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Role of T7 phage lysozyme affected sequence-specific transcription termination by T7 RNA polymerase (염기서열 특이적 전사종결에 영향을 주는 T7 파아지 lysozyme의 역할)

  • Kim, Dong-Hee;Lee, Sang-Soo
    • The Journal of Natural Sciences
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    • v.14 no.1
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    • pp.51-61
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    • 2004
  • T7 RNA polymerase is a single subunit RNA polymerase able to accomplish whole transcription process without auxiliary factors. T7 phage lysozyme involcing in destruction of host cell wall repress T7 transcription and affects transcription termination process. Therefore expression vector pT7lys containing T7 phage lysozyme gene was constructed and expressed. T7 phage lysozyme protein was purified to homogeneity by Ni-NTA column chromatography. Also amidase activity of the purified lysozyme was identified. In order to understand the effect of the lysozyme on the sequence specific transcription termination. T7 transcription elongation complexes at the site rrnB T1 transcription termination signal were made in the presence the lysozyme. The results shows that the transcription elongation complexes are unstable in the presence of T7 phage lysozyme.

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Funcyional Studies on Gene 2.5 Protein of Bacteriophage T7 : Protein Interactions of Replicative Proteins (박테리오파아지 T7 의 기능에 관한 연구;복제단백질간의 단백질 상호작용)

  • 김학준;김영태
    • Journal of Life Science
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    • v.6 no.3
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    • pp.185-192
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    • 1996
  • Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

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T7 RNA Polymerase Is Expressed in Plants in a Nicked but Active Form (T7 RNA polymerase 유전자의 담배식물에서의 발현)

  • Caviedes, Miguel A.;Thornburg, Robert W.;Park, Sang-Gyu
    • Applied Biological Chemistry
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    • v.40 no.4
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    • pp.271-276
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    • 1997
  • We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)

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Efficacy of Cyanobacterial Biofertilizer (CBB) on Leaf Yield and Quality of Mulberry and its Impact on Silkworm Cocoon Characters

  • Dasappa D.M. Ram Rao;Ramaswamy S.N.
    • International Journal of Industrial Entomology and Biomaterials
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    • v.13 no.1
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    • pp.15-22
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    • 2006
  • An experiment was conducted to study the efficiency of cyanobacterial biofertilizer (CBB) with chemical (NPK) fertilizer on quantitative and qualitative characters of mulberry variety Kanva-2. Their influences on silkworm growth and cocoon characters were also studied. Ten different CBB and NPK fertilizer treatments were given to 5000 plants of established mulberry garden. Treatments were of four types viz., (i) T1 to T7: single and combination dose of CBB+50% NPK (ii) T8: combination dose of CBB + 25%NPK, (iii) T9: CBB only and (iv) T10: control-l00% NPK. Soil pH decreased and nutrients status increased in CBB (T1- T9) treated plots. Average of ten crops data on quantitative traits revealed that T7 (CBB [N. muscorum (1.0 g), A. variahilis (1.0) and S. millei (1.0 g)] + 50% NPK) was very effective in improving growth parameters. Leaf yield was also found high in treatment T7 (32.12 tons/ha/yr.) followed by T10 (31.17 tons/ha/yr.) and T8 (27.67 tons/ha/yr.). Leaf quality characters were found high in T7 and low in T9. Most of the quality traits in T7 are on par with control no. The results revealed that reduction in the dose of chemical fertilizers in T7 did not affect the leaf yield and leaf quality traits of mulberry. This clearly indicates that the efficiency of CBB (T7) provides nitrogen, increases essential nutrients available in soil, maintain soil pH and supply growth substances required for the improvement of leaf yield and leaf quality of mulberry. Bioassay study also revealed no significant difference in silkworm growth and cocoon characters between treatments T7 and T10. Economics calculated revealed that T7 is highly economical and beneficial over T10 by gaining an amount of Rs. 660/-/acre/crop. Thus, treatment T7 containing N. muscorum (1.0 g), A. variahilis (1.0 g) and S. millei (1.0 g) + 50% NPK fertilizers can be recommended to sericulturists mainly to reduce the use of NPK fertilizers, by saving 50% of its cost and to improve soil fertility conditions, which in turn improves leaf yield and quality of mulberry.

