• 제목/요약/키워드: T4 phage

검색결과 46건 처리시간 0.036초

K11 RNA 중합효소의 Cloning 및 발현 (Cloning and Expression of K11 Phage RNA Polymerase)

  • 이상수
    • 자연과학논문집
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    • 제9권1호
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    • pp.19-24
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    • 1997
  • PCR 방법을 이용하여 K11 RNA 중합효소를 coding하는 Klebsiella phage gene 1을 cloning 하였고 lac 전사촉진제 조절 하에 발현시켰다. K11 RNA 중합효소는 DAEA-sephacel과 Affigel blue column chromatographies를 사용하는 상용 방법으로 분리하였다. DAEA-sephacel의 0.2-0.3 M $NH_4Cl$ 분획에서 K11 RNA 중합효소의 활성을 보였고, 다음 단계의 Affigel blue column에서 SDS-polyacryl amide gel 상의 단일 band로 분리되었다. K11 RNA 중합효소는 T7 그룹 phage RNA 중합효소로 다른 T7 그룹phage RNA 중합효소와 많은 상동성을 보인다. (대장균 phage T7, T3과 Salmonella tyhimurium phage SP6 RNA 중합효소). 이미 우리는 T7과 SP6 전사촉진제 변이체를 제조한 바 있고 T7과 SP6 RNA 중합효소의 전사촉진제 특이성을 연구한 바 있다 (이상수와 강창원, 1993). K11 RNA 중합효소의 전사촉진제 특이성을 알아보기 위해 SP6 전사촉진제 변이체를 사용하여 in vitro K11 RNA 중합효소의 활성을 측정하였다. 이 변이체 중 K11 전사촉진제와 가장 유사한 것이 가장 높은 K11 RNA 중합효소 활성을 보였다.

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The Effect of Environmental Factors on Phage Stability and Infectivity on Their Host Bacteria: a Case Study for an Escherichia coli Phage (T7), a Listeria Phage (A511), and a Salmonella Phage (Felix O1)

  • Kim, Kwang-Pyo
    • Food Science and Biotechnology
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    • 제16권3호
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    • pp.398-403
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    • 2007
  • The effectiveness of phage biocontrol depends on the activity of bacteriophage in a given environment. In order to investigate the infectivity and the stability of bacteriophages in representative environments, three virulent phages, Listeria phage A511, Salmonella phage Felix O1, and Escherichia coli phage T7, were subjected to different temperatures, pHs and salt concentrations (NaCl). Phage infectivity was also determined in the presence of divalent cations ($Mg^{2+}$ or $Ca^{2+}$). As a result, three phages exhibited a wide range of survival rates under various environments. Phage infectivity was directly correlated with bacterial growth under the applied conditions. One exception was Felix O1 that did not kill Salmonella grown in low pH (4.5). The failure was attributed to defective adsorption of Felix O1. This finding is significant as it provides an explanation for the inefficient phage biocontrol. Therefore, such information is crucial to improve phage biocontrol of pathogens.

L-글루타민산 생산균 Brevibacterium lactofermentum의 Bacteriophag에 관한 연구 (Studies on the Bacteriophages of Brevibacterium lactofermentum)

