revelou que, uma benzofenona, a Gutiferona A (GA), é um dos seus compostos majoritários, que segundo estudos recentes, apresenta importante atividade antioxidante e antimicrobiana. Considerando o interesse em se aprofundar as análises do potencial farmacológico da GA e a inexistência de estudos que avaliem a sua toxicidade genética, o presente estudo foi elaborado visando investigar o potencial genotóxico e mutagênico da GA em diferentes células de camundongos in vivo, utilizando alguns dos testes tradicionais na área de mutagênese, como o Ensaio Cometa (EC) para a verificação da genotoxicidade e o Teste do Micronúcleo (TM) para a verificação da mutagenicidade. O experimento foi conduzido com camundongos Suíços albinos machos (Mus musculus) de 12 semanas, divididos em cinco grupos, constituídos cada um por seis animais. O grupo controle negativo recebeu, via gavagem, 0,3 mL de DMSO 1%. O grupo controle positivo, recebeu intraperitonealmente, 80 mg/kg de doxorrubicina. Os grupos tratados receberam, via gavagem, 0,3 mL da GA nas doses de 15, 30 e 60 mg/kg. Para a avaliação da genotoxicidade foi coletado sangue da veia caudal dos camundongos (4 e 24 horas após o tratamento), células do fígado, medula óssea, cérebro e testículos (coletadas 24 horas após o tratamento). Para a avaliação da mutagenicidade, foram coletadas células da medula óssea 24 horas após o tratamento. A citotoxicidade foi avaliada pela contagem de 200 eritrócitos policromáticos (PCE) e normocromáticos (NCE) e determinação de sua razão (PCE/NCE). Na amostra de sangue de 4h, analisadas pelo EC, os resultados obtidos mostraram que nas doses de 30 mg/kg e 60 mg/Kg. A análise ...
Garcinia achachairu (GAC) is a native plant from Bolivia that has been used in folk medicine for the treatment of gastric disorders, rheumatism, inflammation and as a healing. The phytochemical characterization of this plant extract revealed that the benzophenone guttiferone A (GA) is one of its major compounds, which according to recent studies, has important antioxidant and antimicrobial activity. Considering the interest in deepening the analysis of the pharmacological potential of GA and the lack of studies assessing its genetic toxicity, the present study was designed in order to investigate the genotoxic and mutagenic effects of GA in different cells of mice in vivo, using some of the traditional tests in the mutagenesis area, the Comet Assay (CA) for genotoxicity evaluation and the Micronucleus Test (MT) for the mutagenicity assessment. The experiment was conducted in Swiss albino male mice (Mus musculus) with 12 weeks, divided into five groups with six animals each. The negative control group received, by oral gavage, 0.3 mL of 1% DMSO. The positive control group received, intraperitoneally, 80 mg/Kg of doxorubicin. The treated groups received 0.3 ml of GA at 15, 30 and 60 mg/kg, by gavage. For the genotoxicity evaluation, blood was collected from the tail vein of the mice (4 and 24 hours after treatment), and liver, bone marrow, brain and testicular cells were collected 24 hours after treatment. For the mutagenicity assessment, bone marrow cells were collected 24 hours after treatment. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes and their ratio (PCE/NCE) determined. For the 4 h blood sample, the results with GA at doses of 30 and 60 mg/kg showed that was a statistically significant increase in DNA damage in comparison to the negative control. For the 24 h blood sample, only 60 mg/kg dose showed significant genotoxicity. The analysis of ther ...
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Sheng, Mei. "Site Directed Mutagenesis of β-Ketoadipate Succinyl-Coenzyme A Transferase II from Acinetobacter Calcoaceticus." Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc279281/.
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The role of specific amino acid residues in β-ketoadipate succinyl-coenzyme A transferase II from Acinetobacter calcoaceticus was investigated. A 1412 base pair BamiHI-EcoRI fragment carrying the catIJ genes was amplified by polymerase chain reaction and inserted into pUCl9 to generate the plasmid pCATEl9. Escherichia coli DH5α (pCATEl9) carrying only the catlJ genes expressed 3-fold higher enzyme activity than the parent strain. Two mutants were constructed by site directed mutagenesis so that glutamate was replaced by a glutamine at positions Gln155 and Gln193 in the ß subunit of the primary amino acid sequence of the CoA transferase. Both mutants produced transferase that was catalytically active suggesting that Glu155 and Glu193 do not participate directly in catalysis.
