• 미생물학회지
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• 제37권1호
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• pp.42-48
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• 2001

출아 효모인 Saccharomyces cerevisiae의 베타-1,3-글루칸 생합성의 결함을 초래하는 돌연변이 유전자(sool-1)를 분리하여 돌연변이 부위의 염기서열을 결정하고 그 특성을 분석하였다. cool-1 유전자의 염기서열 분석 결과, 681번의 염기인 G가 A로 치환되어 Soolp의 $Gly^{227}$이 Asp로 치환되는 결과를 나타내는 것으로 판명되었고, cool-1 유전자는 기존에 보고된 retl-1 유전자와 동일한 돌연변이 유전자로 판명되었다. 그러나, ret1-1이 나타내는 온도감수성 형질은 배지에 1.2 M sorbitol 등의 삼투안정제를 첨가하면 극복될 수 있으며, cool-l/retl-1의 돌연변이 부위가 세포벽합성 관련 단백질의 번역 후 수식과정에 영향을 미친 것임을 확인하였다. 한편, Soolp/$\alpha$-COP의 N-말단에 존재하는 6개의 WD40 domain중 5번째 WD40 domain이 효모의 세포벽 합성이나 구조유지에 중요한 역할을 담당할 것임을 시사하는 결과를 얻었다.

• 한국균학회지
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• 제26권4호통권87호
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• pp.450-457
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• 1998

Cryparin은 Cryphonectria parasitica의 세포벽에 풍부한 소수성 단백질에 속한다. cryparin은 비록 하나의 유전자에 의해 발현되지만 액체배양 후 48시간이 지나면 발현된 전체 유전자중에서 22%를 차지할 정도의 높은 발현 양상을 나타낸다. 또한 cryparin은 RNA mycovirus인 Cryphonectria hypovirus 1의 감염에 의해 발현이 현저히 억제되는 유전자로 알려졌다. 이미 지난 실험(Kim et al., 1999)에서 상동염색체간의 재조합을 이용하여 cryparin 유전자를 항생제 hygromycin B 저항성 유전자로 치환한 치환체를 제조하였다. 발현율이 매우 높으면서도 virus에 의해 밀접하게 영향받는 cryparin 유전자의 promoter 분석을 위하여서는 대상이 되는 유전자 치환을 위한 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 어느 한곳에만 삽입되도록 하는 성질을 가진 균주의 개발이 필요하다. 그러나 지난번 실험의 결과 얻어진 cryparin 치환체는 치환용 vector외에도 무작위로 삽입된 vector가 존재하고 나아가 새로운 vector들이 어느 한곳에만 삽입되도록 하는 성질을 갖지 못하였다. 따라서 본 실험에서는 cryparin 유전자 치환체와 영양요구성 돌연변이체인 균주간의 교잡을 이용하여 분석 대상이 되는 유전자의 치환에 이용된 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 genome내의 어느 한곳에만 삽입되도록 하는 성질을 가진 균주를 제조하였다.

• 한국미생물·생명공학회지
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• 제21권3호
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• pp.214-220
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• 1993

The mating type a of yeast, Saccharomyces cerevisiae mutant with hmla2-102 and sir3-8ts was changed to type alpha by changing the growth temperature from 25C to 35C. A temperature-sensitive expression vector system was constructed using mating factor alpha1 (Mfalpha1) gene encoding alpha factor which is expressed in the type alpha cells. Vectors with different copy numbers were constructed by joining the promoter and pre or prepro-secretion single sequence of Mfalpha1 to promoterless PHO5' gene as a reporter gene.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.318-326
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• 2002

The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia. To better understand this organism's strategies to survive osmotic stress, a mutant that was more sensitive to high osmolarity was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, putAP genes encoding a proline dehydrogenase and a proline permease were identified and cloned from V. vulnificus. The amino acid sequences deduced from nucleotide sequences of putAP from V. vulnificus were 38 to $59\%$ similar to those of PutA and PutP reported from other Enterobacteriaceae. Functions of putAP genes were assessed by the construction of mutants, whose putAP genes were inactivated by allelic exchanges. When proline as the sole carbon or nitrogen source was used, the putA mutant was not able to grow to the substantial level, revealing the proline dehydrogenase is the only enzyme for metabolic conversion of proline into other amino acids. Although the growth rate of the putP mutant on proline as the sole carbon or nitrogen source was significantly reduced, the mutant still grew. This indicated that at least one more proline permease is produced by V. vulnificus. The putP mutant decreased approximately $2-log_10$ CFU/ml after a hyperosmotic challenge, while the parent strain decreased approximately $l-log_10$ CFU/ml. This result suggests that the gene product of putP contributes to the osmotic tolerance of V. vulnificus.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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• pp.149-154
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• 2003

Numerous virulence factors such as O antigen have been proposed to account for the fulminating and destructive nature of V. vulnificus infections. To better characterize the role of O antigen, a pmm gene encoding a phosphomannomutase was identified and cloned from V. vulnificus. The deduced amino acid sequence of the pmm was 42 to 71% similar to that reported from other Enterobacteriaceae. Functions of the pmm gene in virulence were assessed by the construction of an isogenic mutant, whose pmm gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of pmm resulted in a loss of more than 90% of phosphomannomutase, and reintroduction of recombinant pmm could complement the decrease of phosphomannomutase activity, indicating that the pmm gene encodes the phosphomannomutase of V. vulnificus. There was no difference in the $LD_50S$ of the wild-type and the pmm mutant in mice, but the $LD_50S$ observed by the mutant complemented with recombinant pmm were lower. Therefore, it appears that PMM is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of injecting purified LPS into animals, but it is not completely dispensable for virulence in mice.

• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• BMB Reports
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• 제40권6호
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• pp.1021-1027
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• 2007

We demonstrated the genetic polymorphism of aldehyde oxidase (AO) in Donryu strain rats: the ultrarapid metabolizer (UM) with nucleotide mutation of (377G, 2604C) coding for amino acid substitution of (110Gly, 852Val), extensive metabolizer (EM) with (377G/A, 2604C/T) coding for (110Gly/Ser, 852Val/Ala), and poor metabolizer (PM) with (377A, 2604T) coding for (110Ser, 852Ala), respectively. The results suggested that 377G > A and/or 2604C > T should be responsible for the genetic polymorphism. In this study, we constructed an E. coli expression system of four types of AO cDNA including Mut-1 with (377G, 2604T) and Mut-2 with (377A, 2604C) as well as naturally existing nucleotide sequences of UM and PM in order to clarify which one is responsible for the polymorphism. Mut-1 and Mut-2 showed almost the same high and low activity as that of the UM and PM groups, respectively. Thus, the expression study of mutant AO cDNA directly revealed that the nucleotide substitution of 377G > A, but not that of 2604C > T, will play a critical role in the genetic polymorphism of AO in Donryu strain rats. The reason amino acid substitution will cause genetic polymorphism in AO activity was discussed.

• 미생물학회지
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• 제30권4호
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• pp.286-290
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• 1992

광주근교 지역의 근권 토양으로부터 cetrimide agar medium을 이용하여 형광성 pseudomonads를 분리하였고, CAS medium에서 siderophore의 생성능력이 우수한 pseudomonads만을 분리하여 생리화학적인 실험을 수행하였다. Kanamycin-sensitive pseudomonads를 Tn5를 이용한 mutagenesis를 실시하여 Kanamycin에 내성을 갖는 transconjugants를 선별하였고, siderophore 생합성을 하지 못하는 돌연변이주를 선별하기 위하여 CAS medium에서 yellow hallow를 형성하지 못하거나 King's B medium에서 형광성을 나타내지 못하는 colony를 선별하였다. 선별된 mutants들의 genomic DNA에 Tn5가 삽입되었는지를 확인하기 위하여 Southern blot hybridization을 실시한 결과 intact Tn5에 homology를 나타내는 하나의 single band를 확인하였다.

• 미생물학회지
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• 제44권1호
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• pp.80-84
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• 2008

mRNA의 핵에서 세포질로의 이동에 중요한 역할을 하는 발아효모 Saccharomyces cerevisiae의 DEAD-box RNA helicase인 DBP5 유전자와 유사한 분열효모 Schizosaccharomyces pombe의 유전자(spDbp5로 명명)의 결실돌연변이주(knockout mutant)를 제조하여 그 특성을 조사하였다. 이배체인 S. pombe 균주에 하나의 spDbp5 유전자만을 결실시킨 후 4분체분석(tetrad analysis)을 수행한 결과, 이 유전자가 결실된 반수체 균주는 생장하지 못했다. mRNA의 핵에서 세포질로의 이동에 있어서 spDbp5의 역할을 알아보기 위해, spDbp5의 발현이 티아민(thiamin)에 의해 억제되는 균주를 제작하여 in situ hybridization을 통해 세포 내의 $poly(A)^+$ RNA 분포를 살펴보았다. spDbp5 유전자의 발현이 억제되면, $poly(A)^+$ RNA가 핵 안에 축적되고세포질에서는 줄어들었다. 이와 같은 결과들은 spDbp5 유전자 역시 mRNA의 핵에서 세포질로의 이동에 매우 중요한 역할을 담당하고 있음을 시사한다.

• BMB Reports
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• 제42권6호
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• pp.315-323
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• 2009

In order to elucidate ultimate biological function of the genome, the model animal system carrying mutations is indispensable. Recently, large-scale mutagenesis projects have been launched in various species. Especially, the mouse is considered to be an ideal model to human because it is a mammalian species accompanied with well-established genetic as well as embryonic technologies. In 1990', large-scale mouse mutagenesis projects firstly initiated with a potent chemical mutagen, N-ethyl-N-nitrosourea (ENU) by the phenotype-driven approach or forward genetics. The knockout mouse mutagenesis projects with trapping/conditional mutagenesis have then followed as Phase II since 2006 by the gene-driven approach or reverse genetics. Recently, the next-generation gene targeting system has also become available to the research community, which allows us to establish and analyze mutant mice carrying an allelic series of base substitutions in target genes as another reverse genetics. Overall trends in the large-scale mouse mutagenesis will be reviewed in this article particularly focusing on the new advancement of the next-generation gene targeting system. The drastic expansion of the mutant mouse resources altogether will enhance the systematic understanding of the life. The construction of the mutant mouse resources developed by the forward and reverse genetic mutagenesis is just the beginning of the annotation of mammalian genome. They provide basic infrastructure to understand the molecular mechanism of the gene and genome and will contribute to not only basic researches but also applied sciences such as human disease modelling, genomic medicine and personalized medicine.