  • 이태우
    • 미생물학회지
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    • 제17권3호
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    • pp.97-130
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    • 1979
  • Many industrial processes those employ bacteria are subjected to phage infestations. In L-glutamic acid fermentions using acetic acid, the phage infestations of the organisms have been recently recognized. In efforts to elucidate the sources of phage contamination involved in the abnormal fermentation, a series of study was conducted to isolate the phages both from the contents of abnormally fermented tanks and the soil or sewage samples from the surroundings of a fermentation factory, to define major charateristics of the phage isolates, and finally to determine the correlation between the phage isolates and temperate phages originating from the miscellaneous bacterial species isolated from the soil or sewage samples. The results are summarized as follows; 1) All phages were isolated from the irregular fermentation tanks and soil or sewage samples, and they were designated as phage PR-1, PR-2, PR-3, PR-4, PR-5, PR-6, and PR-7, in the order of isolation. These PR-series phages were proved to be highly specific for the variant strains of Br. lactofermentum only, namely, phage PR-1 and PR-2 for Br. lactofermentum No. 468-5 and phage PR-3~PR-7 for Br. lactofemrentum No. 2256. By cross-neutralization test, the 7 phagescould be subdivided into 3 groups, i. e., phage PR-I and PR-2 the first, phage PR-3, PR-4, PR-5, PR-6 the second, and the phage PR-7 the third. 2) The 7 phages were virulent under the experimental conditions. They produced plaques with clear and relatively sharp margins without distinct halo. The mean sizes of plaques were 1.5mm in diameter for phage PR-1 and PR-2, and 1. Omm for phages PR-3~PR-7. Double layer technique modified by Hongo and described by Adams, was applied to assay of the PR-series phages. The factors influencing the plaques were as follows;young age cells of host bacteria cultured for 3-6 hours represented the largest number and size, optimum was pH 7.0, incubation temperature was $30^{\circ}C$, and agar concentration and amount of overlayer medium were 0.6% and 0.2ml, respectively. 3) PR-series phages were stable in 0.05M tris buffer and 0.1M ammonium acetate buffer solution. The addition of $5{\times}10^{-3}M$ magnesium ion effectively increased the stability. Thermostability experiments indicated that PR-series phages were stable at the teinperture between $50^{\circ}{\sim}55^{\circ}C$ in nutrient medium, $45^{\circ}{\sim}50^{\circ}C$ in buffer solution. However, the phages mere completely inactivated at 603C and 65$^{\circ}$C within 10 minutes. The phages were stable at the range of pH6~9 in nutrient medium and of pH 8-9 in buffer solution, respectively. Exposure of the phages to UV for 25, 60 and 100 seconds resulted in the complete loss of infectivily, respectively. 4) Electron microscopy showed that PR-series phage particles exhibited rather similar morphology, differing in the size All of PR-series phages had a multilateral head and had a simple long tiil about three to five times long as compared with head. By the size, phage PR-1 and PR-2, PR-3, PR-4, PR-5, and PR-6 and PR-7 were classified into same groups, respectively. The head and tail size of phage PR-1, PR-5, PR-5(T) and PR-7 were 85nm, 74nm and 235nm and 350mm, and 72nm and 210nm, respectively. 5) Nucleic acids of PR-series phages were double stranded DNA. The G+C contents of phage PR-1, PR-5 and PR-7 were 56.1, 52.9 and 53.7, respectively. The values of G+C contents derived from the $T_m$ were in agreement with the chemically determined values. 6) PR-series phages effectively adsorbed on their host bacteria at the rate of more than 90% during 5 min. K value for phage PR-1, PR-5 and PR-7 were calculated to be $6{\times}10^9 ml$ per minute, respectiveky. The pH of the medium did effect adsorption rate, but both temperature and age of host cells did not. Generally, optimum adsorption condition of phages seemed to be almost same as optimum growth conditions of host bacteria. 7) In one-step growth experiments, the latent periods at $30^{\circ}C$ for PR-1, and PR-7 were about 70, 50 and 55 min, respectively. The corresponding average burst size was 200, 70 and 90, respectively. Lpsis period according to the multiplicity of infection and a phage series. In case of m. o. i. 100, strain No. 2256 (PR-5) and No. 468-5(PR-1) failed to grow and turbidity decreased after 50 and 70min, respectively. 8) In the lysate of a plaque purified phage PR-5 infected bacteria, there observed 2 types ofphage particles, i. e., phage PR-5 and PR-5 (T) of similar morphology but differing at the length of phage tail, and phage tail like particles. The phage taillike particles could be divided into 4 types by the length. Induction experiments of Br. lactofermentum with UV irradiation, mitomycin C or bacitracin treatment produced neither phage PR-5 (T) or phage tail-like particles. 9) No lysis occured when the growth of 7 strains of miscellaneous bacteria, isolated from soil and sewage samples, were inoculated with either phage PR-5 (T) or phage tail-like particles the inoculation of phage PR-5 pellet resulted in the growth inhibition of the orgainsms in the spot test. The lysates obtained from 3 miscellaneous soil derived bacteria following mitomycin C treatment the growth of Br. lactofermentum, but did not lyze the bacterium.

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Yeast two-hybrid 시스템을 통한 K11 phage lysozyme과 K11 phage RNA 중합효소와의 결합에 대한 연구 (Interaction of phage K11 lysozyme with phage RNA polymerase)

