Cloning and expression on a multicopy vector of five invertase genes ofSaccharomyces cerevisiae

✍ Scribed by Stefan Hohmann; Friedrich K. Zimmermann

• Publisher: Springer-Verlag
• Year: 1986
• Tongue: English
• Weight: 837 KB
• Volume: 11
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00420610

✦ Synopsis

Six unlinked loci for invertase structural genes are known in the yeast Saccharornyces cerevisiae: SUC1-SUC5 and SUC7. These genes are similar in structure and expression but not identical. Different yeast strains possess none, one or several of these genes.

We have isolated the genes SUC1-SUC5, subcloned them into the multicopy vector YEp24 and compared the expression of the five SUC genes in one recipient strain.

SUC2 was isolated by transformation of a sucO strain with a gene pool and complementation to sucrose fermentation. SUC4 was cloned from a minipool of chromosomal fragments which were shown to contain SUC4 by Southern hybridization. SUC1, SUC3 and SUC5 were isolated using the method of plasmid eviction. A plasmid containing regions flanking SUC4 was integrated next to these SUC genes. The plasmid together with the SUC genes were then cut out of the chromosome using an appropriate restriction endonuclease.

The length of chromosomal DNA fragments containing the different SUC genes were 4.8 kb for SUC1, 5.2 kb for SUC2, 4.8 kb for SUC3, 12.8 kb for SUC4 and 17.2 kb for SUC5. Fragments containing the complete SUC genes and the sequences controlling their expression were subcloned into YEp24 and transformed into a strain without any active invertase gene. Invertase activity of transformants was measured after growth repressing (8% glucose) and derepressing (2% raffinose) conditions. As expected from results with strains carrying the individual * Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday ** This work was supported by Deutsche Forschungsgemeinschaft