Analysis of DNA sequences homologous with the ARS core consensus in Saccharomyces cerevisiae

We have previously identified an autonomously replicating segment (ARS) near the 3' end of the histone H4 gene at the copy-I H3-H4 locus. We have now searched for additional autonomously replicating segments and sequences homologous with the ARS core consensus sequence near the copy-II histone H4 gene and both of the histone H3 genes. No new ARS elements were identified by functional cloning assays. However, several matches to the ARS core consensus element were found within the DNA sequences of the copy-I and copy-II genes. An exact match to the ARS core consensus was identified in the region downstream from the copy-I histone H3 gene and a set of sequences with weak homology was also located within the copy-II region. However, restriction fragments including these sequences did not demonstrate ARS activity on a plasmid in transformed cells.     

A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains. We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins.     

Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae

Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast.     

Kluyveromyces marxianus small DNA fragments contain both autonomous replicative and centromeric elements that also function in Kluyveromyces lactis

Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences. Centromeric sequences are organized in a similar way to those of budding yeasts: two well-conserved elements: CDEI (5′ TCACGTG 3′) and CDEIII (5′ TNTTCCGAAAGTWAAA 3′), are separated by a 165 bp AT-rich (± 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis. The ARS-core consensus sequence (5′ TTTATTGTT 3′) is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae. A third ARS-containing fragment with a different organization has been isolated and sequenced. The nucleotide sequences of DNA fragments reported in this paper will appear in the EMB data library under the accession numbers: Z31562, Z31563, Z31564.     

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis

The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.     

Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.     

Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae

A detailed characterization of the mak1-3 mutation of Saccharomyces cerevisiae has been made possible by modifying its genetic background. The mak1-3 mutation, which confers temperature sensitivity for growth, was originally identified as one of four mak1 mutations (Wickner and Leibowitz, 1976). Mak1-1, 1-2 and 1-4 mutants are deficient in DNA topoisomerase I activity and thus have been renamed 'top1' (Thrash et al., 1984). Studies presented here show that the map position of MAK1-3 on chromosome XVI distinguishes it from TOP1 which maps on chromosome XV (Wickner and Leibowitz, 1976). An investigation of in vivo macromolecular synthesis in the mak1-3 mutant shows that it is deficient in DNA replication at the restrictive temperature. Experiments in which DNA synthesis was measured in synchronized cell populations indicate that the mak1-3 mutant is deficient in the initiation step of DNA synthesis. Furthermore, crude extracts from the mak1-3 mutant cells support temperature-sensitive in vitro DNA synthesis on yeast chromosomal DNA replication origin containing plasmid pARS1, suggesting that the MAK1 gene product is directly required for in vitro DNA replication. The conclusion that mak1-3 is a newly identified DNA replication mutation is based on the observations that it (1) complements all DNA synthesis mutants examined, (2) maps to a previously undetected chromosomal location and (3) has a distinct terminal morphology. In light of these distinctions and of the role mak1-3 plays in DNA replication, it has been renamed 'dna1'.     

A novel structural form of the 2 micron plasmid of the yeast Saccharomyces cerevisiae

DNA was isolated from cells of Saccharomyces cerevisiae incubated under conditions that enriched for DNA replication intermediates. A novel form of the 2 μm plasmid was detected, in which two monomeric or dimeric circles were joined by a linear double-stranded segment of variable length. We suggest that this molecule is a consequence of site-specific recombination within a dimeric DNA molecule during DNA replication. The existence of this molecule provides supporting physical evidence for a variant of the model of 2 μ plasmid amplification first proposed by Futcher (1986).     

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19‐1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19‐1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. The GenBank Accession Nos of the sequences are AF255332 (K.lactis GDI1) and AY007574 (P. pastoris GDI1). Copyright © 2001 John Wiley & Sons, Ltd.     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe

We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations.     

Isolation of GCR1, a major transcription factor of glycolytic genes in Saccharomyces cerevisiae, from Kluyveromyces lactis

To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391.     

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.     

KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity.     

Factors affecting chemical‐based purification of DNA from Saccharomyces cerevisiae

Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical‐based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10⁸ cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Sequences of Saccharomyces cerevisiae 2 μm DNA improving plasmid partitioning in Hansenula polymorpha

Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24–28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region. © 1998 John Wiley & Sons, Ltd.