  • 전현정;이상수
    • 자연과학논문집
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    • 제14권2호
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    • pp.83-91
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    • 2004
  • 박테리오 파이지 K11 lysozyme은 최근에 우리 실험실에서 클로닝 되었으며, 숙주균주의 세포벽을 분해하는 고유의 lysozyme활성과 박테리오 파아지 K11 RNA 중합효소의 전사반응을 억제하는 활성을 가지고 있는 것으로 확인되었다. 이미 잘 연구된 박테리오 파아지 T7 lysozyme의 경우 클로닝되고 분리 정제된 T7 lysozyme 단백질의 3차 구조 및 T7 RNA 중합효소와의 결합양상에 대하여 밝혀졌다. 따라서 우리 실험실에서는 K11 lysozyme과 K11 RNA 중합효소와의 결합 정도 및 그 특성을 파악할 목적으로 yeast two hybrid 시스템을 통하여 K11 RNA 중합효소와 K11 lysozyme의 단백질-단백질 상호작용을 알아보고자 하였다. LexA 시스템을 이용하여 LexA DNA 결합 부위를 갖고 있는 pLexA에 K11 lysozyme 유전자를 삽입하여 prey로 하였따. 활성 부위로는 B42 융합 단백질을 만드는 pJG4-5에 K11 RNA 중합효소의 결합은 생체 밖에서 reporter 유전자인 lacZ와 leu2의 발현으로 확인되었으며 이들의 결합정도와 결합부위에 대한 연구들은 진행중에 있다.

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한강에서 분리한 이종 coliphage의 동정과 점토질에 대한 흡착 및 용출효과 (Identification of two coliphages from Han-river and its adsorption-elution effect on soil materials)

  • 홍순우;하영칠;안태석;이영숙
    • 미생물학회지
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    • 제20권4호
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    • pp.210-222
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    • 1982
  • Coliphages isolated from Han-River from September 1980 to August 1981 were classified by morphological and physiological characteristics. Effects of soil metrial on the fate of coliphage in nature were investigated. 1. The correlation coefficient between coliphage and E.coli which was host of coliphages in nature was 0.7173 (p=0.004). 2. Coliphage I isolated from Han-River of which DNA molecular weight was $27{\times}10^6$ daltons was identified as $T_1$ phage and coliphage II of which DNA molecular weight $72{\times}10^6$ daltons was classified as $T_5$ phage. 3. Soil material SW was composed of 63.65% silt and 21.92% clay. Clay was consisted of illite, kaolinite and chlorite evenly. Soil material J was composed of 68.92% silt and 11.67% clay. Clay consisted of smectite only. 4. Coliphage was absorbed to soil material J more than soil material SW, and $T_1$ coliphage was absorbed to soil material more than $T_5$ coliphage was. 5. The phage adsorption efficiency to soil material was enhanced at lower pH : the phage adsorption efficiency at pH 4 was 27 time higher than at pH 7. 6. Divalent $(Ca^{2+})\;and\;trivalention\;(Al^{3+})$ enhanced the phage adsorption efficiency to soil material from 4 to 39 and from 17 to 91 times higher than monovalent $ion(Na^+)$, respectively. 7. The concentration of organic compound was inversely related to the phage adsorption efficiency to soil. 8. Adsorption of phage onto soil material, and elution efficiency of elutants was in the order of D.D.W>tap water>river water>seawater. 9. The higher the concentration of organic compound was, the more were adsorbed phages to soil eluted. 10. Coliphages survived longer in sterile soil suspension than in nonsterile soil material suspension.

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대장균의 회분식 발효에 의해 생산된 덱스트란 결합 파아지를 활용한 설탕 제조공정 오염 검출 (Detection of Sugar Process Contamination Using Dextran Binding hages Produced by Batch Fermentation of Escherichia coli)

  • 김두운
    • 한국식품저장유통학회지
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    • 제15권5호
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    • pp.617-621
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    • 2008
  • Sequential passes through $Sephadex^{TM}$ columns were used to select phages that displays ligands for dextran ($\alpha$-1,6 linked linear chains) from a phage antibody library. Those phages that bound to the $Sephadex^{TM}$ in each iteration were replicated in E. coli. A phage preparation isolated on the third round selection produced 5.4 nephelos turbidity units (NTU) in a dextran specific immunonephelometric assay, a 2.2 fold higher value than the phage preparation from the first round selection. This phage gave $72\;{\pm}\;10$ normalized intensity (N.I.) in a dip-stick assay against high molecular size dextran (T2000, $2\;{\times}\;10^6) and significantly lower color ($30\;{\pm}\;6$ N.I.) against low molecular size dextran (T10, $10^4$). The presence of an Fab insert in each of these phages was confirmed using a $\beta-galactosidase linked assay and polymerase chain reaction.

