IMPROVED EFFICIENCY AND STABILITY OF MULTIPLE CLONED GENE INSERTIONS AT THE DELTA-SEQUENCE OF SACCHAROMYCES-CEREVISIAE

Two delta-integration vectors were evaluated for the insertion of an i nducible expression cassette (the yeast CUP1 promoter fused to the Esc herichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomyc in-resistance gene (neo) into the genome of Saccharomyces cerevisiae v ia homologous recombination. Cells containing integrations were select ed by resistance to the aminoglycoside G418. The first vector was a tr aditional construct containing only one delta sequence; with this vect or, the transformation efficiency and the number of integrations per c ell were quite low. The second carried two delta sequences flanking th e desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-g alactosidase levels exceeded those obtained with the 2 mu-based plasmi d. Furthermore, the integrations appeared more stable in long-term seq uential culture (both with and without induction of the lacZ gene) tha n those obtained via the single delta vector.