심포지움 초록 ; MRSA 의 β - 락탐 내성기구와 그 유래

송민동 한국생화학분자생물학회 (구 한국생화학회) 1991 추계학술대회집 Vol.- No.-

임상분리된 Staphylococcus aureus의 항생제 내성에 관하여

송민동,장현규,정해만 中央醫學社 1990 中央醫學 Vol.55 No.2

This study was carried out to study the resistant gene evolution of the methicillinresistant Staphylococcus aureus (MRSA) and the mechanism of vancomycin-resistant Staphylococcus aureus in MRSA strains. Minimal inhibitory concentration (MIC) test of 48 strains which were isolated from two general hospitals at Kwang Ju City, Korea to 9 antibiotics; five 0-lactams, kanamycin, tetracyclin, chloramphenicol and vancomycin, was evaluated by using agar dilution method. The results obtained are as following; According to test of MIC distribution on 9 antibiotics against S. aureus, -lactams ranged from 0.39 to > 500 g/ml, chloramphenicol ranged from 3.13 to 250 g/ml, tetracyclin ranged from 6.25 to 500 g/ml, kanamycin ranged from 12.5 to > 500 pg/ml and vancomycin ranged from 3.13 to > 500 g/ml. Among these antibiotics, vancomycin showed excellent antimicrobial activities to clinical isolates of S. aureus. Vancomycin-sensitive strain was isolated from vancomycinresistant strain which was isolated from clinical isolates of MRSA strain with treatment at 43.50C. MIC distributions of the vancomycin-sensitive S. aureus strain are as followings; MIC of vancomycin-resistant strain to -lactams ranged from 6.25 g/ml to > 500 g/m1, but vancomycin-sensitive strain ranged from 0.39 g/ml to 1.56 g/ml. MIC of vancomycin-sensitive strain against chloramphenicol was 3.13 g/ml, tetracyclin was 0.78 pg/ml, kanamycin was 1.56 g/ml and vancomycin was 1.56 g/ml respectively.

PCR을 이용한 MRSA균주의 비유도성 β-락탐항생물질 내성유전자의 클로닝

宋泯東,金香,朴台奎,李廣鎬,李知英 건국대학교 1997 學術誌 Vol.41 No.2

All clinical strains of Methicillin-Resistant Staphylococcus aureus(MRSA) examined so far contain the MRSA-PBP gene(mecA) and its gene product, a novel penicillin-binding protein (PBP)-2'or 24 which has the very low affinity to β-lactam antibiotics such as penicillin, ampicillin, and β-lactamase-resistant β-lactam antibiotic such as methicillin. MRSA- PBP (Mw. 75,000 Dal) is responstable for the resistant to β-lactams and have a characteristics of inducible production by contact with β-lactams. Expression of merA is regulated by regulatory genes such as mecRI and mecI, which are located upstream of the MRSA-PBP structural gene in the opposite direction. MRSA TK731 strain produced constitutively MRSA-PBP, whether present β-lactams or not. In this report, we performed to study the mechanism of MRSA-PBP production at the molecular level. Therefore, we amplified the interested DNA fragments contained MRSA-PBP gene and iris upstream region by polymerase chain reaction(PCR) and subcloned into E. coli plasmid vector.

황색포도상구균에 있어 비유도성 β-락탐 항생물질 내성원인 유전자의 클로닝과 발현

송민동,김향,이광호,이지영 中央醫學社 1997 中央醫學 Vol.62 No.4

Methicillin-Resistant Staphylococcus aureus(MRSA) isolated clinically from hospitals has a characteristics of highly resistant to not only β-lactam antibiotics such as penicillin, but also amino glycosides, macrolides which have differences in the mechanism of targets. All of the MRSA strains examined so far contain the MRSA-PBP gene(mecA) of 2,010 base pairs(bp) in size and its gene product-MRSA-PBP(Mw.76,462), a novel penicillin-binding protein (PBP)-2' or 2A that has very low affinities to the most β-lactam antibiotics. In most of MRSA strains such as TK784, MRSA-PBP produced inducible by contact of the cells with β-lactams. Expression of mecA is regulated by some regulatory genes such as mecRi and mecI1011'1, which are located upstream of the MRSA-PBP structural gene in the opposite direction. However, some strains such as TK731 produced constitutively MIRSAPBP, whether β-lactams is being or not. In this report, we amplified the interested DNA fragments contained MRSA-PBP gene and its upstream region by polymerase chain reaction(PCR) and sub cloned into E. coli plasmid vector and confirmed the expression of MRS-PBP in E. coli to study the uneducable mechanism of MRSA-PBP production in MRSA strains at the molecular level.