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Gong, Shanzhong. "Evaluation of targetron based mutagenesis in Ehrlichia chaffeensis." Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4127.
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White, Malcolm F. "Yeast phosphoglycerate mutase studied by site-directed mutagenesis." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/24419.
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Wright, Tom. "Post-translational mutagenesis : radical methods for protein modification." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:774aa93a-489e-4051-aa01-b33781215968.
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The naturally occurring post-translational modification of proteins expands the chemical and structural diversity available for protein function. Enzymatic processes are known to modify proteins after translation, and in many cases these modifications have been demonstrated to be critical for biological function. However, the paucity of methods for introduction of site-specific modifications to proteins remains a key obstacle to their biochemical study. Access to these naturally modified proteins has long been complicated by their low abundance, difficulties in purifying homogeneous samples from cellular samples and the intractability of bacterial expression systems towards post-translational modification. In this thesis, we report an important step towards circumventing these issues by using a novel 'tag and modify' chemical strategy, termed 'post-translational mutagenesis'. We describe the development and application of C(sp3)-C(sp3) bond-forming reactions on proteins under biocompatible conditions, exploiting unusual free-radical chemistry to form a wide diversity of protein side-chains. Natural, unnatural, posttranslationally-modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically-labeled) side-chains have been installed on a number of proteins. Extensive characterisation of modification site, connectivity and modified protein structure and function confirm the benign nature of our protocols. The modified proteins thus produced have been used to investigate novel aspects of epigenetic regulation of histone proteins, in the context of free histones and in nucleosome particles, and as novel 'isotope PTMs' for protein characterisation by NMR. We believe this approach, a form of 'protein editing', will have wide application in biochemistry and synthetic biology for accessing diverse modified proteins for research.
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Lee, Jong-min. "Mutagenesis of the Yeast ALR1 Mg Transport Gene." Thesis, University of Auckland, 2006. http://hdl.handle.net/2292/5822.
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Magnesium is an essential element and the most abundant divalent cation in eukaryotic and prokaryotic cells, but its transport is not well understood. Mutagenesis was used to study the function of the ALR1 (aluminium resistance) gene, which encodes the major Mg2+ uptake system in Saccharomyces cerevisiae. Random PCR mutagenesis was undertaken of the C-terminal part of ALR1 that is homologous to the bacterial CorA magnesium transport family. The mutants with the most severe phenotype all had amino acid changes in a small region of Alr1 containing the putative transmembrane (TM) domains. Eighteen single amino acid mutants in this critical region were classified into three categories: no, low and moderate activity. One ‘no activity’ mutation, M762L, affected the GMN motif that is a characteristic of the CorA super-family genes. Two other conservative mutations that reduced or inactivated uptake led me to identify Ser729 and Ile746 as critical amino acid residues in Alr1. High expression of inactive mutants inhibited the capability of the wild-type Alr1 protein to transport magnesium, consistent with the idea that Alr1 may form homo-oligomers. The results confirm the classification of ALR1 as a member of the CorA family of magnesium transport genes Random mutagenesis was also undertaken of the critical region of Alr1 containing the TM domains, in order to find important residues for Al3+ toxicity. Two types of Altolerant mutants were obtained: one with increased sensitivity to Co2+ and a second with no change in sensitivity to Co2+ ions. The former class was shown to have an increased rate of Mg2+ uptake, consistent with the hypothesis that Al3+ toxicity results from Mg2+ deficiency via inhibition of Alr1 activity. The latter class of mutants was shown to have normal rates of Mg2+ uptake but with a reduced sensitivity to inhibition by Al3+ ions. The three individual mutants in the latter class were combined in all combinations and the results indicated that their Al3+ tolerance was likely to be additive and that the mutants operate independently. The most tolerant mutant in this class, I746L, involved a conservative change (alteration of the relative location of methyl groups on the amino acid side chain), to a residue that is located within a TM and that was shown above to be critical for Mg2+ uptake. Therefore, Ile746 plays a very important role in both Mg2+ uptake and Al3+ tolerance in Alr1. These results indicate that Al3+ may inhibit Mg2+ uptake by directly competing for binding sites within the pore of the Alr1 protein. Truncation of N-terminal extension of Alr1 showed that the N-terminal 239 amino acids and the C-terminal 53 amino acids are not essential for magnesium uptake. They might be serving some other functions such as protein regulation. In conclusion, these mutagenesis results firmly establish ALR1 as a magnesium transport gene belonging to the CorA super-family and provide direct experimental support for the hypothesis that Al3+ toxicity in yeast occurs by direct inhibition of Mg2+ uptake via the Alr1 protein.
