DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae

A 6·8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22⁺ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5′ end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl-carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNA^Leu (UAA) isoacceptor located near a delta sequence.     

Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe,which encodes a putative phosphoinositide-specific phospholipase C

Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe. Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa. This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca²⁺-binding site (an E-F hand motif). The structure of the putative protein is most similar to that of the δ class of PLC isozymes. To investigate the role of this gene, designated plc1⁺, gene disruption was carried out by interrupting the coding region with the ura4⁺ marker. Growth of plc1 cells was temperature-sensitive in rich medium, and cells could not grow in synthetic medium. Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1^? cells, a strong suggestion that the plc1⁺ gene encodes PLC. The PLC1 sequence appears in the public data libraries, DDBJ GenBank, EMBL under the following Accession Number: D38309.     

Nucleotide sequence of the Schizosaccharomyces pombe lys1 + Gene and Similarities of the lys1+ protein to peptide antibiotic synthetases

The 4·2 kbp lys1⁺ gene of Schizosaccharomyces pombe encoding the large subunit of α-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H. The S. pombe lys1⁺ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155·8 kDa. The encoded protein converts α-aminoadipic acid to α-aminoadipate-δ-semialdehyde by an ATP-mediated adenylation. Analysis of the sequence showed that the putative protein encoded by lys1⁺ shares strong homology with the peptide antibiotic synthetases which also use an adenylation step. The sequence data reported in this paper have been submitted to GenBank database (Washington DC, USA) under the Accession Number U15923. © 1998 John Wiley & Sons, Ltd.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.     

Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.     

New cassettes for single‐step drug resistance and prototrophic marker switching in fission yeast

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein–protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4⁺ gene or the G418‐resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single‐step exchange protocols of markers are a time‐saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single‐step marker swapping by homologous gene targeting. To expand this very useful toolset for single‐step marker exchange, I constructed MX cassettes containing the nutritional markers arg3⁺, his3⁺, leu1⁺ and ura4⁺. Furthermore, a set of constructs was created to enable single‐step exchange of ura4⁺ to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single‐step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4⁺‐ and MX‐deleted and ‐tagged strains. Copyright © 2015 John Wiley & Sons, Ltd.     

A fission yeast gene encoding a protein that preferentially associates with curved DNA

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42 000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1·9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41 597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.     

RESTRICTION MAPPING OF THE POF I GENE

The gene (POFI) which imparts to certain yeasts the ability to decarboxylate phenolic acids to corresponding phenolic compounds has been analysed by restriction mapping. New restriction sites have been used to examine differences between Pof⁺ and Pof^? Saccharomyces cerevisiae strains. Southern Blot analysis of selected yeast strains has demonstrated that the POFI gene sequence is highly conserved between the Pof⁺ strain from which the gene was cloned, two Pof^? lager brewing strains and one Pof⁺ Saccharomyces brewery isolate. However, sequence differences have been found between the original Pof⁺ strain, a Pof^?laboratory strain and a Pof^? ale brewing strain.     

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4⁺ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.     

IV. Yeast sequencing reports. GUF1, a gene encoding a novel evolutionarily conserved gtpase in budding yeast

While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a gene we call GUF1 (G TPase of U nknown F unction), which predicts a 586 amino acid GTPase of the elongation factor-type class. The predicted Guf1p protein bears striking sequence similarity to both LepA from Escherichia coli (43% identical) and LK1236·1 from Caenorhabditis elegans (42% identical). Analysis of both a guf1δ deletion and a putative constitutive-activating mutant (GUF1HG) revealed that GUF1 is not essential nor did mutant cells reveal any marked phenotype. The GenBank Accession Number for the GUF1 sequence is U22360.     

Discovery of genes involved in mitosis,cell division,cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini‐chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non‐essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.     

An 18·3 kb DNA Fragment from Yeast Chromosome VII Carries Four Unknown Open Reading Frames,the Gene for an Asn Synthetase,Remnants of Ty and Three tRNA Genes

An 18·3 kb DNA segment from yeast Saccharomyces cerevisiae chromosome VII encompasses the previously characterized MEP1, NUP57 and PPT1 genes as well as seven new open reading frames (ORFs) of at least 100 residues. G6358 is an ubiquitous glutamine-dependant asparagine synthase. G6362 is a membrane protein highly homologous to a protein of unknown function in the yeast Schizosaccharomyces pombe. Three ORFs (G6324, G6335 and G6365) have no significant homology with previously reported proteins or characteristic motifs. G6321 and G6359, enclosed in longer ORFs, are not likely to be coding. The segment also contains tRNA genes for Asn, Arg and Ile as well as a sigma element and two solo deltas. ORFs and genetic elements are named according to a preliminary working nomenclature. The sequence is recorded in GenBank^TM/EMBL under Accession Number X83099.©1997 John Wiley & Sons, Ltd.     

Schizosaccharomyces pombe reporter strains for relative quantitative assessment of heterochromatin silencing

The fission yeast Schizosaccharomyces pombe has been emerging as an important model organism for studying the formation and repression of heterochromatin. To enable simple and relative quantitative assessment of heterochromatin silencing, we have created bioluminescence‐based reporter strains. A green‐emitting click beetle luciferase (CBG68) gene was inserted within pericentromeric heterochromatin or at the silent mating‐type locus via homologous recombination. In the same strains, a red‐emitting click beetle luciferase (CBR) gene is expressed from the euchromatic leu1⁺ locus and can be used as a reference in dual‐colour assays. Our reporter strains are suitable for performing Chroma‐Glo? assays, which can be carried out directly in the culture medium without prior cell lysis and in a multiwell format. Our reporter system reliably reflects the state of chromatin and can be easily adapted for use in high‐throughput screening approaches. Copyright © 2012 John Wiley & Sons, Ltd.