Effect of Cutting Times according to Growth Stage in Sorghum X Sudangrass Hybrid on Frequence of Use, Growth Characteristics, Forage Production and Crude Protein Yield (생육단계별 예취 시기가 수수 X 수단그라스 교잡종의 이용회수, 생육특성, 수량 및 조단백질수량에 미치는 영향)

  • Jeon Byong Tae;Lee Sang Moo
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.25 no.1
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    • pp.33-42
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    • 2005
  • A field experiment was conducted to evaluate growth characteristics, forage production and crude protein yield according to cutting time of Soghum ${\times}$ Sudangrass Hybrid, and decide ideal harvesting time for use of soiling and silage. Experiment design was arranged with 7 different treatment T1(150 m), T2(200 cm), T3(boot), T4(heading), T5(milk), T6(dough) and T7(yellow stage), as a randomized block design. The results were as fellows : Cutting times of utilization during the course of a year was 4 times at T1 and T2, 3 times at T3 and T4, and 2 times at T5, T6 and T7. Accumulative plant length was the highest at T2(666cm), but T3 was the lowest as 402 cm. Mean Leaf length was the highest at T5(82.1 m) and lowest at T7(T1.8 m). Mean leaf width was the highest at T2 and lowest at T6. Stem diameter was orderly ranked as T3(10.7 mm)>T1(9.5)>T2, T5(9.3>T6(8.9)>T7(8.6)>T4(8.5). Stem hardness was orderly ranked as $T7(3.2 kg/cm^2$>T5, T6(2.3)>T3, T4(1.5)> T2(0.6)>T7(8.6)>T1(0.5). Mean of leaf number and leaf ratio was the highest at $T3(8.1\%)$ and $T2(45.3\%)$, respectively. The highest yield of fresh and dry matter was obtained at T4 and T6 as 113,246 and 24,249 kg/ha, respectively(P<0.05), and e lowest at T7 and T1 as 82,675 and 13,006 kg/ha, respectively(P<0.05). Crude protein yield was highest at T6(1.456 kg/ha) and lowest at T3 as 1,189 kg/ha. As mentioned above the result T1, T2 and T3 could be recommended as use of soiling, and T5, T6 and T7 as silage.

Selective overproduction of chloramphenicol acetyltransferase in the T7 expression system (T7 발현체계에서 chloramphenicol acetyltransferase의 선택적 과잉생산)

  • 김한복;강창원
    • Korean Journal of Microbiology
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    • v.27 no.4
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    • pp.317-322
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    • 1989
  • A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

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Analyzing the Significance of T1 Slope minus Cervical Lordosis in Patients with Anterior Cervical Discectomy and Fusion Surgery

  • Lee, Ho Jin;You, Soon Tae;Sung, Jae Hoon;Kim, Il Sup;Hong, Jae Taek
    • Journal of Korean Neurosurgical Society
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    • v.64 no.6
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    • pp.913-921
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    • 2021
  • Objective : Accurate measurement of T1 slope (a component of T1s minus cervical lordosis [CL]) is often constrained by anatomical limitations. In this situation, efforts should be made to find the exact meaning of T1s-CL and whether there are any alternatives to it. Methods : We enrolled 117 patients who received two-level anterior cervical discectomy and fusion (ACDF). Occipital slope, C2 slope (C2s), C7 slope (C7s), T1, O-C2 angle (O-C2A), C2-7 angle (C2-7A), O-C7 angle (O-C7A), T1s-CL, C7-T1 angle (C7-T1A), and C2-7 sagittal vertical axis were measured. We determined 16° (T1s-CL) as the reference point for dividing subjects into the mismatch group and the balance group, and a comparative analysis was performed. Results : The mean value of C7-T1A was constantly maintained within 2.6° peri-operatively. In addition, C2s and T1s-CL showed the same absolute change (Δ|0.8|°). The mean values of T1s-CL of the mismatch and balance groups were 23.0° and 7.6°, respectively. The five factors with the largest differences between the two groups were as follows : C2s (Δ13.3°), T1s-CL (Δ15.4°), O-C2A (Δ8.7°), C2-7A (Δ14.7°), and segmental angle (Δ7.9°) before surgery. Only four factors showed statistically significant change between the two groups after ACDF : T1s-CL (Δ4.0° vs. Δ0.2°), C2s (Δ3.2° vs. Δ0.7°), O-C2A (Δ2.6° vs. Δ1.3°), C2-7A (Δ6.3° vs. Δ1.3°). A very strong correlation between T1s-CL and C2s was also found (r=|0.88-0.96|). Conclusion : C2s itself may be the essential key to represent T1s-CL. The amounts and directions of change of these two factors (T1s-CL and C2s) were also almost identical. The above phenomenon was re-confirmed once again through the correlation analysis.

Analysis of $^1H$ MR Spectroscopy of parietal white matter material Phantom (두정부 백질 물질을 이용한 수소 자기 공명 분광 분석)