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Themis, Michael. "Retrovirus insertional mutagenesis : experiences at the hprt locus." Thesis, Brunel University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307483.
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Bryant, J. M. "Expression, mutagenesis and properties of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597038.
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A full length endoE gene construct has been generated from the partial clones used to sequence the gene. Recombinant endoE has been expressed and purified as a fusion protein that is catalytically active and possesses the same kinetic characteristics as the bacteriophage enzyme. The mature, active endoE is 76 kDa in size and is probably produced by the post-translational cleavage of a 90 kDa primary translation product. Recombinant endoE has been purified to homogeneity, in milligram quantities, with the fusion protein partner removed for further studies of the enzyme. The role of the C-terminus of endoE was studied by expression of C-terminally truncated proteins. These were expressed from 3' truncated endoE gene constructs generated either by exonuclease III-mediated nested deletion or by PCR. The site of proteolytic processing has been located to within 18 amino acids and further truncation beyond this cleavage site renders the enzyme inactive and more susceptible to proteolytic degradation. The endoE sequence contains two copies of the sialidase motif and one P-loop motif. The importance of these motifs in enzyme activity was studied by site-directed mutagenesis, replacing part or all of the motifs with alanine residues. These indicated that the P-loop motif was not involved either in binding or hydrolysis of polysialic acid and that the sialidase motifs appear to be necessary to allow production of correctly folded, soluble protein but are not involved directly in catalysis. The kinetic characteristics of the binding interaction of the endoE fusion protein and polysialic acid have been studied and compared to those of C-terminally truncated endoE fusion proteins. As a first step in investigating the use of endoE as a reagent in cell biological studies, the subcellular localisation of polysialic acid and the effects of chloroquine and brefeldin A treatment on its localisation in cultured MCF-7 breast tumour cells have been studied.
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Morais, Francisco Silverio. "Mutagenesis of cytochrome b-559 in Chlamydomonas reinhardtii." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484183.
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Moseley, Gregory William. "Investigation of tetraspanin structure and function by mutagenesis." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391041.
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Woodgate, R. "A mechanism for UV mutagenesis in Escherichia coli." Thesis, University of Sussex, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375858.
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Warburton, Johnny. "Selection and mutagenesis in cultured African violet plantlets." Thesis, University of Edinburgh, 1986. http://hdl.handle.net/1842/11945.
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Basran, Amrik. "Refolding, mutagenesis and characterisation of secretory phospholipase A2." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/35251.
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Bovine pancreatic phospholipase A2 (bPLA2) is a 14 kDa (123 amino acids) enzyme with seven disulphide bonds. Its primary function in vivo is the hydrolysis of dietary phospholipids. A T7 RNA polymerase based expression system was used to overexpress a synthetic gene encoding bovine pancreatic pro-phospholipase A2 in E. coli. The expressed protein was directed to the periplasmic space via an outer membrane secretory signal (OmpT) where, after translocation the protein formed insoluble inclusion bodies. Translocation efficiency was significantly increased (from about 25% to over 90%) when protein expression was induced after the cells had reached the stationary phase of growth (O.D.600~2.8). Optimal conditions for refolding of bPLA2 were found to be by rapid dilution under anaerobic conditions, in the presence of 2 mM oxidised glutathione, 4 mM reduced glutathione, 5 mM potassium EDTA and 25 mM sodium borate pH 8.7, with a final protein concentration of approximately 45 mg/L. The final amount of active recombinant protein produced was 22 mg per litre of bacterial culture. Site directed mutagenesis was used to investigate the role of the 58-71 surface loop of bovine pancreatic PLA2 in interfacial binding to a variety of aggregated phospholipids and surfactants. The surface loop was mutated so that the amino acid sequence was similar to that found in porcine pancreatic PLA2. Three mutants were made,Val-63Phe-63 (V63F), Asn-71Glu-71 (N71E) and finally the double mutant (V63FN71E). The mutants were overexpressed in E. coli and refolded in vitro. V63F had an increased affinity for lipid-water interfaces so that its binding to zwitterionic micelles was more like that of the porcine enzyme but catalysis was still similar to the bovine enzyme. Both N71E and V63FN71E showed a reduction in binding to zwitterionic micellar interfaces at pH 8 compared with bPLA2. The affinity for the lipid-water interface could be restored by the addition of a negative charge (via an anionic detergent) to the micellar interface.
