Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains.
AUTOR(ES)
Sakai, Y
RESUMO
A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=207139Documentos Relacionados
- Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.
- An efficient one-step site-directed and site-saturation mutagenesis protocol
- A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.
- Directed termination PCR: a one-step approach to mutation detection.
- One-step random mutagenesis by error-prone rolling circle amplification
