Controlled expression of homologous genes by genomic promoter replacement in the yeast Saccharomyces cerevisiae.
Exchange of the promoter of a gene in the genome for another promoter whose expression can be controlled easily can overcome problems associated with the expression of the same gene from a promoter on a plasmid. Some genes are difficult or impossible to clone in plasmid-based vectors and often a stable expression and maintenance of the gene during cell proliferation is desirable. We present a method by which any genomic promoter can be replaced by a promoter of choice to achieve controlled (or constitutive and strong) expression of the gene concerned. The new promoter and a marker gene of choice are amplified by PCR using primers with a tail homologous to the regions adjacent to the site of integration in the genome and primers with a restriction site allowing ligation of the promoter and marker PCR products. After ligation of these PCR products, the ligated construct is transformed into yeast cells and allowed to exchange for the original promoter by homologous recombination. The transformants are selected based on the presence of the marker gene and proper exchange of the original promoter for the new promoter is checked by means of PCR amplification using primers in the new promoter and in the gene under its control.
Verstrepen
Kevin J
KJ
Leiden Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Thevelein
Johan M
JM
D003001:Cloning, Molecular / Q000379:methods*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D017930:Genes, Reporter; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism