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Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae
Author(s) -
Akada Rinji,
Shimizu Yoshirou,
Matsushita Yuji,
Kawahata Miho,
Hoshida Hisashi,
Nishizawa Yoshinori
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.797
Subject(s) - cerulenin , selectable marker , biology , cycloheximide , saccharomyces cerevisiae , yap1 , auxotrophy , ura3 , yeast , transformation (genetics) , marker gene , mutant , genetics , gene , cell culture , fatty acid synthase , transcription factor
Drug‐resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug‐resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter‐driven YAP1 expression cassette, called PGKp–YAP1 . Yeast cells containing PGKp–YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp–YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non‐transformed drug‐resistant colonies did appear on the low‐concentration cerulenin plates. However, these non‐transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp–YAP1 could be used as an effective selection for PGKp–YAP1 recipient cells. The PGKp–YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain. Copyright © 2002 John Wiley & Sons, Ltd.