A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains.
We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains. The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996). Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population. Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated. Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S. cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss. Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS.
Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany.
J H
JH
Hegemann
S
S
Klein
S
S
Heck
U
U
Güldener
R K
RK
Niedenthal
U
U
Fleig
D020090:Chromosome Segregation; D015825:Chromosomes, Fungal / Q000235:genetics*; D005434:Flow Cytometry; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins