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 and basic phenotypic analysis of six novel genes from the right arm of chromosome XII of Saccharomyces cerevisiae

Six open reading frames (ORFs) of unknown function from the right arm of Saccharomyces cerevisiae chromosome XII were deleted in two genetic backgrounds by disruption cassettes with regions of short flanking homology. This work was carried out within the framework of the EUROFAN consortium. The SFH disruption cassettes, obtained by PCR, were made by amplification of the kanMX marker module with oligonucleotides containing approximately 40 bp of homology to either the promoter or translation terminator regions of the relevant ORF. Transformants resistant to geneticin (G418) were selected. The SFH disruption cassettes were cloned into a bacterial vector. Each cognate gene was also cloned into a yeast centromeric plasmid. Sporulation and tetrad analysis of the disrupted heterozygous strains revealed that ORF YLR153c (now known as ACS2) is essential. Basic phenotypic analysis was performed on haploid deletants of both mating types of the five non-essential ORFs, YLR082c (now known as SRL2), YLR149c, YLR151c, YLR152c and YLR154c. Plate growth tests on different media at 15 degrees C, 30 degrees C and 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.     

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.     

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 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.     

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.     

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 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.     

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 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.     

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.     

Inactivation of six genes from chromosomes VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains

Within the frame of the EUROFAN project, aimed at the functional analysis of the novel ORFs revealed by the systematic sequencing of the Saccharomyces cerevisiae genome, we have inactivated six ORFs encoding putative proteins with unknown function in the two S. cerevisiae strains FY1679 and W303-1B. Five ORFs are located on chromosome VII (YGR250c, YGR251w, YGR260w, YGR262c, YGR263c) and one on chromosome XIV (YNL234w). The genes have been inactivated in the FY1679 strain by a strategy that makes use of deletion cassettes containing the kanMX4 module, which confers resistance to geneticin to yeast cells, and short flanking regions homologous to the target locus (SFH). Tetrad dissection of heterozygous mutants and basic phenotypic analysis of the spores revealed that ORF YGR251w is an essential gene, while the disruption of YGR262c causes a severe slow-growth phenotype. Deletion of the remaining ORFs did not give rise to a detectable phenotype in the mutant strains. For each ORF we have cloned, in the pUG7 plasmid, a replacement cassette that possesses long flanking regions homologous to the target locus (LFH) and, in the pRS416 plasmid, the cognate wild-type gene. The LFH replacement cassettes were used to inactivate the respective genes in the W303-1B strain. This work has been performed in the framework of the B0 Consortium of the EUROFAN I project.     

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.     

Disruption of six novel genes from chromosome VII of Saccharomyces cerevisiae reveals one essential gene and one gene which affects the growth rate

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 flanked by short homology regions, whereas ORF YGR255c was inactivated with a long flanking homology (LFH) disruption cassette (Wach et al., 1994). Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identified as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cDelta heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was confirmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al., 1999).     

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).     

Gene disruption and basic phenotypic analysis of nine novel yeast genes from chromosome XIV

In this work, we describe the disruption of nine ORFs of S. cerevisiae (YNL123w, YNL119w, YNL115c, YNL108c, YNL110c, YNL124w, YNL233w, YNL232w and YNL231c) in two genetic backgrounds: FY1679 and CEN.PK2. For the construction of the deletant strains, we used the strategy of short flanking homology (SFH) PCR. The SFH-deletion cassette was made by PCR amplification of the KanMX4 module with primers containing a 5' region of 40 bases homologous to the target yeast gene and with a 3' region of 20 bases homologous to pFA6a-KanMX4 MCS. Sporulation and tetrad analysis of heterozygous deletants revealed that YNL110c, YNL124w and YNL232w are essential genes. The subcellular localization of the protein encoded by the essential gene YNL110c was investigated using the green fluorescent protein (GFP) approach, revealing a nuclear pattern. Basic phenotypic analysis of the non-essential genes revealed that the growth of ynl119w delta haploid cells was severely affected at 37 degrees C in N3 medium, indicating that this gene is required at high temperatures with glycerol as a non-fermentable substrate. The ynl233w delta haploid cells also showed a particular phenotype under light microscopy and were studied in detail in a separate work.     

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.     

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.