Functional analysis of six novel ORFs on the left arm of Chromosome XII inSaccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been ana lysed using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell gro wth; yll035w spores arrested after two or three cell divisions, while the m ajority of yll034c spores stopped growth within two cell cycles after germi nation. Complementation of the yll035w deletion with its cognate clone, and a promoter-substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonst rated that YLL035w is under cell-cycle regulation. Bioinformatic analyses p roduced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious p henotypes resulting from deletion of any of the four nonessential ORFs. Rep lacement cassettes, comprising the kanMX marker flanked by each ORF's promo ter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copy right (C) 2000 John Wiley & Sons, Ltd.