• 생명과학회지
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• 제15권2호
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• pp.182-185
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• 2005

IS1096 transposon을 사용하여 결핵균군에 작용하는 항생제인 PA-824에 내성을 나타내는 Mycobacterium bovis BCG의 돌연변이 균주를 얻고자 하였고, 그 결과 24종류의 서로 다른 돌연변이 균주를 얻을 수 있었다. Transposon이 insertion된 부위를 알아내기 위해서 각각의 돌연변이 균주로 inverse PCR을 수행하였고, PPE 유전자를 포함한 여러 가지 부위에 insertion된 transposon의 위치를 확인할 수 있었다. PPE 유전자에 insertion돌연변이가 발생한 5개 균주의 세포 추출물을 HPLC로 분석한 결과, 3개에서는 야생균주에서 관찰되는 coenzyme $F^{420}$이 존재하지 않았고, 그 생합성 경로의 중간산물인 F0만 존재하였다. 또한 나머지 2개에서는 F0 또는 $F^{420}$어느 것도 존재하지 않았다. 이 결과는 PPE 유전자들의 산물들이 coenzyme $F^{420}$에 어떤 식으로든 관여하고 있음을 나타낸다고 할 수 있다.

• Molecules and Cells
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• 제27권5호
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• pp.563-570
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• 2009

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1987년도 식물생명공학 심포지움 논문집 Proceedings of Symposia on Plant Biotechnology
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• pp.323-347
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• 1987

The R locus of maize in one of several genes that regulate the anthocyanin pigments throughout the body of the plant and seed. The R gene product may regulate pigment deposition by controlling the expression of the flavonoid biosynthetic gene pathway in a tissue-specific manner. To understand the basis for tissue specific regulation and allelic variation at R, the molecular study has been done by cloning a portion of the R complex by transposon tagging with Ac. R specific probe were cloned from the R-nj mutant induced by Ac insertion mutagenesis. From southern analysis of R-r complex using the R-nj probe, the structure of R-r was proposed that R-r containes the three elements, (P)(Q)(S). These elements may organize as the inversion triplication model which (S) sequence was inverted in relation to (P) and (Q). The R-sc derivated from R-mb or R-nj was cloned with R-nj probe, and molecular genetical data showed that R-sc containes tissue specific and tissue nonspecific area, and the sequencing of R-sc are progressed now.

• 생명과학회지
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• 제15권3호
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• pp.415-422
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• 2005

Mycobacterium hovis BCG 균주에 transposon을 사용하여 mutagenesis를 수행함으로써 mutant library를 제조하였다. 이 mutant library를 screening하여 항결핵제인 PA-824에 내성을 갖는 mutant들을 얻었고, M. bovis wild type에서는 정상적으로 생성되는 coenzyme $F_{420}$이 대부분의 이들 mutant들에서는 생성되지 않는다는 것을 알게 되었다. 세포 추출액을 HPLC로 분석해본 결과, 그 중에서 한 mutant는 $F_{420}$은 생성하지 않으나 그 전구물질인 F0는 생성하고 있음이 밝혀졌다. 따라서 이 mutant 에서는 $F_{420}$생합성 회로의 마지막 단계가 차단되어있음을 알 수 있다. 이 mutant를 inverse PCR을 통해 분석해본 결과, transposon이 Rv2435c유전자에 삽입되어있는 것을 확인할 수 있었다. Rv2435c유전자는 세포막에 결합되어있는 80.3 kDa의 단백질을 암호화하는 것으로 추정되고, 이 단백질의 N-말단은 periplasm에 존재하고 C-말단은 원형질에 존재하는 것으로 추정되고 있다. 원형질에 존재하는 C-말단은 원핵생물과 진핵생물들의 adenylyl cyclase들과 높은 유사성을 나타낸다. Adenylyl cyclase는 ATP로부터 cAMP를 생합성하는 효소이다 M. tuberculosis나 M. bovis의 genome에는 class III adenylyl cyclase를 암호화하는 것으로 추정되는 유전자가 모두 15개나 존재한다 특히 이들 중에서 Rv1625c 와 Rv2435c는 포유류의 adenylyl cyclase들과 높은 유사성을 가지는 것으로 알려져 있다 이 Rv2435c 단백질이 진정한 adenylyl cyclase인지를 확인하기 위하여 우리는 이 단백질 중에서 원형질에 존재하는 부분을 말단에 6개의 histidine을 첨부한 채로 대장균에서 발현시켰다. 대장균에서 이 단백질이 생성되는 것은 histidine이 첨부된 단백질을 Ni-NTA resin을 사용하여 대장균으로부터 분리함으로써 확인하였다. 그러나 이 단백질이 대장균에서 cya mutation을 complementation하지 못하였고, 따라서 이 단백질이 adenylyl cyclase 활성을 갖지 않음을 알 수 있었다. 자외선이나 hydroxylamine을 사용한 mutagenesis 또는 Rv2435c와 Rv1625c간의 토sion단백질을 만들어서, 이 단백질이 adenylyl cyclae로서의 활성을 획득하도록 하는 모든 시도는 실패하였다. 따라서 Rv2435c단백질이, F0가 $F_{420}$으로 변환되는 데에 영향을 미치는 방법이 cAMP를 생성함으로써가 아니라 다른 방법으로 영향을 미치고 있다는 것을 알 수 있었다.

