• 약학회지
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• 제29권6호
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• pp.109-117
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• 1985

합성 DNA는 분자 생물학의 모든 분야의 연구에 사용될 수 있으며 앞으로도 그 이용도는 더욱 증가할 것으로 전망된다. 합성 기술의 발달로 생리활성을 가진 인조 유전자를 제조하는 것이 가능해졌으며 또한 다른 방법으로는 어려운 유전자의 분리와 cloning, site specific mutagenesis, 질병의 진단, 유전자의 구조 및 기능의 연구등 수많은 분야의 연구에 이용되고 있다. 본 고에서는 현재 여러 군데 분자 생물학 연구실에서 성공적으로 사용되고 있는 자동 합성기에 의한 phosphite합성법의 기초 이론과 합성된 oligonucleotide의 정제법에 대하여 간단히 서술하고 그 응용 방법에 대하여 논하고자 한다.

• Bulletin of the Korean Chemical Society
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• 제20권11호
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• pp.1319-1322
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• 1999

Leucine zipper dynamically tunes the degree of bifurcation of the DNA binding segments in the basic region of the Fos-Jun bZIP complex. Molecular dynamics simulation indicated that site-specific mutagenesis of conserved leucine residues inside the leucine zipper domain caused the change of dynamic behavior of the basic region, and efficient DNA binding occurs only within a certain range of distance between the two DNA binding segments in the basic region. Distribution of α-helices in the hinge region is also suggested to influence the bifurcation of the DNA binding segments.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1827-1834
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• 2015

The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Site-directed mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and p-nitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipid-anchoring mechanism for DAG (L103 and F104 serve as "anchors" to the lipid interface) were proposed.

• 생명과학회지
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• 제22권1호
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• pp.7-15
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• 2012

이전 연구에서 Streptomyces sp. M3 균주로부터 polyguluronate에 기질특이성을 가지는 알긴산분해효소를 cloning하고 활성을 연구하였다. 이번 연구에서는 pColdI vector에 들어있는 M3 알긴산분해효소 유전자를 돌연변이시켜 알긴산분해효소의 활성을 증진시키고자 하였으며, 점-돌연변이 또는 무작위-돌연변이 방법을 사용하여 돌연변이를 실시하였다. Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu 및 Gly194Cys 등 6 종류의 돌연변이 단백질을 얻을 수 있었다. Phe99Leu 및 Lys191Glu 돌연변이 단백질은 알긴산을 분해하는 능력을 완전히 잃었으나 Gly194Cys 돌연변이 단백질의 활성은 원래 단백질에 비하여 10배 증가하였다. 또한 돌연변이된 M3 알긴산분해효소 단백질의 3차 구조는 Swiss-Model 자동모델러를 이용하여 생성하였으며 다른 알긴산분해효소의 결정구조와 비교하였다. 194 번째 아미노산인 글리신은 알긴산의 C-말단 보존서열인 YFKAGXYXQ의 Gly193과 Tyr195 사이에 위치한다. 이 연구에서 돌연변이된 글리신과 페닐알라닌 잔기들은 활성자리로부터 많이 떨어져있음에도 불구하고 돌연변이에 의하여 알긴산 분해활성이 강하게 영향을 받는 것으로 나타났다.

• BMB Reports
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• 제34권5호
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• pp.402-407
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• 2001

Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry $\delta$-endotoxins, their pore-forming activity involves the insertion of the ${\alpha}4-{\alpha}5$ helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteranactive Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.

• Journal of Photoscience
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• 제9권2호
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• pp.33-36
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• 2002

In vertebrate rhodopsins the glutamic acid at position 113 serves as a counterion to stabilize the protonated retinylidene Schiff base linkage and to shift the spectrum to the visible region. Invertebrate rhodopsins and retinochrome have the amino acid residue different from glutamic acid or asparatic acid at this position and therefore, these pigments may have a counterion at different position. We first investigated the counterion in retinochrome by site specific mutagenesis. The results showed that the counterion is the glutamic acid at position 181, where almost of all the pigments including vertebrate and invertebrate rhodopsins in the rhodopsin family have a glutamic acid or an aspartic acid. In vertebrate rhodopsins, however, Glu 181 does not act as a counterion, and the red-sensitive cone pigments have a histidine at this position, which serves as a chloride-binding site for red-shift of the absorption spectrum. These findings suggested that the role of Glu181 as a counterion may be weakened by the newly acquired counterion at position 113. Taken together with our recent studies on an invertebrate-type rhodopsin, the rhodopsin diversity was discussed from viewpoint of counterion.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.534-538
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• 2001

The xynA gene encoding an alikali-tolerant endo-1,4-${\beta}$-xylanase (XYN) was cloned from the alkalophilic Bacillus pumilus A-30. The nucleotide sequence of a 974-bp DNA fragment containing the xynA was determined. An ORF of 684 nucleotides that encoded a protein of 228 amino aicds was detected. Asparagine-71 of XYN from B. Pumilus A-30 showed to be highly conservative in alkaline xylanases of family G/11, upon comparing the amino acid sequences of 17 family G/11 xylanases. Site-directed mutation of N71D of the xynA gene resulted in a decrease of 12.4% in the specific acitivity and a significant decline in the enzyme activity in the alkaline pH range.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.315-320
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• 1996

The role of glyoxylate bypass in a lysine-producing Corynebacterium lactofermentum strain was analyzed. Unlike the wild type, the strain expressed enzymes of glyoxylate bypass during growth in the fermentation broth containing glucose as the carbon source. To evaluate the importance of glyoxylate bypass in the strain, we disrupted chromosomal aceA by using a cloned fragment of the gene. Site-specific disruption of aceA which codes for the isocitrate lyase, the first enzyme of the bypass, was confirmed by Southern blot analysis. The aceA mutant strain completely lost isocitrate lyase activity and ability to grow in a minimal medium containing acetate as the sole carbon source. The mutant strain was similar to its parental strain in growth characteristics and produced comparable amounts of lysine in shake flasks containing glucose as the carbon source. The amount of oxaloacetate accumulated in the fermentation medium was similar for both strains, suggesting that expression of glyoxylate bypass does not necessarily lead to the increase in intracellular oxaloacetate. These data clearly demonstrate that glyoxylate bypass does not function as one of the routes of carbon supply for lysine production in the strain. It appears that the leakiness of the glyoxylate bypass in the strain might be the result of a secondary mutation which arose during previous strain development by random mutagenesis.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.29-34
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• 2001

The mutant enzymes of N-carbamyl-D-amino aicd amidohydrolase (N-carbamylase) from Agrobacterium radiobacter NRRL B11291, showing a negligible activity, were selected from the library generated by random mutagenesis. From the sequence analysis, these mutants were found to contain the amino acids substitutions at Cys172, Glu47, and Glu146. Previously, Cys172 was reported to be necessary for the enzyme catalysis. The chemical modification of the N-carbamylase by carboxyl group specific chemical reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), resulted in a loss of activity. The replacement of glutamic acids with glutamines by site-directed mutagenesis led to aggregation of the enzymes. Mutant enzymes fused with maltose binding protein (MBP) were expressed in soluble form, but were inactive. These results indicate that two glutamic acid residues play an important role in structure and function of the N-carbamylase. Multiple sequence alignment of the related enzymes revealed that Glu47 and Glu146 are rigidly conserved, which suggests that tese residues are crucial for the structure and function of the functionally related C-N hydrolases.