Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants

We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088cΔ and ydl084wΔ haploid strains are viable in the CEN. PK2 genetic background although ydl084wΔ grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects. © 1998 John Wiley & Sons, Ltd.     

Basic functional analysis of six unknown open reading frames from Saccharomyces cerevisiae: four from chromosome VII and two from chromosome XV.

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae from the left arms of chromosomes VII and XV were disrupted by the short-flanking homology method in the diploid strains FY1679 and CENPK2. In each case, the entire ORF, with the exception of the first nucleotide of the start codon, was eliminated and replaced by the kanMX4 cassette. Correct integration of the disrupting marker was checked by colony PCR of the geneticin (G418)-resistant transformants. Sporulation followed by tetrad dissection of the diploids revealed that none of the ORFs encoded a product essential for the viability of either yeast strain. The neutral effect of these disruptions extended to mating and sporulation, since it was possible to create homozygous diploid disruptants that were capable of sporulation. Basic phenotypic analysis was carried out on all strains by growing them on three different media at three different temperatures and revealed no significant differences between disruptants and the parental strains. A cognate clone and a kanMX4 disruption cassette were created for five of the six ORFs by gap repair with specific long-flanking homology cassettes. For experimental reasons, the cognate clone and disruption cassette corresponding to the sixth ORF (YGL161w) had to be created by PCR.     

Disruption and basic phenotypic analysis of six novel genes from the left arm of chromosome XIV of Saccharomyces cerevisiae

We describe here the construction of six deletion mutants and their basic phenotypic analysis in three different backgrounds. The six genes were disrupted in three diploid strains (FY1679, W303 and CEN.PK2) by the long flanking homology (LFH) method (Wach, 1996). Transformants were selected as geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the heterozygous diploids, tetrads were dissected and scored for segregation of G418-resistance and auxotrophic markers. One of the six ORFs (YNL158w) corresponds to an essential gene which has no homology with other genes present in the databases and has two predicted transmembrane domains. Growth tests performed on different media at 15 degrees C, 30 degrees C or 37 degrees C with haploid deletants of the five non-essential genes revealed no apparent phenotype in any of them.     

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae

Deletion via PCR‐mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading‐frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short‐flanking homology (SFH) or a long‐flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non‐growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non‐essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap‐repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright © 1999 John Wiley & Sons, Ltd.     

Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants.

Six ORFs of unknown function from the left arm of chromosome XII of Saccharomyces cerevisiae were chosen for a reverse genetic approach to provide materials to assist in assignment of function. A two-step PCR using long-flanking homology was employed to amplify disruption cassettes consisting of a kanMX gene as selectable marker flanked by 250-350 bp long regions homologous to the target gene. The diploid strains FY1679 and CEN.PK2 were transformed with the replacement cassettes and transformants were selected for geneticin (G418) resistance. Correct targeting of the replacement cassettes at the genomic locus was verified by Southern blot analysis with the kanMX gene as a probe. Disruption cassettes were cloned in pUG7 plasmid for systematic gene inactivation in other yeast strains and the cognate genes were cloned in pRS416 plasmid for gene complementation studies. Sporulation and tetrad analysis of heterozygous disruptants showed that three of the six ORFs [YLR141w (RRN5), YLR145w and YLR147c (SMD3)] were essential genes that were complemented by their cognate genes. ylr146c Delta (spe4) homozygous diploids showed enhanced sporulation efficiency, whereas ylr147c Delta heterozygous diploids failed to sporulate in the FY1679 but not in the CEN.PK2 genetic background. The other two disruptants [ylr143w and ylr144c (acf2)] gave no phenotype.     

Disruption of 12 ORFs located on chromosomes IV,VII and XIV of Saccharomyces cerevisiae reveals two essential genes

We describe the generation of null-mutants of 12 open reading frames (ORFs), discovered during the systematic sequencing of the Saccharomyces cerevisiae genome. These ORFs are located on chromosome IV (YDL183c), on chromosome VII (YGL139w, YGL140c, YGL141w, YGR280c and YGR284c) or on chromosome XIV (YNL006w, YNR004w, YNR007c, YNR008w, YNR009w and YNR013c). Disruptants were generated using the PCR-based short flanking homology (SFH) strategy in yeast strain FY1679. Tetrad analysis, following sporulation of the heterozygous disruptants, revealed that YGR280c and YNL006w are essential genes for vegetative yeast growth in rich medium. The lethality of the two genes was confirmed by gene complementation analysis. The protein encoded by YNL006w (LST8) is now known to be involved in transport of permeases from the Golgi to the plasma membrane. Basic phenotypic analyses were performed on haploid disruptants from both mating types of 10 non-essential genes. One disruptant (YNR004w) revealed a slow growth rate on glucose-minimal medium at 15 degrees C. For each of the individual ORFs, a disruption cassette and the corresponding cognate gene were cloned into appropriate plasmids.     

