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A novel method for in situ screening of yeast colonies with theβ-glucuronidase reporter gene

✍ Scribed by Heribert Hirt

Publisher: Springer-Verlag
Year: 1991
Tongue: English
Weight: 401 KB
Volume: 20
Category: Article
ISSN: 0172-8083
DOI: 10.1007/bf00317075

No coin nor oath required. For personal study only.

✦ Synopsis

Expression of the beta-galactosidase gene in yeast has served as a screening marker for many purposes. Here it is shown that in two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the beta-glucuronidase (GUS) gene can be used as an alternative marker. Since the histochemical substrate can not be taken up by yeast cells, direct colony screening of plates was found to be impossible. However, by a replica plating technique, GUS expression became visibly detectable within 10 min when the GUS gene was strongly expressed. The staining method could still be performed for expression at a 100-fold lower level, but incubation times of several hours were needed. Furthermore, specific GUS expression levels of yeast protein extracts could be quantified by a fluorometric assay which is both very simple to perform and highly sensitive. Since the GUS gene can also tolerate large N-terminal fusions, this method should be particularly attractive for studying such diverse problems as transcriptional and translational regulation or subcellular localization in yeast.

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