CONSTRUCTION OF PCR-LIGATED LONG FLANKING HOMOLOGY CASSETTES FOR USE IN THE FUNCTIONAL-ANALYSIS OF 6 UNKNOWN OPEN READING FRAMES FROM THE LEFT AND RIGHT ARMS OF SACCHAROMYCES-CEREVISIAE CHROMOSOME-XV

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homolo gy method (Wach et al., 1994). Transformants were selected as Genetici n (G418)-resistant colonies and correct integration of the kanMX4 cass ette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-res istant marker. We have developed a widely applicable method for the co nstruction of gap repair plasmids to obtain the cognate clones for eac h of the disrupted ORFs. The 5'- and 3'-flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is c ut at this site it can be used to obtain, by selection for the appropr iate antibiotic resistance, long flanking homology (LFH) cassettes con taining the cognate clone or the disrupted allele. The LFH cassette co ntaining the cognate clone or the disrupted allele can be released fro m the gap-repaired plasmid by cutting at the inserted flanking restric tion sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on differen t media at 15 degrees C, 30 degrees C and 37 degrees C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37 degrees C an d the product of YOL091w appears to play a role in sporulation since t he homozygous diploid disruptant has lost the ability to sporulate. (C ) 1998 John Wiley & Sons, Ltd.