• 한국육종학회지
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• 제40권1호
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• pp.39-47
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• 2008

1. Ds 삽입변이체 계통으로부터 제초제 저항성 1,874계통을 선발하고 농업적 주요 특성으로서 출수일수, 간장, 수수, 수장, 엽장 등 5가지 형질에 대하여 조사한 바, 조사된 5가지 형질에 대하여 원품종인 동진벼에 비하여 매우 다양한 변이폭을 보여주었다. 2. 농업적 유용성과 관련된 수장이 길고, 조기출수, 수수가 많은 변이체 뿐만 아니라 형태학적 변이를 보이는 twin seedling, dwarf, early heading, strip albino, liguleless 등 변이체가 다수 발견됨으로서 육종적 이용 및 유전자 기능해석을 위한 유용한 집단으로서 유용성을 보여주었다. 3. 서던분석 결과 벼 게놈상에서 Ds는 평균 2 copy로 전이되었으며 조직부위별로 GUS의 발현을 조사한 결과 잎, 뿌리 및 화기관등에서 약 3.9%가 발현되었다. 이 삽입변이체에서 나타난 다양한 변이형질의 주요 농업적 특성과 GUS 발현의 재현성을 위해 다음 세대의 전개를 통한 후대분석이 필요하다.

• ALGAE
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• 제33권1호
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• pp.127-141
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• 2018

Low efficiency in microalgal biomass production was largely attributed to the low density of algal cell cultures. Though mutations that reduced the level of chlorophyll or pigment content increased efficiency of photon usage and thus the cell-culture density under high-illumination growth conditions (e.g., >$500{\mu}mol\;photon\;m^{-2}\;s^{-1}$), it was unclear whether algae could increase cell-culture density under low-illumination conditions (e.g., ${\sim}50{\mu}mol\;photon\;m^{-2}\;s^{-1}$). To address this question, we performed forward genetic screening in Chlamydomonas reinhardtii. A pool of >1,000 insertional mutants was constructed and subjected to continuous subcultures in shaking flasks under low-illumination conditions. Complexity of restriction fragment length polymorphism (RFLP) pattern in cultures indicated the degree of heterogeneity of mutant populations. We showed that the levels of RFLP complexity decreased when cycles of subculture increased, suggesting that cultures were gradually populated by high cell-culture density (HCD) strains. Analysis of the 3 isolated HCD mutants after 30 cycles of subcultures confirmed that their maximal biomass production was 50-100% higher than that of wild type under low-illumination. Furthermore, levels of chlorophyll content in HCD mutant strains were similar to that of wild type. Inverse polymerase chain reaction analysis identified the locus of insertion in two of three HCD strains. Molecular and transcriptomic analyses suggested that two HCD mutants were a result of the gain-of-function phenotype, both linking to the abnormality of mitochondrial functions. Taken together, our results demonstrate that HCD strains can be obtained through continuous subcultures under low illumination conditions.

