• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Journal of Microbiology
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• 제44권5호
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• pp.556-561
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• 2006

A differential display for the expression profiles of wild-type Cryphonectria parasitica and its virally-infected isogenic hypovirulent strain revealed several transcripts of interest, which evidenced significant matches with fungal genes of known function. Among which, we have further analyzed an amplified PCR product with significant sequence similarity to the known fungal stress-responsive thioredoxin gene from Neurospora crassa. The product of the cloned thioredoxin gene, CpTrx1, consists of 117 amino acids, with a predicted molecular mass of 13.0 kDa and a pI of 5.4. Sequence comparisons demonstrated that the deduced protein sequence of the CpTrx1 gene evidenced a high degree of homology to all known thioredoxins, with the highest degree of homology with trx1, a thioredoxin gene from Saccharomyces cerevisiae, and evidenced a preservation of the conserved hall markresidues (Trp-Cys-Gly-Pro-Cys) at the active site of thioredoxin. The E. coli-generated CpTRX1 manifested thioredoxin activity, according to the insulin reduction assay, which indicates that the cloned gene does indeed encode for the C. parasitica thioredoxin.

• 원예과학기술지
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• 제32권2호
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• pp.261-268
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• 2014

Senescence of Hosta ventricosa flowers was firstly characterized as ethylene-sensitive since the deterioration of the tepal was accompanied by increased endogenous ethylene biosynthesis. The full-length cDNAs and DNAs of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) involved in ethylene biosynthesis were cloned from H. ventricosa flowers. The HvACS ORF with 1347 bp and two introns, encoded a polypeptide of 448 amino acids showing 79% homology with that in Musa acuminata. The HvACO ORF contained 957 bp and three introns, encoding a 318-residue polypeptide showing 83% homology with that in Narcissus tazetta. The timing of the induction of HvACS expression was in correspond to the timing of the increase in ethylene production, and that the up-regulation of HvACO transcript was closely correlated with an elevated ethylene production, but underwent a down-regulation in wounded leaves with elevated ethylene emission. The results, together with expression analysis in vegetative tissues, suggested that both HvACS and HvACO were specifically regulated by flower senescence.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.256-263
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• 1994

The aceB gene, encoding for malate synthase, one of the key enzymes of glyoxylate bypass, was isolated from a pMT1-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli aceB mutant on an acetate minimal medium. The aceB gene was closely linked to aceA, separated by 598 base pairs, and transcribed in divergent direction. The aceB expressed a protein product of Mr 83, 000 in Corynebacterium glutamicum which was unusually large compared with those of other malate synthases. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 2, 217 base pairs which encodes a protein with the molecular weight of 82, 311 comprising 739 aminoo acids. The putative protein product showed only limited amino acid-sequence homology to its counteliparts in other organisms. The N-terminal region of the protein, which shows no apparent homology with the known sequences of other malate synthases, appeared to be responsible for the protein s unusually large size. A potential calciumbinding domain of EF-hand structure found among eukaryotes was detected in the N-terminal region of the deduced protein.

• 생명과학회지
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• 제10권2호
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• pp.188-195
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• 2000

In order to understand molecular events during silk synthesis and provide genetic resources for molecular breeding, we had analyzed the cDNA library constructed from the posterior silkgland of Antheraea yamamai and partially sequenced 276 randomly selected genes from the cDNA library. Database comparisons of the expressed sequence tags (ESTs) revealed that 26 non-redundant clones showed a high similarity with previously identified genes. Among them, 17 clones exhibited a homology with previously identified insect genes and 9 clones were identical to genes that were previously identified from other organisms. A functional categorization showed that silk synthesis-defense- or stress-related genes, as well as genes involved in the metabolic pathways and in the transcriptional or translational apparatus are represented. In this report, the clone (AY479) which had high similarity with fibroin from A. pernyi was particularly analyzed in detail. The AY479 clone was carboxyl terminal region of fibroin. The 472 bp cDNA has 123 amino acids that shared 85% homology with the fibroin from A. pernyi and its deduced peptide had unique feature, that is, sites of alanine rich residues.

• 미생물학회지
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• 제38권4호
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• pp.260-266
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• 2002

Ralstonia eutropha JMP134로부터 페놀대사의 조절에 관여하는 유전자부위를 cloning하여 염기서열을 파악하였으며 그 특성을 조사하였다. 염기서열 분석 결과 두 개의 open reading frame (ORF1 & ORF2)들을 발견하였다. ORF1은 페놀분해 구조유전자중의 마지막 인자인phlX의 stop codon으로부터 454 bp 아래에서 시작하여 총 501 개의 아미노산으로 구성되었으며 ORF2는 ORF1의 stop codon으로부터 1 bP 위쪽에서 4개의 염기쌍과 중첩된 상태에서 시작하여 총 232개의 아미노산으로 구성되어 있는 것으로 나타났다. 단백질 배열을 분석해본 결과 ORF1은 transcriptional activator로 작용하는 NtrC family에 속하는 것으로 확인되었으며, ORF2는 negative regulator로 알려진 GntR family에 속하는 것으로 나타났다. ORF1과 ORF2를 encoding하는 유전자를 각각 phlR2 와 phlA로 명명하였으며 이들의 가능한 조절기작을 고찰하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.31-32
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• 2005

Neoagaro-oligosaccharides are produced only by enzymatic degradation of agarose by ${\beta}-agarase.^{1)}$ Neoagaro-oligosaccharides inhibit the growth of bacteria, slow the rate of degradation of starch, are used as low-calorie additives to improve food quality, and have macrophage-stimulating activity. Furthermore, neoagarobiose is a rare reagent that has both moisturizing effect on skin and whitening effect on melanoma $cells.^{2)}$ An agar-degrading marine bacterium was isolated from the sea water at the northeast coast in Cheju island, Korea. The strain was gram negative, aerobic, and motile rod. The 16S rRNA of the strain had the closest match of 98% homology, with that from Agarivorans albus. On the basis of several phenotypic characters and a phylogenetic analysis, this strain was designated Agarivorans sp. JA-1. In solid agar plate, Agarivorans sp. JA-1 produced a diffusible agarase that caused agar softening around the colonies. Agarivorans sp. JA-1 was cultured for 36 hr in marine broth 2216 (Difco, USA) and the supernatant that containing an extracellular ${\beta}-agarase$ was prepared by centrifugation of culture media. The enzyme exhibited relatively strong activity at $40^{\circ}C$ and was stable up to $60^{\circ}C$. Using PCR primers derived from the ${\beta}-agarase$ gene of Vibrio sp., the gene encoding ${\beta}-agarase$ from Agarivorans sp. JA-1 was cloned and sequenced. The structural gene consists of 2931 bp encoding 976 amino acids with a predicted molecular weight of 107,360 Da. The deduced amino acid sequence showed 99% and 34% homology to $agaA^{2)}$ and $agaB^{2)}$ genes for ${\beta}-agarase$ from Vibrio sp., respectively. The expression plasmid for ${\beta}-agarase$ gene of Agarivorans sp. JA-1 is being constructed and the recombinant enzyme will be biochemically characterized.

• 한국수산과학회지
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• 제33권6호
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• pp.565-571
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• 2000

기존의 Cx 배열을 참고로 종내${cdot}$종간을 통하여 잘 보존되어져 있는 영역에서 primer를 설계하고, 은어의 난소를 재료로 하여 PCR을 실시하였다. 증폭된 cDNA 단편을 이용하여, 5'RACE 및 3'RACE법에 의해 미지의 영역을 cloning하여 난소에서 발현하는 Cx cDNA의 전염기배열을 결정하였다. 기존의 Cx 배열과 상동성을 비교한 결과, 대서양산 민어의 Cx32.7과 $63.8{\%}$, bovine의 Cx44와 $61.6{\%}$ 및 대서양산 민어의 Cx32.2와는 $56.7{\%}$의 상동성이 나타났다. 본 cDNA는 35,028 Da의 분자량을 code하는 open reading frame (ORF)으로 구성되어 있어, 은어 Cx35로 명명되었다. 또한 아미노산 배열의 친수성${\cdot}$소수성 영역의 분포예측 결과, 4곳의 소수성 영역과 4곳의 친수성 영역을 교차하는 전형적인 Cx의 구조와 일치하였으며, Cx family의 공통${\cdot}$필수적인 배열인 제1세포외 domain의 consensus 배열 및 제2세포외 domain의 consensus 배열도 존재하였다.

