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 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.     

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.     

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.     

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).     

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.     

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).     

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.     

The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: mapping by chromosome engineering.

The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere. In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site-specific recombination system of the yeast plasmid pSR1.     

The sequence of a 24 152 bp segment from the left arm of chromosome XIV from Saccharomyces cerevisiae between the BNI1 and the POL2 genes

In the framework of the European Union programme for sequencing the genome of Saccharomyces cerevisiae we have determined the nucleotide sequence of a region of 24 152 bp located on the left arm of chromosome XIV between the BNI1 and the POL2 genes. The sequence was obtained by directed sequence analysis using a mixture of ExoIII and primer walking strategies. Subsequent analysis revealed 13 open reading frames (ORFs) including four small ORFs completely internal to, or partly overlapping with, other ORFs. Five of these ORFs have been described previously (BNI1, APL1, LYP1, PIK1, POL2) and thus 74·8% of the 24 152 bp were already present in the databases prior to this sequencing effort. Interestingly, all 13 identified ORFs are characterized by a low codon adaptation index (0·04–0·22). In addition, this region of chromosome XIV shows an unusually high gene density with about 88% of coding DNA. This amounts to one gene per 2177 bp, which is significantly above the average gene length (about 1500 bp). For eight ORFs considerable homologies to ‘Expressed Sequence Tags’ derived from human cDNAs located in the XREF database could be identified. The complete nucleotide sequence of the 24 152 bp segment has been deposited in the EMBL data library under the Accession Number X92494.     

Disruption and basic functional analysis of five chromosome X novel ORFs of Saccharomyces cerevisiae reveals YJL125c as an essential gene for vegetative growth

We describe the disruption and basic functional analysis of five novel open reading frames (ORFs) discovered during the sequencing of the Saccharomyces cerevisiae genome: YJL118w, YJL122w, YJL123c, YJL124c, YJL125c, located on chromosome X. Disruptions have been realized using the long-flanking homology-PCR replacement strategy (LFH-PCR; Wach et al., 1996) in the FY1679 diploid strain. Sporulation and tetrad analysis of these heterozygous deletants were performed, as well as a functional analysis on the haploid deleted strains: different growth conditions (complete glucose and glycerol, minimal media) at three temperatures 15, 30 and 37 degrees C were tested. Analysis revealed YJL125c as an essential gene; the four other ORFs were non-essential and showed no particular phenotype. In addition, the five kanMX4 disruption cassettes were cloned in pUG7 vector. Finally, the five ORFs with their promoter and terminator regions were cloned in the centromeric yeast vector pRS416. The vectors containing the disruption cassettes, the cognate wild-type genes, as well as the deletant strains are available at the EU EUROFAN (EUROSCARF, Frankfurt, DE) genetic and stock centre.     

Sequence and analysis of a 26·9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048. The analysis of the 26 857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A). Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work. Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable). O5248 is a probable folylpolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome. O5273 shows homology with a yeast protein required for vanadate resistance. O5268 shows homology with putative oxidoreductases of different organisms. O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast. The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function. The present sequence corresponds to coordinates 772 331 to 799 187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp. mips. embnet. org).     

XV. Yeast sequencing reports. Sequence of a 10·27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene

A 10 270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size. The sequence has been deposited in the EMBL data library under Accession Number X75449.     

Analysis of a 26 kb region on the left arm of yeast chromosome XV

In the framework of the EC programme for sequencing yeast chromosome XV, we have determined the nucleotide sequence of a 26 kb region. Subsequent analysis revealed 13 non-overlapping open reading frames, three of which correspond to known yeast genes. A pair of tRNA genes associated with remnant Ty elements were localized in this region. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 2 kb. Protein coding regions occupy 61% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes. The complete nucleotide sequence of the 26 kb segment as depicted in Figure 1 has been deposited at the EBI data library under Accession Number X91067.