• Journal of Animal Science and Technology
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• 제45권2호
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• pp.169-182
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• 2003

ADRP 유전자가 24개월령 한우 등심조직에서 발현량이 급격히 증가하여 30개월령 등심조직에서는 발현량이 다소 감소하는 발현양상 분석결과로부터 이전 연구에서는 ADRP 유전자를 한우 성장단계 특이발현 유전자로 선정하였다. 본 연구에서는 ADRP 유전자의 발현조절 기작을 분석하기 위하여 promoter 영역을 포함하는 ADRP 유전자 전체영역을 cloning하였으며, 구조를 분석하고 promoter의 특성을 조사하였다. 한우 ADRP cDNA 단편을 probe로 합성하여 Southern blot 분석을 실시한 결과로부터 ADRP 유전자가 한우 genome 상에서 single copy로 존재하고 크기는 대략 12 kb에 해당하는 것을 확인하였다. Genomic DNA library screening을 실시하여 promoter 영역을 포함하는 ADRP 전체 유전자에 해당하는 clone을 확보하고 HwADRPg-1으로 명명한 후, 염기서열을 결정하고 분석하였다. 한우 ADRP 유전자, HwADRPg-1은 8개의 exon과 7개의 intron으로 구성되어 있으며 모든 exon-intron 경계는 GT/AG 원칙을 따르고 있었고, coding 영역은 7,633 bp로서 6개의 intron에 의해 7개의 exon으로 나누어져 있었다. HwADRPg-1의 promoter 영역에서는 TATAA box는 발견되지 않았으며, -70 위치에 근육 특이적 transcription activator인 Myo G 서열이 존재하였고, -629 위치에는 지방세포의 분화를 유도하는 것으로 알려진 C/EBP (CCAAT/enhancer binding protein) 서열이 존재하였다. HwADRPg-1의 조절영역에 있는 Myo G factor가 근육조직에서 ADRP 유전자가 발현될 수 있도록 하며, 근육의 발달정도를 신호로써 감지하여 근육조직에서 성장단계에 따른 ADRP 유전자의 발현량을 조절할 것으로 추정되고, 다른 종류의 지방세포 특이적인 전사인자 및 지방세포의 분화정도를 신호로 인식하는 전사단계 조절인자를 조사하기 위하여 promoter 영역의 추가분석이 이루어져야 할 것으로 사료된다.

• KSBB Journal
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• 제25권2호
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• pp.167-172
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• 2010

농작물에 심각한 피해를 주는 phytotoxin을 생산하는 S. scabiei ATCC 49173의 분자 유전학적인 연구를 위해 대장균으로부터 S. scabiei로 plasmid DNA를 도입하는 접합 전달법을 이용한 형질전환법을 확립하였다. 본 연구를 통해 확인된 S. scabiei의 접합전달용 최적배지는 50 mM의 $MgCl_2$를 첨가한 MS배지이며 접합전달에 사용되는 DNA 수용체인 포자는 $45^{\circ}C$의 열처리와 $5{\times}10^7$이상의 plasmid DNA 공여체가 필요하다는 것을 확인하였다. 또 얻어진 접합전달체에 대하여 Southern blot hybridization과 벡터가 삽입된 염색체부분의 염기서열분석을 통해 attB site의 특성을 분석한 결과 S. scabiei 염색체의 pirin 상동체를 코드하는 ORF내에 단일위치로 존재하고 있으며 이미 밝혀진 다른 방선균유래 attB site의 염기서열에 대해 86.3%~96.1%의 상동성을 보였다.

• 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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• 제41권2호
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• pp.140-145
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• 2005

상업적으로 중요한 macrolide계 항진균 학생물질인 natamycin을 생산하는 Streptomyces natalensis ATCC27448의 환자 유전학적인 연구를 위해 대장균으로부터 S. natalensis로 plasmid DNA를 직접 도입하는 형질전환법을 확립하였다. 이러한 S. natalensis의 형질전환은 oriT와 attP 단편을 가지고 있는, ${\Phi}C31$ 유래의 integration 벡터인 pSET152를 이용하여 Escherichia coli ET12567/pUZ28002을 DNA 공여체(donor)로 이용한 접합전달법(conjugal transfer)을 사용하여 확립하였다. 접합전달의 가장 높은 효율은 10 mM의 $MgCl_2$를 포함한 MS 배지에서, $6.25\times10^8$의 E. coli 공여체와 열처리를 하지 않은 S. natalensis의 포자를 사용하여 얻어졌다. 또 얻어진 접합전달체 (exconjugant)에 대하여 southern blot hybridization과 벡터가 삽입된 염색체부분의 염기서열분석을 통해 attB site와 pseudo-attB site를 확인하다. attB site의 경우에는 다른 방선균들처럼 S. natalensis 염색체의 pirin 상동체를 코드하는 ORF내에 존재하였으나 pseudo-attB site는 염색체내 다른 site (GenBank accession no. $YP\_117731$)에 존재하였고 그 염기서열은 attB 염기서열과 차이를 나타내었다.