  • Lee, Jae-Yeong;Lim, Cheong-Hwan;Kim, Myeong-Soo
    • Journal of radiological science and technology
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    • v.26 no.2
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    • pp.57-61
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    • 2003
  • The purpose of this study is to compare both 1.5T and 4.7T in Praietal White matter material Phantom using the same methodology at both field strengths. Data at both field strengths are compared in terms of $T_2$ relaxation times, line widths and SNRs MR imaging and $^1H$ MR spectroscopy were performed on GE 1.5T SIGNA system and Broker Biospec 4.7T/30 MRI/MRS system. After phantom axial scan $^1H$ MRS was obtained from T2 weighted image by 3-dimensional localization technique(PRESS : Point RE solved spectroscopy Sequence) this phantom is composed of an aqueous solution 36.7 mmol/L of NAA, 25.0 mmol/L of Cr, 6.3 mmol/L of choline chloride, 30.0 mmol/L or Glu, and 22.5 mmol/L of MI(adjusted to a pH of 7,15 in a phosphate buffet). Data processed using software developed inhouse. At 1.5T, T2 relaxation times for Cho, Cr, and NAA were $0.41{\pm}0.07,\;0.26{\pm}0.04,\;0.46{\pm}0.07$ while at 4.7T they were $0.17{\pm}0.03,\;0.14{\pm}0.05,\;0.20{\pm}0.03$ respectively. At 1.5T, line widths for water, Cho, Cr and NAA were $2.9{\pm}0.7,\;1.6{\pm}0.7,\;1.7{\pm}0.8,\;2.2{\pm}0.02Hz$ while at 4.7T they were $5.2{\pm}1.1,\;4.6{\pm}1.9,\;4.01{\pm}1.8,\;4.8{\pm}1.9Hz$ respectively. It can be seen that $T_2$ relaxation times were significantly shorter at 4.7 compared to 1.5T and that the line widths were also broader. The average SNRs for NAA for subjects at short and long TEs were $23.5{\pm}11.3$ at TE=20 msec ; $15.4{\pm}7.7$ at TE=272 msec at 1.5T and $40{\pm}8.3$ and $17{\pm}3.5$ respectively at 4.7T higher field strength is superior because of improved sensitivity and chemical shift dispersion. However these improvements are partially offset by increased line widths and decrease $T_2$ relaxation times, which act to reduce both sensitivity and resolution. In our experiments with the equipment available to us, 4.7T proton spectra at short TEs exhibit moderately improved sensitivity compared to 1.5T.

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Characterization of a Bacteriocin Produced by Enterococcus sp. T7 Isolated from Humans

  • Moon, Hi-Seong;Jeong, Jong-Jin;Ji, Geun-Eog;Kim, Jong-Sang;Kim, Jeong-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.10 no.4
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    • pp.507-513
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    • 2000
  • A bacteriocin-producing organism, Enterococcus sp. T7, was isolated from human fecal samples. Bacteriocin T7, named tentatively as the bacteriocin, was produced by Enterococcus sp. T7 and it inhibited some strains of Lactobacillus. Staphylococcus, Enterococcus, and Streptococcus, but not all the lactococci and gram-negative bacteria tested. Bacteriocin T7 inhibited the growth of Listeria monocytogenes Scott A, but the degree of inhibition was less than those for other sensitive gram-positive vacteria. Bacteriocin T7 in MRS broth started to produce at the middle of the exponential growth phase and the inhibitory activity reached its maximum level during the stationary growth phase. Bacteriocin T7 was stable against heat treatments, pH variations (pH 2-10), and exposure to organic solvents. The molecular weight of bacteriocin T7 was estimated to be 6.500 Da by SDS-PAGe. All these facts, including physico-chemical stabilities, small molecular size, and inhibition of Kisteria monocytogenes, indicate that bacteriocin T7 is likely to be a member of the class IIa bacteriocins.

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Seeing is Believing: Illuminating the Source of In Vivo Interleukin-7

  • Kim, Grace Yoon-Hee;Hong, Chang-Wan;Park, Jung-Hyun
    • IMMUNE NETWORK
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    • v.11 no.1
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    • pp.1-10
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    • 2011
  • Interleukin-7 (IL-7) is an essential cytokine for T cells. However, IL-7 is not produced by T cells themselves such that T cells are dependent on extrinsic IL-7. In fact, in the absence of IL-7, T cell development in the thymus as well as survival of naive T cells in the periphery is severely impaired. Furthermore, modulating IL-7 availability in vivo either by genetic means or other experimental approaches determines the size, composition and function of the T cell pool. Consequently, understanding IL-7 expression is critical for understanding T cell immunity. Until most recently, however, the spatiotemporal expression of in vivo IL-7 has remained obscured. Shortage of such information was partly due to scarce expression of IL-7 itself but mainly due to the lack of adequate reagents to monitor IL-7 expression in vivo. This situation dramatically changed with a recent rush of four independent studies that describe the generation and characterization of IL-7 reporter mice, all utilizing bacterial artificial chromosome transgene technology. The emerging consensus of these studies confirmed thymic stromal cells as the major producers of IL-7 but also identified IL-7 reporter activities in various peripheral tissues including skin, intestine and lymph nodes. Strikingly, developmental and environmental cues actively modulated IL-7 reporter activities in vivo suggesting that IL-7 regulation might be a new mechanism of shaping T cell development and homeostasis. Collectively, the availability of these new tools opens up new venues to assess unanswered questions in IL-7 biology in T cells and beyond.