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Staphylococcus aureus DH1에서 분리된 R-plasmid pSBK203의 복제조절 유전자 cop의 Cloning, 염기서열 결정 및 상동성 분석
박승문,변우현,Park, Seung-Moon,Byeon, Woo-Hyeon 한국미생물학회 1994 미생물학회지 Vol.32 No.2
Staphylococcus aureus DH1에서 분리된 R-plasmid pSBK203상의 복제개시 인자인 rep 유전자산물의 발현이 어떻게 조절되는가를 밝히기 위해 관련 부위를 확인하고 cloning한 후 그 염기서열을 결정하였으며 이를 같은 계열에 속하는 pT181족 plasmid들의 서열과 그 상동성을 비교 분석하였다. 복제 조절 관련 부위에 염기 삽입 및 염기 결손을 유도함으로써 얻어진 변이체들의 copy수를 측정하여 그 복제 조절 기능에 초래된 변화를 확인하였다. Replication control region of pSBK203, a chloramphenicol acetyltransferase conferring plasmid from Staphylococus aureus was cloned and its nucleotide sequence has been determined. Base sequence homology of this copy control region with those of plasmids belonging to pT181 family was obtained and analyzed. Copy number of four copy mutants derived by addtion or deletion of nucleotides in unique XbaI recognition site in copy control region of pSBK203 was also determined.
Staphylococcus aureus DH1에서 분리한 R-plasmid pSBK203상의 복제개시 부위 ori에 관한 연구
민경일,변우현,Min, Kyung-Il,Byeon, Woo-Hyeon 한국미생물학회 1994 미생물학회지 Vol.32 No.3
Staphylococcus aureus로부터 분리한 R-plasmid pSBK203의 복제개시 단백질인 Rep의 작용부위인 ori 및 dsDNA로의 전환을 위해 요구되는 minus origin부위를 밝히고자 시도하였다. Escherichia coli vecotr를 이용하여 pSBK203의 복제관련 부위를 최소한도로 포함하는 재조합 E.coli-Bacillus subtilis shuttle vector를 구성, 분리하고 여기에 포함된 pSBK203부위의 염기 서열을 분석함으로써 ori를 확인하였다. pSBK203의 복제개시 부위 ori는 rep의 구조 유전자 ORF내에서 약 50bp의 크기로 발견되었으며 지금까지 알려진 staphylococcal plasmid들중에서 pT181족 plasmid들의 ori와 높은 상동성을 갖는 것으로 분석되었다. 복제 과정에서 ssDNA로 먼저 만들어진 (+)쇄가 dsDNA로 전환되기 위해 필요한 신호로 작용하는 것으로 알려저 있는 minus origin (M-O)인 긴 palindrome 구조, 즉 pal 부위가 rep 우전자의 상류에서 2개 연이어 존재하는 것이 발견되었다. 이중에서 pOX6, pC194, 및 pE194 등과 같은 다른 staphylococcal plasmid들의 pal 부위와 비교적 높은 상동성을 갖는 paLA 는 plasmid 유지에 별 영향을 미치지 못하는 반면 다른 plasmid에서 유사 서열이 보고되지 않은 palA는 plasmid 유지에 필수적이라는 사실이 밝혀졌다. The origin of the leading strand replication (ori) and of lagging strand replication (M-O) of R-plasmid pSBK203 was identified and its base sequence was determined. About 50 bp of ori sequence residues overlapped with the structural gene of rep. Sequence comparison reveals that pSBK-ori shares obvious identities with those of pT181 family and consists of two regions, one is conserved and the other is variable region. Of two palindrome sequence located one after another in upstream region of rep gene, palA' instead of palA which shares sequence homology with diverse family of plasmids such as pOX6, pC194, and pE194 seems to act as a signal for conversion of primarily replicated ssDNA to dsDNA (minus origin (M-O)).
Staphylococcus aureus에서 분리된 유발성 ${\beta}$-Lactamase 유전자의 유전적 구성
김영선,민경일,변우현,Kim, Young-Sun,Min, Kyung-Il,Byeon, Woo-Hyeon 한국미생물학회 1994 미생물학회지 Vol.32 No.1
An inducible ${\beta}$-lactamase gene (bla) was identified and isolated from the chromosomal DNA of multiple drug resistant strains of Staphylococcus aureus. Determined base sequence of bla and of its flanking region was compared with those of bla genes identified on the staphylococcal plasmids pPC1, pI258, pI1071, and pUB101. Base sequence of 843 base-long structural gene of our bla was same as that of pPCl-, pI258-, and pS1-bla. However, HindIII recognition site Which is found in most of the bla genes at 140 base upstream from the structural gene was moved to the site of 370 base upstream from the structural gene. And one of the two direct repeat sequence found in downstream flanking region of pI1071-bla was deleted in our bla. Amino acid sequence homology analysis of the ORF located around HindIII recognition site reveals that this 80 amino acids-long polypeptide is C-terminus of transposase of Tn4001. 항생물질에 대한 다중 저항성을 갖는 Staphylococcus aureus 균주의 chromosomal DNA로부터 유발성 발현을 하는 ${\beta}$-lactamase(bla) 유전자를 확인, 분리하였다. Cloning에 이어 결정된 염기서열을, S. aureus 에서는 지금까지는 plasmid상에서만 분리, 보고되어 있는 bla 유전자들의 염기서열과 비교하였다. 본 bla 유전자의 구조유전자 부분인 843base의 염기서열은 기 발표된 pPC1, pl258, pS1, pI1071, pUB101, pl3796 및 pI3804 유래의 bla 유전자들 중 pPC1, pI258 및 pS1상에 존재하는 bla 구조유전자의 염기서열과 완전히 일치하였고 나머지 것들과도 매우 높은 상동성(99%)을 유지하고 있었다. Bla구조 유전자의 상류 370base 및 하류 220base까지 결정된 염기서열을 비교한 결과에서는 다른 모든 bla구조유전자의 상류 150base에 위치하는 HindIII 인식부위가 약 230base 이상 더 윗쪽으로 옮겨가 있었고 이 HindIII 인식부위를 포함하는 염기서열에서 ORF의 C말단이 발견되었다. 하류 서열에서는 pI1071 유래 bla가 갖는 두개의 직접반복 염기서열 중 하나가 결손된 형태를 보이고 있다. 구조 유전자 상류에 존재하는, 80개 아미노산으로 구성된 ORF의 상동성 검색 결과 Tn4001 의 transposase 의 C 말단과 일치함이 발견되었다.
낮은 복제수 플라스미드를 활용한 일본뇌염바이러스 SA14-14-2주의 Full-Length cDNA 클론의 제작및 안정적 유지
민경일(Kyung-Il Min),김영민(Young-Min Kim),추미성(Mi-Sung Choo),백선영(Sun-Young Baek),김재옥(Jae-Ok Kim),류승렬(Seung-Rel Ryu),민복순(Bok-Soon Min),김연희(Yeonhee Kim),박미경(Mi-Kyung Park),변우현(Woo-Hyeon Byeon),허숙진(Sook-Jin Hu 대한미생물학회 2004 Journal of Bacteriology and Virology Vol.34 No.4
Salmonella typhimurium 내의 plasmid pKM101 존재와 유전자 uvrB 결손이 화학물질의 돌연변이 유발성과 독성에 미치는 영향
이세영,변우현 한국유전학회 1979 Genes & Genomics Vol.1 No.1
The effects of uvrB deletion and plasmid pKM101 on mutagenesis and cell death of Salmonella typhimurium were studied using 10 potent known chemical mutagens (MNNG, MMS, EMS, DES, Sodium azide, 4-NQO, AF-2, Niridazole, MT-C, and Ad-m). MNNG-, EMS-, and DES-induced mutagenesis were not affected in the presence of pKM101 gene product. MMS-induced mutation, however, was plasmid pKM101 dependent. R- strains were not mutagenized by the MMS treatment. Generally, alkylating agent-induced cell death was not affected by uvrB gene product. 4-NQO and AF-2 induced UV-mimic DNA damage. The effects of pKM101 and uvrB deletion on 4-NQO-induced and AF-2-induced mutagenesis and toxicity were similar to those of UV-induced mutagenesis and toxicity. Niridazole showed both UV-mimic and highly lethal effect on uvrB^- strains while MT-C and Ad-m showed less lethal effect. Plasmid pKM101 gene product showed some protection effect on MNNG, MMS, 4-NQO, and AF-2 cytotoxicities. On the other hand, no effect was observed on the toxicity caused by EMS, DES, Niridazole, MT-C, and Ad-m. All the tested compounds except alkylating agents and MT-C enhanced mutagenesis by uvrB deletion and plasmid pKM 101. The uvrB gene produce seemed to be needed in mutagenesis by MT-C.
李敏載,河永七,李光雄,邊宇玄 서울대학교 1975 서울대학교 論文集 Vol.25 No.-
Serveral physiological characterstics of sulfuroxidizing bacteria such as Thiobacillus thiooxidans and T. concretivorus are described. 1) Effect of pH on inhibitory function of pyruvate. The rate of oxygen uptake was reduced in accordance with decrease of pH. And on the other hand inhibition rate of pyruvate was more severe in lower pH than in higher pH. High concentration of hydrogen ion seemed to accelerate inhibitory function of pyruvate. Limiting concentration of pyruvate which absolutely inhibit oxygen uptake was lowered with time. In O time, 10^-2M of pyruvate absolutely inhibited oxygen uptake in pH 5. After 24 hours limiting concentration was lowered to 10^-3M. 2) Effect of organic compounds on the oxygen uptake of T. concretivorns. Effect of glucose, fructose, xylose, glutamate, succinate, malate, glycine, lactate, acetate, pyruvate, citrate, formate, and cisaconitate on thiosulfate oxidation and availability of these compounds as sloe source of energy by T. concretivorus were observed. A 0.5% concentration of malate and glycine accelerated thiosulfate oxidation almost react somewhat inhibitory. Pyruvate and citrate inhibited thiosulfate oxidation. In thiosulfatefree medium, organic compounds except formate and pyruvate affected no significant influences on oxygen uptake. 3) Enzyme assay. Of enzymes concerned TCA cycle and glycolysis, T. concretivorus had most of those enzyme activities even though they were low. Activities of hexokinase and succinic dehydrogenase were somewhat high and that of aconitase was very low on the contrary. 4) Optimum condition of mass culture of sulfur oxidizing bacteria. In a large jar fermenter opimum conditions including agitations speed, volume of supplying gases, and constitution rate of oxygen and carbon dioxide, etc. were experimented. The sulfuroxidier, T. thiooxidans was grown most effectively under the condition of automatically controlled pH of 1.0, and with the aeration of oxygen and carbon dioxide mixture (5 : 1) at the flow rate of 0.05 vvm, and at the agitation velocity of 200 rpm in this laboratory scale fermenter.