• 한국응용곤충학회지
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• 제38권2호
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• pp.145-149
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• 1999

야생주 핵다각체병 바이러스의 낮은 병원성에 대해 살충제로서의 파밤나방 핵다각체병 바이러스(Spodoptera exigua nucleopolyhedrovirus: SeNPV)의 병원성 향상을 꾀함과 동시에 재조합 바이러스가 다각체 내에 매립됨으로써 살충제로의 적용시 야외에서의 안정성을 확보할 수 있는 p10 유전자의 프로모터를 이용한 새로운 발현벡터를 개발하기 위하여 국내분리주 SeNPV의 p10 유전자 구조를 분석하였다. 그 결과, SeNPV p10 유전자의 프로모터와 구조유전자 부위를 포함한 545염기서열을 결정하여 기존에 보고된 SeNPV p10 유전자(Zuidema et al., 1993)와 비교한 결과 구조유전자 부위에서는 100%의 상동성을 보였으나 5` 및 3` flanking region의 4개의 염기서열에서 차이를 보였다. Southern hybridization에 의하여 SeNPV전체 genomic DNA상에서 p10 유전자는 Sph I 2.4Kb와 Cla I 4.0Kb 단편내에 각각 존재함을 확인하였으며, p10 유전자를 포함하는 이들 단편을 각각 클로닝하여 제한효소 지도를 작성한다.

• 한국육종학회지
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• 제40권4호
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• pp.387-393
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• 2008

Ds 삽입변이체로 부터 5,400개의 FST를 분석한 결과, intragenic FST가 48.1%로 2,597개, intergenic FST가 25.6%로 1,383개였으며 hot spot을 포함한 origin insertional sequence는 1,350개로 25%로 나타났다. Intragenic FST로서 선발된 2,597개를 이용하여 Ds와의 유전자형을 분석한 결과 53.6%인 1,393개가 heterozygous 혹은 homozygous 계통으로 나타났으며 이들에 대한 염색체상의 분포도는 3번 염색체에서 422 계통으로 가장 많은 분포도를 보여주었고 다른 염색체는 56 계통에서 157 계통 범위 내에 포함되었다. 1차적으로 유전자형 분석이 끝난 1,393개의 유전자들 중에서 expressed protein 등 알려지지 않은 것은 40.6%로 566개였으며 TIGR DB에서 염기서열의 유사성 검색을 통해 유전자의 명칭이 알려진 것은 59.4%인 827개로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1189-1196
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• 2005

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

• Fisheries and Aquatic Sciences
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• 제14권1호
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• pp.43-54
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• 2011

Gene structure of copper/zinc-superoxide dismutase (Cu/Zn-SOD; sod1) was characterized in Hemibarbus mylodon (Teleostei; Cypriniformes), an endangered freshwater fish species in Korean peninsula. Full-length cDNA of H. mylodon SOD1 consisted of a 796-bp open reading frame sequence encoding 154 amino acids, and the deduced polypeptide sequence shared high sequence homology with other orthologs, particularly with regard to metal-coordinating ligands. Genomic structure of the H. mylodon sod1 gene (hmsod1; 1,911 bp from the ATG start codon to the stop codon) was typical quinquepartite (i.e., five exons interrupted by four introns); the lengths of the exons were similar among species belonging to various taxonomic positions. The molecular phylogeny inferred from sod1 genes in the teleost lineage was in accordance with the conventional taxonomic assumptions. 5'-flanking upstream region of hmsod1, obtained using the genome walking method, contained typical TATA and CAAT boxes. It also showed various transcription factor binding motifs that may be potentially involved in stress/immune response (e.g., sites for activating proteins or nuclear factor kappa B) or metabolism of xenobiotic compounds (e.g., xenobiotic response element; XRE). The hmsod1 transcripts were ubiquitously detected among tissues, with the liver and spleen showing the highest and lowest expression, respectively. An experimental challenge with Edwardsiella tarda revealed significant upregulation of the hmsod1 in kidney (4.3-fold) and spleen (3.1-fold), based on a real-time RT-PCR assay. Information on the molecular characteristics of this key antioxidant enzyme gene could be a useful basis for a biomarker-based assay to understand cellular stresses in this endangered fish species.

• The Plant Pathology Journal
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• 제30권4호
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• pp.375-383
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• 2014

Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5'-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3627-3629
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• 2016

Background: Activation and inactivation of nuclear factor of kappa light chain gene enhancer in B cells (NFKB) is tightly regulated to ensure effective onset and cessation of defensive inflammatory signaling. However, mutations within NFKB, or change in activation and inactivation molecules have been reported in a few cancers. Although oral squamous cell carcinoma is one of the most prevalent forms of cancer in India, with a development associated with malignant transformation of precancerous lesions, the genetic status of NFKB and relative rates of change in oral precancerous lesions remain unknown. Hence in the present study we investigated all twenty four exons of NFKB gene in two precancerous lesions, namely oral submucous fibrosis (OSMF) and oral leukoplakia (OL) to understand its occurrence, incidence and assess its possible contribution to malignant transformation. Materials and Methods: Chromosomal DNA isolated from twenty five each of OSMF and OL tissue biopsy samples were subjected to PCR amplification with intronic primers flanking twenty four exons of the NFKB gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Sequence analysis identified a novel heterozygous mutation, c.419T>A causing substitution of leucine with glutamine at codon 140 (L140Q) in an OL sample. Conclusions: The identification of a substitution mutation L140Q within the DNA binding domain of NFKB in OL suggests that NFKB mutation may be relatively an early event during transformation. To the best of our knowledge, this study is the first to have identified a missense mutation in NFKB in OL.