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접합전달을 이용한 Streptomyces natalensis ATCC27448의 형질전환 최적화 및 attB-site의 특성연구
이강무,최선욱,박해룡,황용일,Lee Kang-Mu,Choi Sun-Uk,Park Hae-Ryong,Hwang Yong-Il 한국미생물학회 2005 미생물학회지 Vol.41 No.2
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. Streptomyces natalensis ATCC27448 produces natamycin, a commercially important macrolide antifungal antibiotic. For molecular genetic study of S. natalensis, we have developed a system for introducing DNA into S. natalensis via conjugal transfer from Escherichia coli. An effective transformation procedure for S. natalensis was established based on transconjugation from E, coli ET12567/pUZ8002 using a ${\Phi}C31$-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 10 mM $MgCl_2$ using $6.25\times10^8$ of E.coli donor cells without heat treatment of spores. In addition, southern blot analysis of exconjugants and the sequence of plasmids containing DNA flanking the insertion sites from the chromosome revealed that S. natalensis contains a single ${\Phi}C31$ attB site and at least a secondary or pseudo attB site. Similar to the case of various Streptomyces species, a single ${\Phi}C31$ attB site of S. natalensis is present within an ORF encoding a pirin-homolog, but a pseudo-attB site is present within a distinct site (GenBank accession no. $YP\_117731$) and also its sequence deviates from the consensus sequences of attB sequence.
이민아(Lee Minah),이강해(Lee Kang Hae),강석규(Kang Seok Kyu) 한국통신학회 2021 한국통신학회 학술대회논문집 Vol.2021 No.6
본 논문은 국제공식표준화기구인 ITU-T 표준화자문그룹(TSAG; Telecommunication Standardization Advisory Group)에서 논의되고 있는 ITU-T 작업방법 표준화 관련 동향을 분석하고 이에 따른 향후 논의 방향 예측 및 대응 방안을 제시하였다.
Streptomyces griseus IFO13350 유래 sprA 및 sprB 유전자를 이용한 Pretense 생산균주 개발
황지환,이창권,이강무,조병기,박해룡,황용일,Hwang Ji-Hwan,Lee Chang-Kwon,Lee Kang-Mu,Jo Byoung-Kee,Park Hae-Ryong,Hwang Yong-Il 한국미생물학회 2005 미생물학회지 Vol.41 No.1
Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp. 방선균 Streptomyces griseus에서 상업적 목적으로 생산되는 protease인 protease는 serine protease, alkaline protease, aminopeptidase및 carboxypeptidase로 구성되어 있는 복합체로서 의 약용 소염제로 널리 사용되어지고 있다. 본 연구에서는 기존에 개발되어 있는 방선균용 integration vector인 pSET152로부터 목적산물의 대량발현을 위해 방선균용 promoter ermE가 cloning된 새로운 integration vector인 pHJ101을 개발하였고, pretense의 생산량 증대에 사용하였다. 새로 개발된 integration vector에 S. griseus protease A를 코드하고 있는 유전자, sprA와 S. griseus pretense B유전자, sprB를 각각 cloning하여 plasmid pHJ201과 pHJ202를 구축하였다. 이들 plasmid들을 S. griseus IFO 13350에 형질전환하여 발현용plasmid가 chromosome에 integration된 재조합 균주 S. gliseus HA와 S. griseus HB를 얻었다. 이들 재조합균주로부터 전체 protease의 생산량을 확인한 결과, 모균주보다 각각 S. griseus HA는 약 5.3 배, S. griseus HB는 약 5 배 정도 생산량이 증대되었다. 이들 결과로부터 특정유전자의 고발현용 integration vector의 제작이 확인되었으며, 전체 protease의 생산량 증대의 가능성이 시사되었다.
갑상선 유두미세암에서 중심 림프절 전이를 예측할 수 있는 인자에 관한 분석
안소은,김준호,이강율,임영아,이연옥,김해성,김이수,So-Eun Ahn,Jun Ho Kim,Kang Yool Lee,Young-Ah Lim,Younok Lee,Hae Sung Kim and Lee Su Kim 대한갑상선-내분비외과학회 2012 The Koreran journal of Endocrine Surgery Vol.12 No.4
Purpose: Despite the excellent overall prognosis for patients with papillary thyroid microcarcinoma (PTMC), the rate of central lymph node (CLN) metastasis has been reported to be as great as 60% and the optimal surgical extent of PTMC has been controversial. The aim of this study is to identify factors for predict CLN metastasis in patients with PTMC. Methods: We conducted a retrospective study of 535 patients with PTMC who underwent total thyroidectomy with prophylactic CLN dissection between Jan. 2008 and Aug. 2011. We analyzed the association of CLN metastasis and clinicopathologic characteristics. Results: CLN metastasis was found in 181 patients (33.8%). Results of univariate analysis showed an association of younger than 45 years of age, male gender, a tumor size greater than 5 mm, bilaterality, multiplicity, extrathyroidal extension, and positivity of resection margin with CLN metastasis. Of these, results of multivariate analysis showed that age (P=0.003), gender (P=0.004), tumor size (P<0.001), extrathyroidal extension (P=0.001), and status of resection margin (P=0.002) were independent predictive factors for CLN metastasis. Conclusion: A large tumor size (>5 mm), male gender, young age (<45 yr), extrathyroidal extension, and positive resection margin were determined as the predictive factors for CLN metastasis, which occurred in approximately one third of patients with PTMC. Therefore, prophylactic CLN dissectionshould be considered in patients with PTMC who have these factors through investigation before surgery