• Korean Journal of Microbiology
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• v.35 no.4
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• pp.315-321
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• 1999

This study has been performed to deveope a yeast strain having high ${\alpha}$-amylase production ability using nuclear transfer method. Hybrids formed between the strains of Saccharomyces fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-)were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic mutants S. cerevisiae and selected the hybrids showing an increased starch degrading capability were selected (MN-16). This transformant grew best and produced maximal ${\alpha}$-amylase activity on the medium containing 2% (V/V) soluble starch. ${\alpha}$-Amylase from MN-16 was purified electrophoretically homogenety and its properties were investigated. The enzyme was purified about 10.6 fold with an overall yield 9.7% from the culture medium by ammonium sulfate fractionation. DEAE-Sephacel column chromatography, and Sephacryl S-200 column chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight of the ${\alpha}$-amylase was estimated to be 53,000 daltons by SDS-PAGE and by gel permeation chromatography on Sephacryl S-200. The purified enzyme showed the maximum activity at pH 5.5 and 40${\circ}C$. The km value for soluble starch was 2.5㎎/㎖. The enzyme activity increased in the presence of $Ca^{2+}, Co^{2+}, EDTA, Mg^{2+}, Mn^{2+}, Zn^{2+}$, but inhibited by $Cu^{2+}, Fe^{2+}$, and $Ni^{2+}$

• The Korean Journal of Mycology
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• v.19 no.4
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• pp.266-275
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• 1991

The gene CDC70 encoding the${\alpha}-subunit$ of G protein has been known to be a component involved in mating pheromone signalling in the yeast, Saccharomyces cerevisiae. To isolate mutations of the genes involved in the signal transduction, Saccharomyces cerevisiae the strain bearing the cdc70-5 mutation was mutagenized to be forced to recover the ability of colony-formation at restrictive temperature, which means the new mutation can suppress the temperature sensitivity of the cdc70-5 phenotypes. Among these suppressors, $sir^-$ and $mat{\alpha}2^{-}$ mutations are excluded because of no relationship to signal transducer. And the selected suppressors were analyzed for the linkage relationships by the tetrad analysis. Out of fifteen suppressors isolated, twelve were classified into four linkage groups, designated as sga1, sga2, sga3, sga4 by the tetrad analysis. The other three genes were determined for the linkage.

• Proceedings of the Microbiological Society of Korea Conference
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• 2007.05a
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• pp.120-122
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• 2007

All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.

• Journal of Life Science
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• v.29 no.8
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• pp.916-921
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• 2019

In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase ${\delta}$ and ${\varepsilon}$ encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures ($38^{\circ}C$ and $40^{\circ}C$), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than $38^{\circ}C$. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126-Ht strains that proliferated at $40^{\circ}C$ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.