• Molecules and Cells
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• 제28권2호
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• pp.105-109
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• 2009

We reported previously that overexpression of a salicylic acid (SA) methyltransferase1 gene from rice (OsBSMT1) or a SA glucosyltransferase1 gene from Arabidopsis thaliana (AtSAGT1) leads to increased susceptibility to Pseudomonas syringae due to reduced SA levels. To further examine their roles in the defense responses, we assayed the transcript levels of AtBSMT1 or AtSAGT1 in plants with altered levels of SA and/or other defense components. These data showed that AtSAGT1 expression is regulated partially by SA, or nonexpressor of pathogenesis related protein1, whereas AtBSMT1 expression was induced in SA-deficient mutant plants. In addition, we produced the transgenic Arabidopsis plants with RNAi-mediated inhibition of AtSAGT1 and isolated a null mutant of AtBSMT1, and then analyzed their phenotypes. A T-DNA insertion mutation in the AtBSMT1 resulted in reduced methyl salicylate (MeSA) levels upon P. syringae infection. However, accumulation of SA and glucosyl SA was similar in both the atbsmt1 and wild-type plants, indicating the presence of another SA methyltransferase or an alternative pathway for MeSA production. The AtSAGT1-RNAi line exhibited no altered phenotypes upon pathogen infection, compared to wild-type plants, suggesting that (an)other SA glucosyltransferase(s) in Arabidopsis plants may be important for the pathogenesis of P. syringae.

• Molecules and Cells
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• 제27권6호
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• pp.673-680
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• 2009

Conversion of the normal soluble form of prion protein, PrP ($PrP^C$), to proteinase K-resistant form ($PrP^{Sc}$) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from ${\alpha}$-helix to ${\beta}$-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

• 한국식물병리학회지
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• 제11권4호
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• pp.304-311
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• 1995

To determine whether chitinolytic enzymes from Chromobacterium violaceum C-61 play an important role in the suppression of Rhizoctonia damping-off, Tn5 insertion mutants deficient in chitinolytic activity (Chi a- mutants) were selected and their chitinolytic and disease suppression were compared with those of the parental strain. Four Chi a- mutants selected from about 2,000 transconjugants did not inhibit mycelial growth of Rhizoctonia solani on nutrient agar-potato dextrose agar (BA-PDA) and their abilities to suppress Rhizoctonia damping-off were much lower than the parental strain. However, population density in the eggplant rhizosphere did not differ significantly between the parental strain and four Chi a- mutants. The crude enzyme of the parental strain inhibited growth of R. solani on NA-PDA and its chitinase activity was much higher than that of Chi a- mutants. But the N,N' -diacetylchitobiase activity between these isolates were not significantly different. The chitinase of Chi a- mutants was defective in 2 isoforms of 52- and 37-kDa among four isoforms of 54-, 52-, 50- and 37-kDa. A Tn5 element was inserted into one site of 10 kb EcoRI fragment of chromosomal DNA in three Chi- mutants, C61-C1, -C2, and -C3. In C61-C4 mutant, a Tn5 element was inserted into two sites of 10 kb and 4.4 kb EcoRI fragments. These results suggest that the chitinase of C. violaceum C-61 play an important role in the suppression of Rhizoctonia damping-off of cucumber and eggplant.

• The Plant Pathology Journal
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• 제21권3호
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• pp.275-282
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• 2005

A chitinolytic bacterium, Chromobacterium sp. strain C-61, was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of eggplant. In this study, the biocontrol activity and enzymatic characteristics of strain C-61 were compared with its four Tn5 insertion mutants (C61-A, -B, -C, and -D) that had lower chitinolytic ability. The chitinase activity of a 2-day old culture was about $76\%,\;49\%\;and\;6\%$ level in C61-A, C61-B and in C61-C, respectively, compared with that of strain C-61. The $\beta-N-acetylhexosaminidase$(Nahase) activity was little detected in strain C-61 but increased largely in C-61A, C61-B and C61-C. Activities of chitinase and Nahase appeared to be negatively correlated in these strains. Another mutant, C-61D, produced no detectable extracellular chitinase and Nahase. The in vitro and in vivo biocontrol activities of strain C-61 and its mutants were closely related to their ability to produce chitinase but not Nahase. No significant differences in population densities between strain C-61 and its mutants were observed in soil around eggplant roots. The results of SDS-PAGE and isoelectrofocusing showed that a major chitinase of strain C-61 is 54-kDa with pI of approximately 8.5. This study provides evidence that the biocontrol activity of Chromobacterium sp. strain C-61 against Rhizoctonia solani depends on the ability to produce chitinase with molecular weight of 54-kDa and pI of 8.5.