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Wilson, Mark Steven Michael. "Mutagenesis of nifE and nifN from Azotobacter vinelandii." Thesis, Virginia Tech, 1988. http://hdl.handle.net/10919/43071.
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The products of nifE and nifN from Azotobacter vinelandii, which
are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co)
co) from nitrogenase, have been analyzed using a variety of mutagenic techniques.
NifE was the object of several site-specific, amino acid
substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer
of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A
model of metal cluster binding; regions within the nifEN-products is
discussed insofar as it relates to the rationale for the targeting of
particular amino acids for-substitution.
A translational fusion between nifN and lacZ was constructed and
used to study the regulation of nifEN. This gene fusion was regulated
in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of β-galactosidase.
Results from mutant strains which carry lesions in nifH or nifA in
addition to the nifN::lacZ fusion are presented and discussed.Master of Science
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Romier, Christophe. "Crystallographic and mutagenesis studies of tRNA-guanine transglycosylase." Aix-Marseille 2, 1997. http://www.theses.fr/1997AIX22011.
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L'enzyme procaryote trna-guanine transglycosylase (tgt) catalyse l'echange, sur les arnt specifiques pour asn, asp, his et tyr, de la guanine a la position wobble avec la 7-aminomethyle-7-deazaguanine (preq1), un precurseur de la base hypermodifiee queuine. La tgt de zymomonas mobilis, une proteine de 43 kda, a ete purifiee et cristallisee pour son etude structurale par cristallographie aux rayons x. Sa structure a ete resolue par la methode de remplacement isomorphe multiple a l'aide de six derives aux atomes lourds independants et a ete affinee a une resolution de 1,85 angstroms. La tgt adopte une structure en tonneau-(beta-alpha)#8 irreguliere avec differentes insertions entre les huit brins composant le tonneau, notamment un sous-domaine liant le zinc. La structure de la tgt en complexe avec son substrat preq#1, qui a aussi ete determinee, suggere un mode de reconnaissance de l'arnt ou les phosphates seraient reconnus par le domaine de liaison du zinc, tandis que la sequence specifique u#3#3g#3#4u#3#5 serait reconnue par le tonneau. De plus, par analyse des complexes tgt-arnt en conditions denaturantes, il a ete demontre que la reaction enzymatique catalysee par la tgt comprenait la formation d'une liaison covalente avec l'arnt. Par mutagenese dirigee, il a ete prouve que l'aspartate 102 etait le nucleophile du site actif de la tgt de z. Mobilis et que le site de fixation le plus probable pour la guanine wobble est la poche de fixation de la molecule de preq#1
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Shah, Nehal Rajendra. "Towards Novel Methods of Mutagenesis for Histophilus somni." Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/43708.
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Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases in bovines, although nonpathogenic commensal strains also exist. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. To identify genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and may be enhanced by the presence of uptake signal sequences (USS) within the genome. We hypothesized that natural transformation occurs in H. somni because its genome is over-represented with USS and contains all the necessary genes for competence, except that ComD and ComE are mutated. For natural transformation, H. somni was grown to exponential phase, and then transferred to a non-growth defined medium to induce competence. H. somni strain 2336 was successfully transformed with homologous linear DNA (lob2A) containing an antibiotic marker gene, but at low efficiency. Shuttle vector pNS3K was also naturally transformed into H. somni at low efficiency. To attempt to improve transformation efficiency, comD and comE from H. influenzae were cloned into shuttle vector pNS3K to generate the plasmid pSScomDE. Although introduction of pSScomDE into H. somni was expected to increase the number and breadth of mutants generated by natural transformation, multiple attempts to electroporate pSScomDE into H. somni were unsuccessful. A native plasmid (pHS649) from H. somni strain 649 may prove to be a more efficient shuttle vector. Due to inefficiency in generating mutants by allelic exchange, transposon (Tn) mutagenesis with EZ::Tn5â ¢Master of Science
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Wang, Xiaoshan. "Site-Directed Mutagenesis in Francisella Tularensis by Allelic." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/36440.
Full textAbstract:
Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention.
The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy.
In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence.
Master of Science