Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S...

Full description

Saved in:
Bibliographic Details
Published in:Yeast (Chichester, England) England), 2013-06, Vol.30 (6), p.243-253
Main Authors: Fukunaga, Tomoaki, Cha‐aim, Kamonchai, Hirakawa, Yuki, Sakai, Ryota, Kitagawa, Takao, Nakamura, Mikiko, Nonklang, Sanom, Hoshida, Hisashi, Akada, Rinji
Format: Article
Language:English
Subjects:
DNA, Recombinant - genetics
Gene Expression Regulation, Fungal
Gene Targeting - methods
Genetic Loci
Homologous Recombination
overlap extension PCR
Polymerase Chain Reaction
Promoter Regions, Genetic
recombinant DNA
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - genetics
ura3
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.
ISSN:0749-503X
1097-0061
DOI:10.1002/yea.2957