HETEROLOGOUS HIS3 MARKER AND GFP REPORTER MODULES FOR PCR-TARGETING IN SACCHAROMYCES-CEREVISIAE

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TE F gene promotor of the filamentous fungus Ashbya gossypii. Both chimer ic modules and the cognate S. kluyveri HIS3 gene were tested in transf ormations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (H IS3MX6) performed best. With less than 5% incorrectly targeted transfo rmants, it functions as reliably as the widely used geniticin resistan ce marker kanMX. The rare false-positive His(+) transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluore scent protein gene from Aequorea victoria into our pFA-plasmids with H IS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-typ e GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. c erevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could est imate that only about 10% of the transformants carried inactivating mu tations in the GFP reporter. (C) 1997 John Wiley & Sons, Ltd.