Bacteriophage T7의 유전자 복제기작에 관한 생화학적, 분자생물학적 특성 연구 (Biochemical and Molecular Biological Studies on the DNA Replication of Bacteriophage T7)

  • KIM Young Tae
    • 한국수산과학회지
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    • 제28권2호
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    • pp.209-218
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    • 1995
  • 본 연구에서는 유전자 복제기작을 생화학적, 분자생물학적 방법을 사용하여 bacteriphage T7을 대상으로 연구하였다. Bacteriophage T7의 유전자 복제, 재조합, 수선시 필수 단백질로 작용하는 gene 2.5 단백질의 생체내 기능에 대한 유전학적 연구와 단백질을 분리 정제하여 복제 단백질들과의 상호작용에 대한 연구를 수행하였다. 연구결과 gene 2.5 단백질은 DNA복제시 필수 구성단백질로 작용하며, 복제과정에서 유전자 복제에 관여하는 핵심 단백질들인 DNA polymerase, helicase/primase와 직접 단백질-단백질 상호 협동 작용을 하는 r것을 증명하였다. 특히 gene 2.5 단백질의 C-terminal domain이 절편된 변이체의 경우 복제 단백질들과 상호작용이 결여되었다. 따라서 C-terminal domain이 gene 2.5 단백질의 기능에 필수적으로 관여함을 입증하였다.

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C형 간염바이러스 E2 단백질에 결합하는 추정 세포수용체 cDNA의 클로닝 (Cloning of cDNA Encoding Putative Cellular Receptor Interacting with E2 protein of Hepatitis C Virus)

  • 이성락;백재은;석대현;박세광;최인학
    • 생명과학회지
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    • 제13권4호
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    • pp.541-550
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    • 2003
  • 본 실험에서는 C형 간염바이러스 (HCV)의 외피 단백질인 E2 당단백질에 결합하는 세포단백질들을 클로닝하기 위해 간세포 cDNA를 phage 표면에 발현시킨 phage library를 제작하였고, 12-mer peptide library와 함께 E2 단백질에 대해 panning을 실시하였다. 검색결과 세포내 신호전달과 cytoskeleton 구성에 관여하는 tensin, membrane protein band 4.1 등 세포질내 단백질과 CCR7, CKR-L2, insulin-like growth factor-1 receptor 등 세포막 단백질 등이 확인되었다. 이들 단백질들을 발현하는 phage들은 수용성 E2단백질을 이용한 결합중화반응 결과 E2 단백질에 특이적으로 결합함이 확인되었다. 사람 T 세포에서 주로 발현되는 CCR7 유전자를 PHA로 활성화된 사람 T 세포의 total RNA를 이용하여 증폭하고 클로닝하였다. 293T 세포에 transfection시켜 단백질 발현양상을 flow cytometer로 분석하여 70% 이상의 세포들이 CCR7을 발현하고 있음을 관찰하였다. 수용성 E2 단백질을 CCR7이 transfection된 세포와 mock transfection된 대조군 세포에 각각 반응시킨 결과 dose-dependent 양상으로 CCR7에 결합하였다.

Mitomycin C에 의해 유도되는 Bacillus cirulans F-2의 Bacteriophage-like 입자 (Bacteriophage-like Particles Induced by Mitomycin C in Bacillus circulans F-2)

  • 김철호;권석태;이대실;타니구치하지메
    • 한국미생물·생명공학회지
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    • 제18권3호
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    • pp.221-226
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    • 1990
  • Bacillu circulans F-2를 포함한 B.circulans의 prophage와 bacteriocin을 검출하기 위하여, mitomycin C를 처리하고 전자현미경상, plaque 형성능(plaque-forming activity), 세균세포살균능(killing activtiy)을 실험했다. killing activity 양성균으로부터 서당밀도균배원심(sucrose gradient centrifugation)을 통해 bacteriophage-like particle의 존재를 확인하였다. 이들 입자들은 형태학적으로 phage tail 또는 T4 phage와 닮은 구조를 가지고 있었다.

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Effects of $K^+$ lon on in vitro RNA Splicing of T4 Phage Thymidylate Synthase Gene

  • Sung, Jung-Suk;Park, In-Kook
    • Journal of Microbiology
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    • 제34권1호
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    • pp.49-53
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    • 1996
  • The effects of K$^{+}$ ion on the activity of RNA splicing of T4 phage thymidylate synthase gene have been investigated. The splicing activity was stimulated within the range of 5 to 20 mM concentration of KCI. When the concentration of KCI in the splicing reaction was brought to 100 or 200 mM a small amount of the exonl-intron product (1, 4 kb) was formed with large proportion of primary RNA transcript not undergoing splicing. This observation strongly suggests that there may exist come kinds of interferences with transesterification at the first step of splicing. Overall it can be concluded that K$^{+}$ ion exhibits very unique roles in RNA splicing of tdd gene depending on its concentration.ion.

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