MRSA 균주의 mec B 유전자의 염기배열

송민동 건국대학교 생명과학연구원 1994 생명과학지 Vol.1 No.-

The nucleotide sequence of the mec B, which may encode the repressor or regulatory protein for the expression of the structural gene of MRSA-PBP(or PBP-2') that is responsible for the highly β-lactam resistance in methicillin-resistant Staphylococcus aureus(MRSA) strain, has been determined. The expression in in vitro translation system of mec B gene had been reported(Song, 1992). The size of the longest open reading frame(ORF) is 507 bases encoding a protein of predicted size 19,572 daltons. A segment of the mec B gene sequence(including the promoter region) was found in inverted directions at 100 bases upstream of the mec A gene in MRSA(Song et al, 1987) and the proposed promoter for mec B gene overlaps mec A gene.

안면견갑상완형 근 디스트로피의 3.3kb 반복배열의 결실에 관하여는 단백질 검색을 위한 PCR-primer의 합성

이지영,송민동 建國大學校 自然科學硏究所 1997 建國自然科學硏究誌 Vol.8 No.-

Facioscapulohumeral muscular dystrophy(FSHD) is a autosomal-dominant muscular disorder initially affecting facial and shoulder-girdle muscles. The major FSHD locus(FSHD1) was assigned to the distal long arm of chromosome 4(4q35-ter.) by linkage analysis. DNA-rearrangements within a polymorphic EcoRI restriction fragment were detected in FSHD patients using probe p13E-11 or pFR-1. These rearrangements were found to be due to deletion of an integral number of 3.3kb tandemly repeated units(D4Z4). In this paper, we report the preparation of the PCR primer sets for the detection of the proteins which may be responsible for the delection of 3.3 kb tandem repeated sequences within a polymorphic EcoRI restriction fragment.

안면견갑상완형 근 디스트로피(FSHD)환자 염색체중 10q26 좌위로부터 p13E-11 EcoRⅠ 특이적 DNA 단편 유전자 클로닝

김대영,Arahata, Kiichi,송민동 건국대학교 자연과학연구소 1999 建國自然科學硏究誌 Vol.10 No.2

안면견갑상완형 근 이양증(facioscapulohumeral muscular dystrophy ; FSHD)은 상 염색체 우성의 근육질환으로, 안면근 및 견갑근이 약화되면서 심한 경우에는 하지대까지 약화하여 휠체어생활을 하기도 한다. FSHD 원인유전자는 현재까지 밝혀져 있지 않으나, 유전자 연관분석에 의하여, 그 유전자 위치(gene locus)는 4번 염색체 장완 말단(4q35-ter)에 위치하고 있음이 알려져 있다. 또한 탐침 DNA (p13E-11, pFR-1)을 이용한 게놈 분석 (Southern blot)에서는 특이적인 EcoRI 패턴을 보이고 있다. 본 연구에서는 FSHD가계의 염색체중 4q35와 상동성이 높은 10q26 염색체로부터 p13E-11- EcoRI 특이적인 DNA 단편을 클로닝하여 4q35와의 상동성 등 유전자 해석을 실시하였다. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder initially affects facial and shoulder-girdle muscles. The gene locus for major FSHD (FSHDI) has been assigned to the distal long arm of chromosome 4(4q35-qter) by linkage analysis. The DNA rearrangements within the polymorphic EcoRI restriction fragments were detected in patients with FSHD using the probes p13E-11 and/or pFR-1. These rearrangements were found due to deletions of the putative integral numbers of 3.3kb tandemly repeated units (D4Z4) within the EcoRⅠ fragment. In the present study, some researchers performed genomic Southern blot analysis using the probe p13E-11 in one FSHD family (SU, OH) where some members were clinically diagnosed as having FSHD1 which is derived from 4q35. In this paper, we cloned the p13E-11 EcoRⅠ-specific DNA fragments (OH21,OH17,OH14, OH10) in 10q26 locus using the in vitro packaging and plaque hybridization and analysed the nucleotide sequences between putative 4q35 FSHD and 10q26 locus.

