TY1-MEDIATED INTEGRATION OF EXPRESSION CASSETTES - HOST STRAIN EFFECTS, STABILITY, AND PRODUCT SYNTHESIS

The yeast retrotransposon Ty1 has been used to insert multiple copies of heterologous genes into the genome of Saccharomyces cerevisiae. Amp lification using a GAL1-regulated Ty1 element carrying a 4.6 kilobase pair expression cassette (Escherichia coli lacZ structural gene under the control of the yeast CUP1 promoter, and the bacterial neo gene) wa s compared with that of a GAL1-regulated Ty1 element carrying only the neo gene. Mobilization of Ty1 was induced from a chromosomal element and a 2 mu-plasmid-based element; similar results were obtained for bo th locations. The two marked Ty1 cassettes were successfully integrate d into the genomes of three different S. cerevisiae strains. Efficienc ies were found to vary significantly between strains. The size of the inserted cassette was also important; the efficiency for the CUP1p-lac Z-neo cassette was much lower than that for the neo cassette in the sa me host. All amplified copies were found to be quite stable with or wi thout expression for at least 50 generations in nonselective medium; m oreover, there were no significant effects on the growth of the cells. After integration, beta-galactosidase specific activity from the lacZ construct in the three hosts was found to correlate well with the cop y number of the CUP1p-lacZ expression cassette amplified by the Ty1 re trotransposon.