Disruption of six novel genes from the left arm of chromosome XV of Saccharomyces cerevisiae and basic phenotypic analysis of the generated mutants

Six open reading frames (ORFs) of unknown function from the left arm of Saccharomyces cerevisiae chromosome XV were deleted in two genetic backgrounds by disruption cassettes with long flanking homology (LFH) (Wach, 1996), within the frame of the research project EUROFAN. The LFH disruption cassettes, obtained by PCR, were made by introducing the kanMX4 marker module between two fragments homologous to the promoter and terminator regions of a given ORF. Transformants resistant to geneticin (G418) were selected. The LFH disruption cassettes were cloned in a bacterial vector. Each cognate gene was also cloned in a centromeric plasmid. Correct deletion of each gene was verified by four different PCR reactions. Sporulation and tetrad analysis of heterozygous deletants revealed that ORF YOL102c is essential. The non-growing haploid spores gave rise to microcolonies. Basic phenotypic analyses were performed on haploid deletants of both mating types of the five non-essential ORFs, YOL018c, YOL098c, YOL101c, YOL104c and YOL105c. Plate growth tests on different media at 15 degrees C, 30 degrees C or 37 degrees C did not reveal any significant differences between parental and mutant cells. Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells.     

Deletion of six open reading frames from the left arm of chromosome IV of Saccharomyces cerevisiae

The construction of six deletion mutants of Saccharomyces cerevisiae and their basic phenotypic characterization are described. Open reading frames YDL148c, YDL109c, YDL021w, YDL019c, YDL018c and YDL015c from the left arm of chromosome IV were deleted using a polymerase chain reaction (PCR)‐based disruption technique, introducing the kanMX4 resistance marker into the respective genes. Gene replacement cassettes (pYORCs) for use in other strain backgrounds were cloned by PCR using DNA templates from haploid or diploid deletion mutants, and inserted into episomal plasmids. Cognate clones of all six ORFs were obtained by gap repair. Deletions were carried out in diploid cells and, after sporulation, yielded four viable spores for clones disrupted in YDL109c, YDL021w, YDL019c and YDL018c. Spores harbouring disruptions in ORFs YDL148c and YDL015c germinated but underwent only a few divisions before ceasing growth, suggesting that the respective genes are essential for vegetative growth on YPD complete media. The other deletion mutants grew like wild‐type at different temperatures and on different carbon sources. A brief computational analysis of the six ORFs studied in this work is presented. Copyright © 1999 John Wiley & Sons, Ltd.     

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene.

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the beta-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.     

Generation of null alleles for the functional analysis of six genes from the right arm of Saccharomyces cerevisiae chromosome II

Using PCR‐ligated long flanking homology cassettes, null alleles of six open reading frames (ORFs) from chromosome II have been created in Saccharomyces cerevisiae. Deletants were constructed in three genetic backgrounds: FY1679, W303 and CEN.PK2. Tetrad analysis of heterozygous deletants revealed that none of the ORFs is essential for vegetative growth. Basic phenotypic analysis of haploid deletants showed that deletion of the YBR283c ORF causes a slight growth defect at 30°C and 37°C on glycerol‐complete, glucose‐complete, and glucose‐minimal media only in the FY1679 and W303 backgrounds. Transformation of these deletants with the corresponding cognate gene in a centromeric plasmid complements the defects. Deletion of the YBR287w ORF leads to poor growth on glucose‐minimal medium at 15°C in the FY1679 background. None of the six ORFs seems to be involved in mating or sporulation. Copyright © 1999 John Wiley & Sons, Ltd.     

Generation of Saccharomyces cerevisiae deletants and basic phenotypic analysis of eight novel genes from the left arm of chromosome XIV

The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains. © 1998 John Wiley & Sons, Ltd.     

High-Resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; A first step towards an ordered sequencing approach

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.     

