Please use this identifier to cite or link to this item: https://ir.swu.ac.th/jspui/handle/123456789/13958
ชื่อเรื่อง: Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae
ผู้แต่ง: Yarimizu T.
Nonklang S.
Nakamura J.
Tokuda S.
Nakagawa T.
Lorreungsil S.
Sutthikhumpha S.
Pukahuta C.
Kitagawa T.
Nakamura M.
Cha-aim K.
Limtong S.
Hoshida H.
Akada R.
Keywords: DNA fragment
amino acid metabolism
article
auxotrophic mutant
chromosome
DNA end joining repair
fungal strain
gene amplification
gene library
gene mutation
gene synthesis
genetic code
genetic complementation
genetic identification
genetic transformation
Kluyveromyces marxianus
mating
mutation rate
nonhuman
nucleic acid metabolism
nutritional requirement
phenotype
polymerase chain reaction
priority journal
Saccharomyces cerevisiae
sporogenesis
Kluyveromyces marxianus
Saccharomyces cerevisiae
auxotrophic mutants
Kluyveromyces marxianus
mating
non-homologous end-joining
Saccharomyces cerevisiae
tetrad
DNA, Fungal
Genetic Complementation Test
Kluyveromyces
Mutation
Plasmids
Recombination, Genetic
Saccharomyces cerevisiae
Transformation, Genetic
Transgenes
วันที่เผยแพร่: 2013
บทคัดย่อ: 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. © 2013 John Wiley & Sons, Ltd.
URI: https://ir.swu.ac.th/jspui/handle/123456789/13958
https://www.scopus.com/inward/record.uri?eid=2-s2.0-84890183384&doi=10.1002%2fyea.2985&partnerID=40&md5=94b00440b084482868d374bb931388b9
ISSN: 0749503X
Appears in Collections:Scopus 1983-2021

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