안면견갑상완형 근 디스트로피의 3.3kb 반복배열의 결실에 관여하는 단백질 검색

송민동,김대영,이지영 건국대학교 자연과학연구소 1998 建國自然科學硏究誌 Vol.9 No.1

안면견갑상완형 근 디스트로피(FSHD)는 최초 안면과 견갑근에 영향을 주는 상 염색체(autosomal-dominant)우성의 근육 질환이다. 주요한 FSHD(FSHDI) 유전자의 유전자 좌(locus)는 4번 염색체 장완의 말단(4q35-ter)에 위치하고 있음이 유전자 연쇄해석에 의하여 밝혀져 있다. Probe p13E-11 또는 pFR-1을 이용한 genomic Southem blot결과, FSHD 환자에게는 정상인보다는 짧은 p13E-11EcoRI 단편 다형성(polymorphism[<~30kb]을 나타내고 있다. 이러한 현상은 p13E-11-EcoRI 단편 내에 존재하고 있는 3.3kb 반복배열의 결실에 의해 일어나고 있음이 알려져 있다. 본 연구에서는 이들 3.3kb 반복배열의 결실에 관여하고 있는 특이적인 단백질을 검색하기 위하여, 이들 3.3kb 반복배열단위를 중심으로 primer set를 작성하였으며, 이를 이용하여 3.3kb 반복배열의 연결부위(junction region)[primer set 1,2,3]를 polymerase chain reaction(유전자 중폭반응; PCR)으로 증폭하였다[probe A,B,C]. 이들과 결합하는 단백질을 검색하기 위해, E.coli(XL1-Blue MIRA) 와 phage의 막을 분리하여 mobility shift assay를 실시한 결과, 본 연구에서는 probe B 와 E.coli(XL1-Blue MRA) 의 crube extract에서 특이적으로 결합하는 단백질을 검출하였다. Facioscapulohumeral muscular dystrophy(FSHD) is an autosomal-dominant muscular disorder initially affects facial and shoulder-girdle muscles. The gene locus for major FSHD(FSHDI) has been assigned to the distal long arm of chromosome 4 (4q35-qter) by linkage analysis. The DNA rearrangements within the polymorphic EcoR I restriction fragments were detected in patients with FSHD using the probes p13E-11 and/or pFR-1. These rearrangements were found due to deletions of the putative integral numbers of 3.3kb tandemly repeated units(D4Z4) within the EcoR I fragment. In the present study, we could 21kb-EcoRI fragment(OH21) which is related to FSHD 4q35 and consists of four 3.3kb-Kpn I repeated units. We packaged 21kb-EcoR I fragment in the EB phage particle and found that the size of OH21 clone in the EB phage vector was decreased to 17 kb, 14kb, 10kb during the amplification period of the phage. From these results, we assumed that in the phage or in the host cell(XL1-Blue MRA), there must be proteins that may be responsible for the putative deletion of 3.3kb-Kpn I tandemly repeated units. To investigate this hypothesis, we performed PCR using synthesized PCR primer sets that can amplify sites of the junction of 3.3kb-Kpm I tandemly repeated sequences, and then obtained three kinds of PCR products. And then we subclone. At this study, we detected the DNA-binding proteins in crude extract from XLI-Blue MRA.

황색포도상구균의 β - 락탐 항생물질의 내성기구 발현에 관한 연구

송민동 ( Min Dong Song ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.4

MRSA(methicillin-resistant Staphylococcus aureus) has a unique novel protein, Mec A protein, which is responsible for methicillin-resistance and has a characteristic inducible-synthesis system by β-lactam antibiotics. Previously, DNA sequences of 4 kb HindIII fragment containing mec A gene were determined, anaylsed. In this fragment, two open reading frame (ORF) had been reported; one is mec A structural gene, which encoded Mec A protein (670 amino acids residues), and another one which encodes 169 amino acids residues, what we called, is mec B gene which located on 100 base pairs (bp) upstream of the mec A structural gene, which also may encode for some regulatory protein, but not yet identified mec B gene product. And the functions of mec B gene are poorly understood. This present work has shown that the inverted-directed ORF (mec B) for the mec A structural gene encoded the protein of about 18,000 daltons using in vitro translation system.

Xylanase와 CMCase의 생산성이 높은 Bacillus sp.의 분리 및 동정

김창원,송민동,정원형,양시용 건국대학교 동물자원연구센터 2000 動物資源硏究誌 Vol.21 No.-

CMCase와 xylanase를 동시에 분비하는 미생물을 얻기 위해 1차적으로 111균주를 분리한 후, 활성이 가장 우수한 1-5균주를 선발하였다. 선발된 균을 API 20과 API 50 CHB kit를 이용하여 동정한 결과 Bacillus licheniformis로 동정되었다. Nutrient broth를 배양배지로 하여 growth와 효소역가를 측정한 결과 cell growth growth는 20시간 후에 stationary phase에 도달하였으며 xylanse는 8시간 배양 후 최대의 역가를 나타냈고, CMCase는 28시간 후 최대의 역가를 나타냈다. A bacterium producing xylanase and CMCase was isolated from soil, has been identified as Bacillus sp. The isolate, named Bacillus sp.1-5, was shown to be very similar to Bacillus licheniformis on the basis of its biochemical and physiological properties. In culture time, xylanase and CMCase production reached to the plateau after 8hr and 28hr of cultivation in the nutrient broth.