Biotechnology and Bioengineering, Vol.49, No.1, 45-51, 1996
G418 Selection and Stability of Cloned Genes Integrated at Chromosomal Delta-Sequences of Saccharomyces-Cerevisiae
The chromosomal delta sequences of the yeast Saccharomyces cerevisiae were employed as recombination sites to integrate the bacterial neo(r) gene and the yeast SUC2 gene into the yeast genome. A dominate selection method employing the aminoglycoside antibiotic G418 was used. Transformation efficiencies and growth behaviors of the transformants were studied. Transformants were obtained with more than 40 integrations; the majority of insertions were tandem with a maximum of three different insertion sites utilized at one time. After 70-100 generations of growth in nonselective medium, the high copy number SUC2-neo(r) integrants were found to be unstable; only minor instability was observed for the neo(r) and low copy number SUC2-neo(r) integrants.
Keywords:COPY-NUMBER INTEGRATION;NERVE GROWTH-FACTOR;RIBOSOMAL DNA;YEAST;EXPRESSION;SECRETION;TRANSPOSITION;TRANSFORMATION;RECOMBINATION;CONSTRUCTION