• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.328-333
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• 2008

A series of pep tides binding to the HCV NS5B polymerase was selected from phage display peptide libraries. A conserved motif of Ser-Arg-X-Arg/Leu was identified among the selected peptides, and Pep2 (Trp-Ser-Arg-Pro-Arg-Ser-Leu) was chosen for further characterization. The binding of Pep2 to HCV NS5B in vivo was shown by a yeast two-hybrid assay and by subcellular colocalization analysis using immunofluorescence confocal microscopy. The in vitro interaction was also confirmed by GST pulldown assay. The replication of the HCV 1b subgenomic replicon was efficiently inhibited by the presence of the peptide. By using a subtractive biopanning against Pep2, the binding site of the peptide was mapped at the pocket of Pro388 to Pro391 in the thumb subdomain of the polymerase. A yeast two-hybrid analysis using Pro388Ala and Pro391Ala mutants of NS5B confirmed the binding.

• BMB Reports
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• v.41 no.6
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• pp.455-460
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• 2008

Calcineurin (Cn) is a serine/threonine phosphatase implicated in a wide variety of biological responses. To identify proteins that mediate Cn signaling pathway effects, we used yeast two-hybrid assays to screen for Cn interacting proteins, discovering a protein encoded by the gene, cnp-2 (Y46G5A.10). Utilizing serially deleted forms of Cn as baits, we demonstrated that the catalytic domain of Cn (TAX-6) binds with CNP-2, and this physical interaction was able to be reconstituted in vitro, supporting our yeast two-hybrid results. cnp-2 is a nematode-specific novel gene found in C. elegans as well as its closest relative, C. briggsae. CNP-2 was strongly expressed in the intestine of C. elegans. To study the function of cnp-2, we performed cnp-2 RNAi knock-down and characterized phenotypes associated with Cn mutants. However, no gross defects were revealed in these RNAi experiments. CNP-2 was proven to be a Cn binding protein; however, its role remains to be elucidated.

• Journal of Applied Biological Chemistry
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• v.55 no.1
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• pp.67-70
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• 2012

Plant MYB transcription factors regulate secondary metabolism, cellular morphogenesis, and plant hormone signaling pathway. MYB proteins in plants consist of two repeats of 50 amino acid residues, which are referred to as R2R3 and they interact with WD40 or basic helix loop helix (bHLH) proteins. Yeast two hybrid assay was determined whether rice MYB protein interacts with either OsTTG1, which contains a WD40 domain, or with OsGL3, which contains a bHLH domain. Among 30 OsMYB proteins, three interacted with OsTTG1 and five interacted with OsGL3. A series of MYB mutants were created to determine the MYB domain important for the interaction with OsTTG1 or OsGL3. By using the yeast two hybrid assay, we found that the R3 motif of OsMYB10 and the R2 motif of OsMYB16 were required for interaction with OsTTG1 and OsGL3 proteins, respectively.

• BMB Reports
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• v.41 no.8
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• pp.581-586
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• 2008

The pre-replication complex (pre-RC), including the core hexameric MCM2-7 complex, ensures that the eukaryotic genome is replicated only once per cell division cycle. In this study, we identified a rice $\underline{m}ini\underline{c}hromosome$ $\underline{m}aintenance$ (MCM) homologue (OsMCM2) that functionally complemented fission yeast MCM2 (CDC19) mutants. We found OsMCM2 transcript expression in roots, leaves, and seeds, although expression levels differed slightly among the organs. Likewise, the OsMCM2 protein was ubiquitously expressed, but it was downregulated when nutritients were limiting, indicating that MCM2 expression (and therefore cell cycle progression) requires adequate nutrition. Yeast two-hybrid and GST pull-down assays demonstrated that OsMCM2 interacted with the COP9 signalosome 5 (CSN5). Taken as a whole, our results indicated that OsMCM2 functions as a subunit of the rice MCM complex and interacts with CSN5 during developmental regulation.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.149-152
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• 2006

The sp%pl null mutant was constructed to study the function of fission yeast Schizosaccharomyces pombe spThp1, which is homologous to budding yeast Saccharomyces cerevisiae THP1. Tetrad analysis showed that the spThp1 is not essential for vegetative growth. The spThp1 null mutant also showed no massive poly(A)+ RNA export defect. However, spThp1 null is genetically associated with spMex67 null. These results suggest that spThp1 is involved in mRNA export out of the nucleus.

