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Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae
Author(s) -
Hartzog Phillip E.,
Nicholson Bradly P.,
McCusker John H.
Publication year - 2005
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.1245
Subject(s) - cytosine deaminase , cytosine , biology , saccharomyces cerevisiae , ura3 , selectable marker , plasmid , genetics , orfs , gene , open reading frame , biochemistry , genetic enhancement , peptide sequence
We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae . The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5‐fluorocytosine (5FC) is toxic to cytosine deaminase‐producing cells, fcy1 strains are resistant to 5FC. FCY1 MX and FCA1 MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1 , respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1 PR, FCY1 PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1 ‐ and FCA1 MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter‐selection on both synthetic and rich media. Copyright © 2005 John Wiley & Sons, Ltd.