• Food Science and Biotechnology
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• v.16 no.3
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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.

• BMB Reports
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• v.35 no.6
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• pp.637-641
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• 2002

Only one natural promoter that interacts with bacteriophage K11 RNA polymerase has so far been identified. To identify more, in the present study restriction fragments of the phage genome were individually assayed for transcription activity in vitro. The K11 genome was digested with two 4-bp-recognizing restriction enzymes, and the fragments cloned in pUC119 were assayed with purified K11 RNA polymerase. Eight K11 promoter-bearing fragments were isolated and sequenced. We report that the nine K11 promoter sequences (including the one previously identified) were highly homologous from -17 to +4, relative to the initiation site at +1. Interestingly, five had -10G and -8A, while the other four had -10A and -8C. The consensus sequences with the natural -10G/-8A and -10A/-8C, and their variants with -10G/-8C and -10A/-8A, showed nearly equal transcription activity, suggesting residues at -10 and -8 do not regulate promoter activity. Using hybridization methods, physical positions of the cloned promoter-bearing sequences were mapped on SalI-and KpnI-restriction maps of the K11 genome. The flanking sequences of six cloned K11 promoters were found to be orthologous with T7 or T3 genomic sequences.

• Bulletin of the Korean Chemical Society
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• v.27 no.10
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• pp.1618-1622
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• 2006

Bacteriophage T7 gp4A' is a ring-shaped hexameric DNA helicase that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. To investigate the effect of single-stranded DNA (ssDNA) on the kinetic pathway of dTTP hydrolysis by the T7 DNA helicase complexed with ssDNA, we have first determined optimal concentration of long circular M13 single-stranded DNA and pre-incubation time in the absence of $Mg^{2+}$ which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of $Mg^{2+}$ by the helicase-ssDNA complex provided $k_{cat}$ of $8.5\;{\times}\;10^{-3}\;sec^{-1}$. Pre-steady state kinetics of the dTTP hydrolysis by the pre-assembled hexameric helicase was monitored by using the rapid chemical quench-flow technique both in the presence and absence of M13 ssDNA. Pre-steady state dTTP hydrolysis showed distinct burst kinetics in both cases, indicating that product release step is slower than dTTP hydrolysis step. Pre-steady state burst rates were similar both in the presence and absence of ssDNA, while steady state dTTP hydrolysis rate in the presence of ssDNA was much faster than in the absence of ssDNA. These results suggest that single-stranded DNA stimulates dTTP hydrolysis reaction by T7 helicase by enhancing the rate of product release step.

• The Plant Pathology Journal
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• v.33 no.6
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• pp.608-613
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• 2017

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

• BMB Reports
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• v.33 no.3
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• pp.242-248
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• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• The Plant Pathology Journal
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• v.22 no.4
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• pp.386-390
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• 2006

Extensive research of the Potato virus X(PVX) has been performed in in vitro transcription system using the bacteriophage T7 promoter. We constructed an efficient T-DNA based binary vector, pSNU1, and modified vectors carrying PVX replicons. The suitability of the construct to transiently express PVX RNA using Agrobacterium tumefaciens was tested by analysis of infectivity in plants. The expressed PVX RNA was infectous and systemically spread in three plant species including Nicotiana benthamiana, N. tabacum cv. Xanthi-nc, and Capsicum annuum cv. Chilsungcho. The PVX full length construct, pSPVXp31, was caused severe mosaic symptoms on N. benthamiana, severe necrotic lesions on C. annuum while milder symptoms and delayed mosaic symptoms were appeared on the systemic leaves on N. tabaccum. RT-PCR analysis confirmed the presence of PVX RNAs on both inoculated and systemic leaves in all three plant species tested. Our results indicated that PVX replicons were efficiently expressed PVX RNA in at least three tested species. Further investigation win be needed to elucidate the mechanism of PVX replication, translation, movement and assembly/disassembly processes.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.415-422
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• 1996

A gene coding for a pyruvyl transferase enzyme involved in exopolysaccharide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of BamHI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindIII DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORFI and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (ExoV product) of Rhizobium meliloti (36%) as well as the ketalase of X campestris (23%) than that of ORF3. A gene product of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.265-265
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• 1994

본 연구는 간염 바이러스의 X 및 elongated X 유전자를 클로닝하여 E. coli에서 대량 발현시진 후, 그 기능을 여러 측면에서 연구하교 지금까지 알려진 oncogene products, tumor suppressor, 그리고 그 밖의 다른 암 유발인자와의 interaction에 대해 분석함으로써 간암 생성의 분자적 기작을 이해하고 더 나아가 간암의 예방 및 치료제의 개발을 목표로 하였다. 그 일차적 연구로서 이전에 플로닝된 mutant hepatitis Bvirus genome으로부터 X 및 elongated X 유전자를 클로닝하였으며, E. coli에서 대량 발현시키기 위하여 T7 bacteriophage promoter아래에 재 클로닝하였다. 이러한 X 및 ebngated X 유전자를 E. coli에서 대량 발현시킨 후, rabbit anti-X antibody를 이용하여 western blotting을 수행함으로서 이를 확인하였으며 DEAE-cellulose와 heparin-agarose chromategraphy를 이용하여 순수분리하였다. 순수분리된 X 및 etongated X 단백질을 highly differentiated hepatoma cell인 HepG$_2$ cell에 처리하여 transactivation activity를 측정하였다. 그 결과 순수분리된 단백질들이 SV4O promoter를 transactivation 함을 할 수 있었으며, 이로부터 클로닝된 유전자들이 모두 정상적인 기능을 가짐을 확인하였다. 그러고 X 유전자의 작용기작을 규명하기위하여 restriction endonuclease를 이용하여 5 개의 mutant X 유전자를 구성하였으며 현재 이를 HepG2 cell에 transfection 하여 그 기능을 연구하고 있다.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.89-93
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• 1990

To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.

• Applied Biological Chemistry
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• v.38 no.5
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• pp.382-386
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• 1995

The potato proteinase inhibitor II (PI-II) protein contains chymotrypsin and trypsin inhibitory site. Among several PI-II genes isolated from genomic library, amino acid sequence deduced from PI-IIT gene has 84% identity with that of the polypeptide chymotrypsin inhibitor (PCI). Therefore a gene fragment having homology with the PCI was cloned into a vector using polymerase chain reaction(PCR) from the potato proteinase inhibitor IIT gene. Two different primers were utilized for cloning; primer A contains NdeI restriction site and 30 nucleotides, which has AUG N-terminal methionine codon, primer B contains BclI restriction site and 28 nucleotides, which has TAG translation stop codon. After PCR, about 160 bp-long DNA fragment was cloned into pRT146, derivative of pUC118, and sequenced. The sequenced NdeI/BclI fragment was moved to pET3a, containing bacteriophage T7 promoter and terminator. The expressed proteins in E. coli BL2l(DE3) were determined on a polyacrylamide gel containing sodium dodecyl sulfate. The expected size of protein deduced from the sequenced gene fragment is about 6,500 dalton whose size was similar to the IPTG-induced protein (6,000 dalton) on a gel. However the expression level was much lower than expected.