High-frequency cotransformation by copolymerization of plasmids in the fission yeast Schizosaccharomyces pombe.

We have developed a high-frequency cotransformation system which is useful in introducing nonreplicating circular DNA plasmids into the fission yeast Schizosaccharomyces pombe. This system depends on two factors: the ability of the ural-complementing helper plasmids pFYM2 and pFYM225 to propagate autonomously in S. pombe, and the intensive recombination activity intrinsic to this yeast. If cotransformed with a helper plasmid, plasmids such as YIp5 or YIp32, Escherichia coli-Saccharomyces cerevisiae shuttle vectors incapable of replication in S. pombe, can enter S. pombe and express the gene carried on them at a frequency comparable to that of autonomously replicating plasmids (10(3) to 10(4) transformants per microgram of DNA). Even if characters of the nonreplicating DNA are not selected directly, 50 to 70% of Ura+ cells transformed with the helper have also incorporated the nonreplicating plasmid. It is shown that these two plasmids have physically recombined at a site of common DNA sequence to form a heteropolymer in the fission yeast. Since any foreign DNA cloned in pBR322 or ColE1 derivatives can be incorporated into S. pombe by using pFYM2 or pFYM225 as a helper, this cotransformation system will serve as a convenient method to examine functional expression of such cloned DNA in S. pombe. This work also demonstrates that the kanamycin resistance gene carried by the bacterial transposon Tn903 can be expressed in S. pombe, as shown by its ability to inactivate the antibiotic G418.     

A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a popular model organism to study various cellular processes, although research tools available for S. pombe are relatively inadequate. To facilitate genetic and biochemical investigation in S. pombe, we report here a system of vectors for genomic FLAG epitope-tagging. These vectors enable us to amplify gene-targeting fragments for integration into specific loci of the S. pombe genome. All vectors in this report were designed to express FLAG epitope-tagged proteins from their endogenous genomic loci. Vectors for N-terminal FLAG epitope-tagging allow us to control protein expression levels using the wild-type nmt1 promoter, its weaker derivatives, and the urg1 promoter. These vectors are available with various antibiotic markers including kanMX6, hphMX6, natMX6 and bleMX6, and the his3(+) marker. Vectors for C-terminal FLAG epitope-tagging were designed to express FLAG-fusion proteins under the control of their native promoters at their own genomic loci, allowing us to characterize protein functions under physiological conditions. These vectors are available with kanMX6, hphMX6, nat-MX6 and bleMX6 markers. The series of vectors described in this report should prove useful for protein studies in fission yeast.     

Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase

Schizosaccharomyces pombe has an open reading frame, which we named alr1(+), encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1(+) gene in Escherichia coli and purified the gene product (Alr1p), with an M(r) of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K(m) and V(max) values as follows: for L-alanine, 5.0 mM and 670 micromol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 micromol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1(+) gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1(+) gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.     

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.     

Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism

We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.     

Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae.

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14. 5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae. Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits. We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly. We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.     

Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.     

Characterization of the rhp7(+) and rhp16(+) genes in Schizosaccharomyces pombe.

The global genome repair (GGR) subpathway of nucleotide excision repair (NER) is capable of removing lesions throughout the genome. In Saccharomyces cerevisiae the RAD7 and RAD16 genes are essential for GGR. Here we identify rhp7 (+), the RAD7 homolog in Schizosaccharomyces pombe. Surprisingly, rhp7 (+)and the previously cloned rhp16 (+)are located very close together and are transcribed in opposite directions. Upon UV irradiation both genes are induced, reaching a maximum level after 45-60 min. These observations suggest that the genes are co-regulated. Schizo-saccharomyces pombe rhp7 or rhp16 deficient cells are, in contrast to S.cerevisiae rad7 and rad16 mutants, not sensitive to UV irradiation. In S.pombe an alternative repair mechanism, UV damage repair (UVDR), is capable of efficiently removing photolesions from DNA. In the absence of this UVDR pathway both rhp7 and rhp16 deficient cells display an enhanced UV sensitivity. Epistatic analyses show that rhp7 (+)and rhp16 (+)are only involved in NER. Repair analyses at nucleotide resolution demonstrate that both Rhp7 and Rhp16, probably acting in a complex, are essential for GGR in S.pombe.     

