• Journal of Life Science
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• v.11 no.1
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• pp.52-56
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• 2001

The present study intends to characterize the DNA damage-inducible responses in eukaryotic cells. The fission yeast, S. pombe, which displays efficient DNA repair systems, was used in this study as a model system for higher eukaryotes. To study UV-inducible responses in S. pombe, five UV-inducible cDNA clones were isolated from S. pombe by using subtration hybridization method. To investigate the expression of isolated genes, the cellular levels of the transcripts of these genes were determined by Northern blot analysis after UV-irradiation. The transcripts of isolated gene (UV130) increased rapidly and reached maximum accumulation after UV-irradiation. Compared to the message levels of control, the levels of maximal increase were approximately 5 fold to UV-irradiation. In order to investigation whether the increase of UV130 transcripts was a specific results of UV-irradiation, UV130 transcript levels were examined after treating the cells to Methylmethane sulfonate (MMS). The transcripts of UV130 were not induced by treatment of 0.25% MMS. These results implied that the effects of damaging agents are complex and different regulatory pathways exist for the induction of these genes. To characterize the structure of UV130 gene, nucleotide sequences were analyzed. The nucleotide sequence of 1,340 nucleotide excluding poly(A) tail contains one open reading frame, which encodes a protein of 270 amino acids. The predicted amino acid sequences of UV130 do not exhibit any significant similarity to ther known sequences in the database.

• BMB Reports
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• v.34 no.5
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• pp.395-401
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• 2001

Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using $\beta$-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.

• Molecules and Cells
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• v.22 no.1
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• pp.113-118
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• 2006

Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1499-1512
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• 2006

Based on the first 2D reference map of the fission yeast Schizosaccharomyces pombe protein reported previously, we expanded and updated the map using narrower pI ranges. In this paper, 240 protein spots were identified on our reference map. In the pI 4-7 range, 144 spots corresponding to 86 different proteins were identified. In the pI 6-9 range, 43 spots corresponding to 35 different proteins were identified. Fifty-three new spots corresponding to 39 different proteins were further identified in the pI 5-6 range.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.317-320
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• 2003

Genome-wide systematic deletion mutants were generated using a PCR-based targeted mutagenesis of Schizosacchaaromyces pombe. In a drug-sensitivity assay using thiabendazole(TBZ), an inhibitor of microtubule assembly, a heterozygous nda2 mutant ($nda2^+/nda2^-$), deleting one copy of nda2 encoding the microtubule subunit alpha1 demonstrated a distinct sensitivity to TBZ, indicating TBZ-induced haploinsufficiency. This result suggests that profiling drug-induced haploinsufficiency can be exploited to identify target genes for drugs and discover new drugs.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.1
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• pp.46-54
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• 1997

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by malting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB (spo 5)1. We futher analyzed six recombinant plasmids, pDB (spo 5)2, pDB(spo 5)3, pDB(spo 5)4, pDB(spo 5)5, pDB(spo 5)6, pDB(spo 5)7, and found each plasmids is able to rescue the spo 5-2, spo 5-3, spo 5-4, spo 5-5, spo 5-6, spo 5-7, mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB (spo 5)1, and pDB (spo 5)R1 contained the spo 5 gene. Transcipts of spo 5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 25 kb were detected with 5 kb Hind III fragment containing a part of the spo 5 gene as a probe. The small mRNA (2.5 kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2 kb) was produced constitutively. Appearance of a 2.5 kb spo 5-mRNA depends upon the function of the mei1, mei2 and mei3 genes.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.234-247
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• 2021

We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in erg1 transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the tif302 heterozygous strain was attributed to both decreased erg1 mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.162-172
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• 2002

Schizosaccharomyces pombe is suited for the study of cytokinesis as it divides by forming a septum in the middle of the cell at the end of mitosis. To enhance our understanding of the cytokinesis, we have carried out a genetic screen for temperature-sensitive S. pombe mutants that show defects in septum formation and cell division. Here we present the isolation and characterization of a new temperature-sensitive mutant, sun1(septum uncontrolled), which undergoes uncontrolled septation during cell division cycle at restrictive temperature $(37^{\circ}C)$. In sun1 mutant, actin ring and septum are positioned at random locations and angles, and nuclear division cycle continues. These observations suggest that the sun] gene product is required for the proper placement of the actin ring as well as precise septation. The sun] mutant is monogenic recessive mutation unlinked to previously known various cdc genes of S. pombe. In a screen for $sunl^+$ gene to complement the sun] mutant, we have cloned a gene, $susl^+$(suppressor of sun1 mutant), that encodes a protein of 689 amino acids. The predicted amino acid sequence of $susl^+$ gene is similar to the human hMadlp and Saccharomyces cerevisiae Mad1p, a component of the spindle checkpoint in eukaryotic cells. The null mutant of $susl^+$ gene grows normally at various temperatures and has the increased sensitivity to anti-microtubule drug, while $susl^+$ mutant shows no sensitivity to microtubule destabilizing drugs. The putative S. pombe Sus1p directly interacts with S. pombe Mad2p in yeast two-hybrid assays. These data suggest that the newly isolated susr gene encodes S. pombe Mad1p and suppresses sun] mutant defective in controlled septation in a cell division cycle.

• Journal of Microbiology
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• v.42 no.3
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• pp.233-238
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• 2004

Transcriptional regulation of the Schizosaccharomyces pombe y-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively. The negatively-acting sequence was located in the -607 - -447 bp upstream region of the GCS gene. The upstream sequence responsible for induction by menadione(MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 - -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point. Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region. The transcription factor Papl is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide. Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1. These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 - -447 region, and also that the nitric oxide-mediated regulation of the S. pombe GCS gene may share a similar mechanism with its carbon-dependent induction.

• Animal cells and systems
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• v.2 no.4
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• pp.539-543
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• 1998

Hrp1, of Schizosaccharomyces pombe, is a new member of the SW12/SNF2 protein family that contains a chromodomain and a DNA binding domain as well as ATPase/7 helicase domains. This configuration suggests that Hrp1 could be a homolog of mouse CHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. To understand the enzymatic nature of Hrp1 we purified the 6-Histidine-tagged Hrp1 protein (6$\times$His-Hrp1) to homogeneity from a S. pombe Hrp1-overexpressing strain and hen examined its biochemical properties. We demonstrate that the purified 6$\times$His-Hrp1 protein exhibited a DNA-binding activity with a moderate preference to the (A＋T)-rich tract in double-stranded NA via a minor groove interaction. However, we failed to detect any intrinsic DNA helicase activity from the purified Hrp1 like other SW12/SNF2 proteins. These observations suggest that the DNA binding activities of Hrp1 may be involved in the remodeling of the chromatin structure with DNA-dependent ATPase. We propose that Hrp1 may function in heterochromatins as other proteins with a chromo- or ATPase/helicase domain and play an important role in the determination of chromatin architecture.