Functional analysis of six novel ORFs on the left arm of Chromosome XII ofSaccharomyces cerevisiae reveals three of them responding to S-starvation

Six novel Open Reading Frames (ORFs) located on the left arm of the chromos ome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short-flanking homology (SFH) or long-flanking homol ogy (LFH) gene replacement, Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN grow th tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs, Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S-methylmethionine and that YLL060c encodes a g lutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur-containing amino-acid metabolism, YLL057 c in sulphonate catabolism and YLL056c in stress response. The transcriptio n of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase m ore than 10-fold under sulphate starvation. Replacement cassettes, comprisi ng the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7, All the cognate clones, 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). Copyright (C) 2001 John Wiley & Sons, Ltd.