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

✍ Scribed by El-Moghazy, Abdel-Nasser; Zhang, Nianshu; Ismail, Thamir; Wu, Jian; Butt, Amna; Ahmed Khan, Shakeel; Merlotti, Cristina; Cara Woodwark, K.; Gardner, David C. J.; Gaskell, Simon J.; Oliver, Stephen G.

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 197 KB
• Volume: 16
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(200002)16:3<277::aid-yea524>3.0.co;2-g

✦ Synopsis

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 promotersubstitution 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 nonessential 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).