• 대한바이러스학회지
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• 제30권1호
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• pp.71-81
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• 2000

We have determined and analyzed the full-length cDNA sequence of a coxsackievirus B3 (CVB3) Korean isolate (CVB3-Korea/97) which has been known as a general human pathogen. The whole genome contains 7,400 nucleotides and has a single large open reading frame with 6,555 nucleotides that encodes a potential polyprotein precursor of 2,185 amino acids. The genome also contains a 5' non-coding region (NCR) of 741 bases and a 3' NCR of 104 bases followed by poly(A) tail. Sequence homologies of nucleotides and deduced amino acids between the CVB3-Korea/97 strain and the prototype (Nancy strain) were 81.7% and 91.5%, respectively. The genes encoding the functional proteins including viral protease and RNA dependent RNA polymerase showed higher homology than those encoding the structural proteins. We have further analyzed the sequences of 5' NCR, VP1 and VP2 of CVB3-Korea/97, which are known as cardiovirulent determining factors at the nucleotide and amino acid levels. Although the CVB 3-Korea/97 strain was isolated from an aseptic meningitis patient without cardiomyopathy, its 234th nucleotide and 165th amino acid were uracil and Asn as same as those of other cardiovirulent strains one. However, the 155th amino acid of VP1, which closely associated with cardiovirulence, was replaced with $Arg^{155}$ by single nucleotide substitution from $A^{2916}$ to $T^{2916}$. Moreover, additional amino acid substitutions were observed in the flanking region of $Asp^{155}$. Taken together, amino acid(s) substitution in VP1 may playa critical role in determining cardiovirulence of the CVB3-Korea/97 strain rather than individual nucleotide replacements in the 5' NCR and/or an amino acid substitution in VP2.

• 미생물학회지
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• 제40권3호
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• pp.183-188
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• 2004

TNT 분해 세균 Stenotrophomonas sp. OK-5는 세 개의 다른 NAD(P)H-nitroreductase의 활성 fractions (NTR fractions I, II, III)을 갖고 있는 것으로 확인된 바 있다. 본 연구에서는 NTR fractions I, II, III에 대한 생리학적 특성과 분자생물학적 특성을 규명하고자 하였다. TNT에 대한 균주 OK-5의 NTR fractions I, II, 그리고 III의 활성은 억제 물질인 $\beta$-mercaptoethanol의 첨가 시에 효소의 활성 이 모두 억제되는 것으로 확인되었다. TNT와 그 유사 기질을 이용하여 균주 OK-5에서 분리된 NTR의 기질 특이성을 조사한 결과, nitrobenzene, 그리고 RDX에 대 해서는 비교적 활성 이 높게 나타났으나 2,6-DNT와 2,4-DNT에서는 낮은 활성을 나타내는 것으로 확인되었다. 균주 OK-5에서 정제된 NTR fraction I의 N-말단 아미노산 서 열은 $^1MSDLLNADAVVQLFRTARDS^20$로 분석되었고, Xanthomonas campestris의 NTR과 X. axonopodis의 NTR에서 각각 70%와 65%로 비교적 높은 유사성을 가지는 것으로 나타났다. 균주 OK-5의 NTR fraction I의 효소를 암호화하는 SmOK5nrI 유전자의 염기서열을 확인하고 분석된 유전자로부터 유추되는 아미노산 서열을 각각 비교한 결과 X. campestris의 NTR과 81%, X. axonopodis의 NTR과 75%,그리고 Streptomyces avermitilis의 NTR과 30%의 유사성이 있는 것으로 조사되었으나, Pseudomonas putida KT2440의 NTR (pnrB)과는 16%로 낮은 유사성이 있는 것으로 확인되었다.