• Applied Biological Chemistry
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• v.18 no.1
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• pp.16-22
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• 1975

This experiment was carried out to investigate the physiological characteristics of two original yeasts, 5-Y-5 and 6-Y-6, which selected from 24 Takju yeasts and three mutants, 30-24,30-81 and 40-27. induced from two original yeasts by the irradiation of UV light. The results were summarized as follows. 1) Alcohol tolerances of three mutants were decreased in some degree as compared with those of original yeasts. 2) Tolerances of lactic and citric acids of acid producing mutant 30-81, was increased than those of original yeasts. 3) In the case of using ammonium sulfate as a nitrogen source, two original yeasts and three mutants required Ca-pantothenate as a essential growth factor and four strains of yeasts except the mutant, 30-81, required biotin as a stimulated growth factor, When asparagine was used as a nitrogen source, two original yeasts and three mutants showed the same as above result but the stimulated effect of biotin was far less. 4) Propagation powers of the mutants were weaken than those of original yeasts, particular that of acid producing mutant, 30-81, was the weakest in the three mutants. 5) The optimum temperature for fermentation of original yeasts were $30^{\circ}C\;to\;35^{\circ}C$ but three mutants were $25^{\circ}C\;to\;30^{\circ}C$. 6) The optimum pH for fermentation of original yeasts were pH 5 to 6, and there is no appreciable difference between original yeasts and three mutants. The fermentation power of mutant,30-81, was decreased more rapidly than those of other mutants according to approach neutral. Three mutants were more sensible to heat than original yeasts. 7) Two original yeasts and three mutants were inhibited more over 20 percent of sugar for fermentation and three mutants were more sensible to sugar concentration than original yeasts.

• Applied Biological Chemistry
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• v.31 no.1
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• pp.26-32
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• 1988

This study was performed to construct killer wine yeasts which might suppress the growth of wild yeasts, reduce the consumption of starter and condense the fermentation period. Saccharomyces cerevisiae M524, a commercial wine yeast, was treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce auxotrophic mutants, i.e., CHM $2(thr^-)$, CHM 3 $(asp^-)$ and CHM 6 $(tyr^-)$. These auxotrophs were fused successfully with a killer yeast, S. cerevisiae $1368R({\alpha}\;his\;4\;kar\;1-1(kil-k)\;(k_0)$, respiratory deficient) using sphoroplast techniques and the fusants were designated as CHF 21$(th^-\;kil^+)$, CHF 22$(thr^-\;kil^+)$, CHF 31$(asp^-\;kil^+)$ and GHF 61$(tyr^-\;kil^+)$. Combined cultivation of CHF 31 with 1368R or S. cerevisiae $5{\times}47$ (killer sensitive) proved out that CHF 31 had the characteristic of killing and produced the same amount of ethanol as the prototroph, M524.

• BMB Reports
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• v.41 no.6
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• pp.448-454
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• 2008

Two full-length cDNAs, PutPMP3-1 and PutPMP3-2, encoding PMP3 family proteins were isolated from Puccinellia tenuiflora, a monocotyledonous halophyte. Expression of both genes was induced by low temperature, salt stress, dehydration, ABA, and $NaHCO_3$. Transcripts of PutPMP3-2 were more strongly induced by these stresses relative to those of PutPMP3-1, particularly under low temperature and dehydration conditions. Expression of PutPMP3-1 and PutPMP3-2 in yeast mutants lacking the PMP3 gene can functionally complement the membrane hyper-polarization and salt sensitivity phenotypes resulting from PMP3 deletion. To compare the functions of PutPMP3-1 and PutPMP3-2, the orthologous genes in rice (OsLti6a and OsLti6b) were isolated. Both OsLti6a and OsLti6b could functionally complement the loss of PMP3 in yeast. PutPMP3-2 and OsLti6a were more effective in reversing membrane hyperpolarization than PutPMP3-1 and OsLti6b. However, the four yeast transformants each showed similar levels of salt tolerance. These results imply that these PMP3 family members don't function identically under different stress tolerance conditions.

• Journal of Life Science
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• v.13 no.4
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• pp.383-389
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• 2003

A cDNA (SeMIPS) encoding myo-inositol 1-phosphate synthase has been isolated from developing sesame (Sesamum indicum L. cv. Dan-Baek) seeds and its structure and function analyzed. The SeMIPS protein was highly homologous with those from plant species (88-94%), while a much lower degree of sequence homology (60%) was found with that of human. The functional domains commonly found in MIPS protein were identified and their amino acid residues were compared with each other. Northern blot indicated that the expression of the SeMIPS gene might be organ-specifically regulated. A complementation assay based on a yeast mutant system confirmed that the SeMIPS gene encodes a myo-inositol 1-phosphate synthase (MIPS) of sesame by showing functional expression of the SeMIPS cDNA in the yeast mutants containing the disrupted INO1 gene.