Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins.
We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).
Grava
S
S
Mécanismes Moléculaires de la Division Cellulaire et du Développement, UPR 9005 du CNRS, IBMC, 15 rue Descartes, F-67084 Strasbourg, France.
Dumoulin
P
P
Madania
A
A
Tarassov
I
I
Winsor
B
B
D000199:Actins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics*; D003001:Cloning, Molecular; D004260:DNA Repair; D017353:Gene Deletion; D020043:Genes, Essential; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014404:Tubulin / Q000378:metabolism
D000199:Actins; C000609267:Arp5 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins