Yeast Functional Analysis Report Functional analysis of six novel ORFs on the left arm of Chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control ABDEL-NASSER EL-MOGHAZY 1 , NIANSHU ZHANG 1 , THAMIR ISMAIL 1 , JIAN WU 1 , AMNA BUTT 1 , SHAKEEL AHMED KHAN 1 , CRISTINA MERLOTTI 1 , K. CARA WOODWARK 1 , DAVID C. J. GARDNER 1 , SIMON J. GASKELL 2 AND STEPHEN G. OLIVER 1 * 1 Department of Biomolecular Sciences, UMIST, PO Box 88, Sackville Street, Manchester M60 1QD, UK 2 Department of Chemistry, UMIST, PO Box 88, Sackville Street, Manchester M60 1QD, UK Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short-¯anking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. 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 demonstrated that YLL035w is under cell-cycle regulation. Bioinformatic analyses produced signi®cant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non- essential ORFs. Replacement cassettes, comprising the kanMX marker ¯anked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products ampli®ed from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright # 2000 John Wiley & Sons, Ltd. KEY WORDS Ð functional genomics; Saccharomyces cerevisiae; cell cycle; EUROFAN; INTRODUCTION The analysis of the Saccharomyces cerevisiae genome sequence has revealed nearly 6000 open reading-frames (ORFs) which are likely to encode protein products (Goffeau et al., 1996). A large number of these ORFs (ca. 45%) do not have any known function. While bioinformatic analysis can shed light on the possible functions of some of these genes (see Mewes et al., 1997), a systematic experimental approach is required to group the genes into initial functional categories and then obtain more detailed functional information about each of them (Oliver, 1996). As a ®rst step towards this aim, the EUROFAN network (Oliver, 1997) has embarked upon a large-scale gene deletion effort targeted on 1000 ORFs of unknown function. This collection of deletant strains will be exploited, in the second phase of EUROFAN, in order to obtain more detailed functional assignments (Oliver, 1997; Oliver et al., 1998). As a contribution to this joint effort, we have generated speci®c deletion mutants for YLL044w, *Correspondence to: S. G. Oliver, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Manchester M13 9PT, UK. E-mail: steve.oliver@man.ac.uk YEAST Yeast 2000; 16: 277±288. Received 25 July 1999 Accepted 1 November 1999 CCC 0749-503X/2000/030277±12$17.50 Copyright # 2000 John Wiley & Sons, Ltd.