• 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.

• 미생물학회지
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• 제27권4호
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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.

• 미생물과산업
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• 제14권1호
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• pp.7-11
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• 1988

Efficient in vitro RNA synthesis can be easily accomplished from cloned DNA using bactrio-phage SP6, T7 or T3 RNA polymerase. Despite its popularity as in vitro transcription system, molecular mechanisms of bacteriophage transcription has not been studied, although physical and catalytic properties of several phage RNA polymerases have well been documented (1). Only recently the T7 promoter has been physically mapped by footprinting of the T7 RNA polymerase (2,3). These simple phage systems, however, could be useful for detailed molecular studies of transcription.

• Applied Biological Chemistry
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• 제45권4호
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• pp.190-194
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• 2002

Bacillus subtilis의 glutamyl-tRNA synthetase(GluRS)는 대장균에서 발현될 때 숙주세포의 $tRNA_1^{Gln}$에 glutamate를 잘못 아실화하여 독성을 나타내는 것으로 추정되고 있다. 이러한 B. subtilis GluRS를 대장균에서 과발현 시키기 위하여 B. subtilis 168 균주의 chromosomal DNA에서 GluRS의 유전자(gltX)를 PCR을 이용하여 증폭하고 T7 promoter에 의해 발현이 조절되는 pET11a expression vector에 클로닝하였다. 이 재조합된 pEBER plasmid DNA로 T7 RNA polymerase를 갖는 대장균 NovaBlue(DE3)에 형질전환하였다. 형질전환된 대장균에 IPTG를 처리하여 과량 생성된 GluRS 단백질은 ammonium sulfate 분별침전 후 EPLC를 이용한 Source Q column anion exchange chromatography, Superdex 200 column gel filtration, Mono Q column anion exchange chromatography로 정제하였다. 정제된 B. subtilis의 GluRS 분자량은 약 55 kDa이었으며 효소의 활성도는 조효소액에 비해 18배로 증가하였다.

• Applied Biological Chemistry
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• 제40권4호
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• pp.271-276
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• 1997

박테리오파아지 T7 RNA polymerase 유전자를 식물체내에서 이용할 수 있을지 알아보기 위하여 상처유발인 감자 단백질 분해효소 억제제 유전자의 프로모터에 박테리오파지 T7 RNA polymerase 유전자를 연결시킨 후 담배에 도입시켰다. 형질전환 식물체의 DNA에 대한 Southern hybridization에 의하면 T7 RNA polymerase 유전자가 식물체내에 1-2 copy가 존재하며, Northern hybridization에 의하면 T7 RNA polymerase의 RNA가 상처에 따라 생성되는 것을 확인하였다. 또한 Western hybridization에 의하면 식물체내 T7 RNA polymerase 단백질이 생성되는데 그 크기는 대장균에서 생성되는 단백질 크기와 유사한 80 kDa 이었으며 시험관내에서 전사체에 뉴클레오타이드를 결합시키는 능력이 있음도 확인하였다. 따라서 T7 RNA polymerase 유전자를 이용하여 식물체내에서 원하는 유전자의 발현을 증대시킬 수 있을 것으로 사료된다.

• 생명과학회지
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• 제10권4호
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

• BMB Reports
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• 제35권6호
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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.

• 미생물학회지
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• 제30권4호
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• pp.316-321
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• 1992

Catechol 2, 3-dioxygenase (C230) catalyses the oxidative ring cleavage of catechol to 2-hydroxymuconic semialdehyde. This is one of the key reactions in the metabolism of the widespresd pollutant aniline. We have cloned a gene encoding C230 from cells of the aniline degrading bacteria, Pseudomonas acidovorance KCTC2494 strain and expressed in E. coli, A 11.3-kilobase Sau3A partial digested DNA fragment from KCTC2494 was cloned into phagemid vector pBluescript and designated as pLP201. The C230 gene was mapped to a 2.8-kb region, and the derection of transcription was determined. The cloned C230 gene contains its own promoter which can be recognized and employed by E. coli transcriptional apparatus. C230 activities of subclones were identified by enzyme assay and activity staining. The T7 RNA promoter/polymerase system and maxicell analysis showed that a polypeptide with Mw of 35 kDa is the C230 gene product.

• Journal of Plant Biology
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• 제39권4호
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• pp.265-271
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• 1996

Full-length cDNA for RNA2 of cucumber mosaic virus strian Kor (Kor-CMV) was cloned downstream of synthetic T7 promoter by reverse transcriptase-polymerase chain reaction (RT-PCR). The clone could generate a full-length transcript corresponding to RNA1 in size when synthesized by T7 RNA polymerase. The complete nucleotide sequence has shown that the RNA2 is composed of 3,049 nucleotides and contains one functional open reading frame (ORF) of 2,574 nucleotides encoding 2a protein. The deduced translation product of the 2,574 nucleotides contains GDD motif which is a characteristic of RNA-dependent RNA polymerase (RdRp). The amino acid sequence analysis of the 2a protein has shown that the homology is found in decreasing order with O-CMV (98.8%), Y-CMV (98.7%), Fny-CMV (98.3%), KCMV (94.9%), Ix-CMV (91.9%), and Q-CMV (74.9%). Kor-CMV is suggested to belong to subgroup Ⅰ in the aspect of nucleotide sequence homology of RNA2.

• The Plant Pathology Journal
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• 제33권6호
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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.