Journal
YEAST
Volume 23, Issue 11, Pages 825-831Publisher
WILEY-BLACKWELL
DOI: 10.1002/yea.1397
Keywords
site-directed mutagenesis; PCR; homologous recombination; plasma membrane ATPase; yeast
Ask authors/readers for more resources
The introduction of defined mutations into open reading frames (ORF) or nontranslated regions of the genome is important to study of the structure -function relationship of amino acid residues in proteins or that of sequence motifs at the genome level. We describe a simple two-step method for the introduction of defined single or multiple point mutations into the genome of Saccharomyces cerevisiae. This method circumvents the need for plasmid-based mutagenesis and thus ensures homogenous expression of the gene of interest within the cell population. It is based on the introduction of a selectable marker downstream of the gene of interest. This marker is then amplified with a gene-specific primer that harbours the desired point mutation, creating a selectable marker-tagged mutant version of the gene of interest. The mutant fragment is then integrated into the genome of a wild-type strain through homologous recombination. Successive rounds of amplification of the mutant loci with primers that introduce additional point mutations upstream of existing mutations will generate multiple defined mutations within a single gene. As a proof of principle, we have employed this method to generate a temperature-sensitive mutant version of the plasma membrane ATPase,pmal-7, which bears two point mutations (Pro434Ala and Gly789Ser). Phenotypic analysis of a pmal-7 haploid strain indicates that this allele has the same characteristics as the original pmal-7 allele. It confers a temperaturesensitive growth phenotype and the newly synthesized Pmal-7 protein is unstable and rapidly degraded. Copyright (c) 2006 John Wiley & Sons, Ltd.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available