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Master's Dissertation
DOI
Document
Author
Full name
Nirlei Aparecida Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 1995
Supervisor
Title in Portuguese
Clonagem e mapeamento molecular do gene de resistência ao Metolachlor em Saccharomyces cerevisiae
Keywords in Portuguese
CLONAGEM
COMPOSTOS ORGÂNICOS
LEVEDURAS
MAPEAMENTO GENÉTICO
RESISTÊNCIA GENÉTICA
Abstract in Portuguese
Este trabalho descreve a clonagem do gene de resistência ao Metolachlor em um plasmídio epissomal bifuncional (YEp 351), o qual pode ser utilizado como marcador seletivo dominante na transformação de Saccharomyces cerevisiae. Clones tranformantes selecionados diretamente em meio YEPD acrescido de 720 mg/L de Metolachlor, tiveram seu DNA genômico extraído e utilizado para transformar Escherichia coli a fim de amplificar e recuperar os plasmídios recombinantes. Outras linhagens de Saccharomyces foram também transformadas e foi possível observar que existe diferença entre linhagens quanto às suas capacidades de receber, manter e expressar DNA heterólogo, pois houve variação considerável nas suas eficiências de transformação. Vários métodos foram aplicados para obtenção do "cariótipo eletroforético” (este termo refere-se ao genoma de um dado organismo expresso em bandas cromossômicas) de Saccharomyces cerevisiae, dentre eles, o tratamento com acetato de lítio ganhou mais destaque. Empregando-se a técnica de eletroforese em campo pulsado e hibridização, foi possível mapear o gene de resistência ao Metolachlor no cromossomo XV da levedura, confirmando-se os dados de análise genética.
Title in English
Cloning and molecular mapping of the Metolachlor resistance gene in Saccharomyces cerevisiae
Abstract in English
This work describes the cloning of the metolachlor resistance gene into an episomal bifunction plasmid (YEp 351) which can used as a dominant selective marker for Saccharomyces cerevisiae transformation. DNA of the transformed clones, directly selected onto YEPD medium added of 720 mg/l metolachlor, was used to transform Escherichia coli in order to amplify and recover recombinant plasmids. Other Saccharomyces strains were also transformed and as far as differences in receiving, maintaining and expression of the heterologous DNA is concerned, considerable variation in transformation was found them. Electrophoretic caryotyping (refers to an organism genome expressed in chromosomal bands) was performed in Saccharomyces cerevisiae according to different methods, and among them lithium acetate was the standing one. Pulsed field and hybridization were performed to map the metolachlor resistance gene into the yeast chromosome XV and these data were confirmed by genetical analysis.
 
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Publishing Date
2019-08-22
 
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