Disruption of six novel ORFs from Saccharomyces cerevisiae chromosome VII and phenotypic analysis of the deletants

We describe the disruption and basic phenotypic analysis of six open reading frames (ORFs) of unknown function located in the left arm of Saccharomyces cerevisiae chromosome VII, namely YGL133w, YGL134w, YGL136c, YGL138c, YGL142c and YGL144c. Disruptions were made using the short flanking homology PCR replacement strategy in the FY1679 and CEN.PK2 diploid strains. Sporulation and tetrad analysis of the heterozygous deletants was performed, as well as phenotypic analysis of the corresponding deleted haploid strains. No obvious phenotypes could be attributed to the strains deleted in any of the genes YGL134w, YGL138c and YGL144c under the conditions tested. YGL142c was shown to be an essential gene. Segregants bearing a deletion in YGL136c grew slowly in complete glycerol medium at 37 degrees C. Cells deleted in YGL133w showed abnormal morphology and reduced mating efficiency, but these phenotypes were observed only when the YGL133w disruption was in a MATalpha background. Ygl133 protein was found to localize to the nucleus.     

Disruption and phenotypic analysis of six open reading frames from the left arm of Saccharomyces cerevisiae chromosome VII

Six open reading frames (ORFs) from Saccharomyces cerevisiae chromosome VII were deleted using the kanMX4 module and the long-flanking homology-PCR replacement strategy in at least two different backgrounds. Among these ORFs, two of them (YGL100w and YGL094c) are now known genes which encode well-characterized proteins (Seh1p, a nuclear pore protein, and Pan2p, a component of Pab1p-stimulated poly(A) ribonuclease, respectively). The other four ORFs (YGL101w, YGL099w, YGL098w and YGL096w) code for proteins of unknown function, although the protein encoded by YGL101w has a strong similarity to the hypothetical protein Ybr242p. Gene disruptions were performed in diploid cells using the KanMX4 cassette, and the geneticin (G418)-resistant transformants were checked by PCR. Tetrad analysis of heterozygous deletant strains revealed that YGL098w is an essential gene for vegetative growth in three backgrounds, whereas the other five genes are non-essential, although we have found some phenotypes in one of them. YGL099wDelta strain did not grow at all at 15 degrees C and showed a highly impaired sporulation and a significantly lower mating efficiency. The other three deletants did not reveal any significant differences with respect to their parental strains in our basic phenotypic tests.     

Disruption and phenotypic analysis of seven ORFs from the left arm of chromosome XV of Saccharomyces cerevisiae

We have disrupted seven open reading frames (ORFs) located in the left arm of chromosome XV of the yeast Saccharomyces cerevisiae. These ORFs, previously discovered by our laboratory during the programme of systematic sequencing of the yeast genome, are YOL152w, YOL151w, YOL149w, YOL130w, YOL128c, YOL125w and YOL124c. In most cases, the short flanking homology (SFH) replacement technique has been used. The mutants were analysed for different phenotypic tests. Disruption of YOL130w (also known as ALR1) produced a lethal phenotype, despite the presence of a highly similar gene in the yeast genome (ALR2/YFL050C). Disruption of YOL149w (also known as DCP1, and encoding an mRNA decapping enzyme) results in lethality in the FY1679 background, although it allows slow growth in the CEN.PK141 background. Disruption of the remaining ORFs did not result in readily detectable phenotypic changes.     

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.     

Sequence and analysis of a 33 kb fragment from the right arm of chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a cosmid (pEOA423) from chromosome XV of Saccharomyces cerevisiae. Analysis of the 33,173 bp sequence reveals the presence of 20 putative open reading frames (ORFs). Five of them correspond to previously known genes (MGM1, STE4, CDC44, STE13, RPB8). The previously published nucleotide sequences are in perfect agreement with our sequence except for STE4 and MGM1. In the latter case, 59 amino acids were truncated from the published protein at its N-terminal end due to a frameshift. The putative translation products of six other ORFs exhibit significant homology with protein sequences in public databases: O50 03 and O50 17 products are homologs of the ANC1 and MIP1 proteins of S. cerevisiae, respectively; O50 05 product is similar to that of a protein of unknown function from Myxococcus xanthus; O50 12 product is probably a new ATP/ADP carrier; O50 13 product shows homology with group II tRNA synthetases; and the O50 16 product exhibits strong similarity with the N-terminal domain of the NifU proteins from several prokaryotes. The remaining nine ORFs show no significant similarity. Among these, two contiguous ORFs (O50 19 and O50 20) are very similar to each other, suggesting an ancient tandem duplication. The 33,173 bp sequence has been submitted to EMBL (Accession Number: X92441).