Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up- and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 48K) could represent ~10,000 up-/down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58℃ for both tags. Intriguingly, up-tags required 3× higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25℃) was optimal for cultivation instead of a normal temperature (30℃) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.
Oligonucleotides ; Polymerase Chain Reaction ; Schizosaccharomyces ; Yeasts

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.
DNA* ; Gene Deletion* ; Mutation Rate ; Open Reading Frames ; Polymerase Chain Reaction ; Saccharomycetales ; Schizosaccharomyces*

The cation-dependent galactose-specific flocculation activity of the Schizosaccharomyces pombe null mutant of lkh1⁺, the gene encoding LAMMER kinase homolog, has previously been reported by our group. Here, we show that disruption of prk1⁺, another flocculation associated regulatory kinase encoding gene, also resulted in cation-dependent galactose-specific flocculation. Deletion of prk1 increased the flocculation phenotype of the lkh1⁺ null mutant and its overexpression reversed the flocculation of cells caused by lkh1 deletion. Transcript levels of prk1⁺ were also decreased by lkh1⁺ deletion. Cumulatively, these results indicate that Lkh1 is one of the negative regulators acting upstream of Prk1, regulating non-sexual flocculation in fission yeast.
Flocculation* ; Phenotype ; Phosphotransferases* ; Schizosaccharomyces*

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without alpha-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an alpha-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70degrees C for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no alpha-crystalline domain in their sequences.
alpha-Crystallins ; Citrate (si)-Synthase ; Electrophoresis ; Heat-Shock Proteins, Small* ; Mass Screening* ; Mass Spectrometry ; Molecular Chaperones* ; Pyridoxine ; Recombinant Proteins ; Schizosaccharomyces*

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.
Cdc20 Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Centromere ; metabolism ; Chromatin ; metabolism ; Chromosomal Proteins, Non-Histone ; metabolism ; DNA Replication ; DNA, Fungal ; metabolism ; Epigenesis, Genetic ; Histones ; metabolism ; Schizosaccharomyces ; genetics ; metabolism ; Schizosaccharomyces pombe Proteins ; antagonists & inhibitors ; genetics ; metabolism

The flocculating yeast strain SPSC01 is a fusant strain of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The use of SPSC01 to absorb Cr(VI) from Cr(VI) containing aqueous solution would greatly reduce the cost of post-adsorption separation, since the superior flocculating property of SPSC01 would allow easy separation of the Cr(VI)-biomass from the solution. In order to investigate the effects of flocculating proteins on Cr(VI) reduction and absorption by SPSC01, the absorption behaviors of SPSC01 and its parental strains were compared. The results showed that Cr(VI) removal rate of SPSC01 was almost the same as that of S. pombe, which also has flocculating ability, but was faster than that of S. cerevisiae, which has no flocculating ability. When the system reached equilibrium, the amount of total Cr adsorbed by S. pombe, SPSC01 and S. cerevisiae were 68.8%, 48.6% and 37.5%, respectively. This showed that flocculation was beneficial to Cr(VI) reduction and adsorption, and suggested that focculating proteins may play a role in enhancing the Cr(VI) adsorption capacity of SPSC01 and S. pombe. We investigated the mechanism of Cr(VI) adsorption by SPSC01 using chemical modification and FTIR. The results indicated that the major functional groups (amino, carboxyl and amide) of surface proteins may contribute to the absorption of Cr(VI).
Adsorption ; Biodegradation, Environmental ; Chromium ; isolation & purification ; Flocculation ; Saccharomyces cerevisiae ; metabolism ; Schizosaccharomyces ; metabolism ; Surface Properties ; Water Pollutants, Chemical ; isolation & purification

This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.
Bioreactors ; microbiology ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Glycerol ; metabolism ; Glycolysis ; Hexokinase ; metabolism ; Industrial Microbiology ; methods ; Phosphofructokinase-1 ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; metabolism ; Schizosaccharomyces ; enzymology ; genetics ; metabolism ; Trehalose ; metabolism ; Triticum ; metabolism ; Zea mays ; metabolism

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1 + null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, beta-glucosidase (Psu1), cell surface protein, glucan 1,3-beta-glucosidase (Bgl2), and exo-1,3 beta-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.
Acrylic Resins ; beta-Glucosidase ; Cell Wall ; Databases, Protein ; Electrophoresis, Polyacrylamide Gel ; Glucan 1,3-beta-Glucosidase ; Phosphotransferases ; Proteins ; Proteome ; Schizosaccharomyces ; Tandem Mass Spectrometry

OBJECTIVEGoal of this study was to test the potential regulatory effects of Vpr on Vif and Vif-mediated degradation of APOBEC3G.

METHODSThe Vpr effect was first tested in a fission yeast RE007 strain that carries a single integrated copy of vpr gene in the chromosome and transformed with a vif-expressing plasmid. Similar tests were also carried out in a muristerone A vpr-inducing HEK293 mammalian cell line that were transfected with the plasmids expressing vif and/or APOBEC3G. Western Blot analyses were used to measure the corresponding protein levels under different experimental conditions.

RESULTSExpression of HIV-1 vpr appears to enhance the protein levels of Vif both in fission yeast and mammalian cells. A similar enhancement effect of APOBEC3G by Vpr was also detected in mammalian cells. Interestingly, however, the increased Vif protein level by Vpr did not result in more APOBEC3G degradation than without Vpr, indicating a potential regulatory effect of Vpr on Vif-mediated proteolysis of APOBEC3G.

CONCLUSIONTo our knowledge, this is the first report describing a potentially conserved and regulatory effect of HIV-1 Vpr on Vif and Vif-mediated protein degradation of APOBEC3G.

APOBEC-3G Deaminase ; Animals ; Cell Line ; Cytidine Deaminase ; biosynthesis ; metabolism ; Gene Expression ; Gene Products, vif ; biosynthesis ; metabolism ; Gene Products, vpr ; metabolism ; HIV-1 ; Humans ; Schizosaccharomyces ; genetics

One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glycosylation ; Hydrogen-Ion Concentration ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; biosynthesis ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Schizosaccharomyces ; enzymology ; genetics ; Temperature