Sequence of the HMR region on chromosome III of Saccharomyces cerevisiae.

A 10,095 base pair DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae has been sequenced and analysed. It encompasses the silent mating-type locus HMR. Both HMRa1 and HMRa2 genes, as well as their flanking regulatory regions, have been identified. Three new open reading frames longer than 80 amino acid residues were found in this fragment. One of them (YCR137) shows features compatible with a membranous localization and a transporter function. The other two do not show a similarity with any known gene. A new gene coding for tRNA(thr)al (ACU) has been identified. It is located in a region coding for several delta sequences.     

Sequence of the sup61-RAD18 region on chromosome III of Saccharomyces cerevisiae.

A 7965 bp DNA segment from the right arm of chromosome III of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them, YCR103, is 51% homologous with the G10 gene product of Xenopus laevis.     

Linguistic analysis of chromosome III DNA sequence of Saccharomyces cerevisiae

The analysis of the Saccharomyces cerevisiae chromosome III DNA sequence by computer (‘in silico’) permits the definition of its linguistic characteristics. These characteristics include the designation of non-randomly occurring oligonucleotides, their distribution along the chromosome, and the distribution of some particular homopolymers. All these elements may contribute to the understanding of the organization of information on the chromosome.     

A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditons. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.     

III. Yeast sequencing reports. Corrected sequence for the right telomere of Saccharomyces cerevisiae chromosome III

A comparison of the sequences of telomere regions from several yeast chromosomes revealed an apparent cloning artifact for the right end of chromosome III. An integrating vector containing G_1–3T telomere sequences was used to clone the right end of chromosome III from a strain related to S288C. The sequence of this clone confirmed that the published sequence was incorrect and demonstrated that the right telomere region of chromosome III is similar to other telomeres.     

Analysis of the MSS51 region on chromosome XII of Saccharomyces cerevisiae.

We have localized gene MSS51 on chromosome XII of Saccharomyces cerevisiae between the RDN1 and CDC42 loci. 'Head to head' with MSS51 is another gene, QRI5, the function of which is unknown. However, the proximity of these genes, the structure of the intergenic region and the presence of an ABF1 binding site right in the middle of this region suggest that the MSS51 and QRI5 expressions are submitted to a common regulatory process.     

Differential chromosome control of ploidy in the yeast Saccharomyces cerevisiae

In Saccharomyces cerevisiae, aneuploidy is well tolerated and stable. We analysed whether the induced loss of a disomic chromosome favours endo-reduplication of the remaining chromosome or the cells prefer to retain the acquired euploidy. Chromosome VIII disomes and trisomes were tagged with GFP (green fluorescent protein), DsRed (red fluorescent protein) and BFP (blue fluorescent protein) integrated at the thr1 locus, using our newly designed STIK (specific targeted integration of kanamycin resistance-associated, non-selectable DNA) plasmid system. A knockout cassette for centromere 8 was constructed with the hygromycin-B marker, which was transformed into the strains. The transformants lost sensitivity to hygromycin, thereby indicating the event of centromere replacement. Quantitative PCR and Southern analysis were performed for chromosome VIII copy number determination by probing the markers located on both the right (ARG4 and THR1) and left (GUT1) arm whereas, for chromosome V, markers such as HIS1, located on right arm, and URA3, on left arm, were used. The loss of an extranumerary chromosome VIII in a disome and trisome leads to stable euploidy. Furthermore, in a wild-type diploid, deletion of a copy of chromosome VIII, leads to monosomy, and restoration of euploidy after 22 generations, by reduplication of chromosome VIII, and consequent loss of heterozygosis (LOH). However, chromosome V knockouts in chromosome VIII trisome, still showed LOH and duplication of chromosome V, with return to the original aneuploid condition. These results suggest that yeast cells could control the integrity of their genetic complement acting at the individual chromosome level.     

The complete sequence of a 10.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae.

The complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarity with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculiar phenotype was observed.     

A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF.

In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S. servazzii. We find that the order of four genes surrounding the RE in S. cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii. However, there is an additional ORF in S. servazzii between PRD1 and KAR4 that is not homologous to any gene in S. cerevisiae or to genes in other organisms. Despite a 75-79% amino acid identity for KAR4 and SPB1, respectively, the S. servazzii sequence did not carry a well-conserved RE sequence and these sequences lacked RE function when introduced into S. cerevisiae. The S. servazzii region contains a sequence that supports autonomous DNA replication in S. cerevisiae and may represent a homologue of ARS304. The S. servazziii sequence has Genbank Accession No. BankIt359091 AF307954.     

A new essential gene located on Saccharomyces cerevisiae chromosome IX

A new 1150 amino acids long open reading frame (ORF), coding for an essential protein of unknown function was found Saccharomyces cerevisiae by sequencing 3754 bp of geonomic DNA. The clone was isolated in a search for a fatty acid-binding protein (FABP) and was localized on chromosome IX. The ORF bears no homology to FABP, but it shows weak similarity to Plasmodium vivax reticulocyte binding protein 1 and to aggregation-specific adenylate cyclase from Dictyostelium discoideum. The new gene is constitutively transcribed regardless of the carbon source used. The nucleotide sequence reported in this paper has been deposited in GenBank (Accession Number U17918).     

Large-scale phenotypic analysis—the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. © 1997 John Wiley & Sons, Ltd.     

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374–376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33·4%, 26·7%, 23·4% and 29·2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68·4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L37111.     

Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae

A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.     

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).