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Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.
Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji.
Afiliação
  • Yarimizu T; Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan.
Yeast ; 30(12): 485-500, 2013 Dec.
Article em En | MEDLINE | ID: mdl-24150815
The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Saccharomyces cerevisiae / Kluyveromyces / DNA Fúngico Tipo de estudo: Diagnostic_studies Idioma: En Revista: Yeast Assunto da revista: MICROBIOLOGIA Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Japão País de publicação: Reino Unido

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Saccharomyces cerevisiae / Kluyveromyces / DNA Fúngico Tipo de estudo: Diagnostic_studies Idioma: En Revista: Yeast Assunto da revista: MICROBIOLOGIA Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Japão País de publicação: Reino Unido