A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe

For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.     

Homologous mRNA 3'' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.     

Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe.

We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe. These can be used for both gene bank construction and subcloning. Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc. pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts. These vectors have been used to construct representative Sc. pombe and Saccharomyces cerevisiae genomic libraries. To assist in the creation of gene deletions, we have constructed another two plasmids. Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene. Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc. pombe transformations. When integrated into the Sc. pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site.     

Detection of a conformational change in G gamma upon binding G beta in living cells

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N-linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1(+) gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated alpha-1,6-linked mannose into pyridylaminated Man(9)GlcNAc(2), indicating that och1(+) encodes an alpha-1,6-mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.     

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S. cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T. reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.     

Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae

We have constructed a variety of vectors for use in both budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). Four of these, pDB262, pWH4, pWH5, and pMAK262, have positive selection for the insertion of cloned DNA, making them convenient for the construction of gene banks. pDB262, pWH4 and pWH5 contain the 2 mu ARS and the LEU2 gene from S. cerevisiae and can be used for gene isolation. They can also be converted into integration vectors for use in the genetic mapping of cloned sequences. pMAK262 contains only the LEU2 gene from budding yeast and can be used to screen for ARS elements or for gene integration. We also describe two other integration vectors, pDAM3 and pDAM6, which have a variety of restriction sites suitable for subcloning.     

The nucleotide sequence of the major glutamate transfer RNA from Schizosaccharomyces pombe.

The nucleotide sequence of glutamate tRNA1 from Schizosaccharomyces pombe was determined to be pU-C-C-G-U-U-G-U-m1G-G-U-C-C-A-A-C-G-G-C-D-A-G-G-A-U-U-C-G-U-C-G-C-U-U-U*-C-A-C-C-G-A-C-G-G-G-A-G-m5C-G-G-G-G-T-psi-C-G-A-C-U-C-C-C-C-G-C-A-A-C-G-G-A-G-C-C-AOH. The sequence differs markedly from that of S. cerevisiae tRNAGlu. S. pombe glutamate tRNA1 can be aminoacylated by the homologous glutaminyl-tRNA synthetase as well as by the corresponding enzyme from S. cerevisiae.     

Saccharomyces cerevisiae--a model to uncover molecular mechanisms for yeast biofilm biology

Microbial biofilms can be defined as multi-cellular aggregates adhering to a surface and embedded in an extracellular matrix (ECM). The nonpathogenic yeast, Saccharomyces cerevisiae, follows the common traits of microbial biofilms with cell-cell and cell-surface adhesion. S.?cerevisiae is shown to produce an ECM and respond to quorum sensing, and multi-cellular aggregates have lowered susceptibility to antifungals. Adhesion is mediated by a family of cell surface proteins of which Flo11 has been shown to be essential for biofilm development. FLO11 expression is regulated via a number of regulatory pathways including the protein kinase A and a mitogen-activated protein kinase pathway. Advanced genetic tools and resources have been developed for S.?cerevisiae including a deletion mutant-strain collection in a biofilm-forming strain background and GFP-fusion protein collections. Furthermore, S.?cerevisiae biofilm is well applied for confocal laser scanning microscopy and fluorophore tagging of proteins, DNA and RNA. These techniques can be used to uncover the molecular mechanisms for biofilm development, drug resistance and for the study of molecular interactions, cell response to environmental cues, cell-to-cell variation and niches in S.?cerevisiae biofilm. Being closely related to Candida species, S.?cerevisiae is a model to investigate biofilms of pathogenic yeast.