A counterselection for the tryptophan pathway in yeast: 5‐fluoroanthranilic acid resistance

• Published in: Yeast (Chichester, England) Vol. 16; no. 6; pp. 553 - 560
• Main Authors: Toyn, Jeremy H., Gunyuzlu, Paul L., Hunter White, W., Thompson, Lorin A., Hollis, Gregory F.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-04-2000
• Subjects: 5-fluoroanthranilic acid; Aldose-Ketose Isomerases; Anthranilate Phosphoribosyltransferase - genetics; Anthranilate Synthase - genetics; antimetabolism; Cell Division - drug effects; Cell Division - genetics; counterselection; Drug Resistance, Microbial - genetics; fluoroanthranilic acid; Fungal Proteins - genetics; Gene Deletion; Genetic Markers; Indole-3-Glycerol-Phosphate Synthase - genetics; Mutation; ortho-Aminobenzoates - chemistry; ortho-Aminobenzoates - pharmacology; Plasmids - genetics; Recombinant Fusion Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; TRP1; TRP1 gene; tryptophan; Tryptophan - biosynthesis; Tryptophan Synthase - genetics; yeast
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/(SICI)1097-0061(200004)16:6<553::AID-YEA554>3.0.CO;2-7

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5‐fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5‐fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5‐fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica‐plating method to rapidly assess genetic linkage in self‐replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5‐fluoroanthranilic acid. Copyright © 2000 John Wiley & Sons, Ltd.