• 한국환경성돌연변이발암원학회지
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• 제15권2호
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• pp.100-105
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• 1995

The effects of DNA polymerase $\beta$ on the somatic chromosome mutations and mitotic recombinations were investigated using the transgenic Drosophila beating chimetic gene consisting of a promoter region of Drosophila actin 5C gene and rat DNA polymerase $\beta$. For detecting the somatic chromosome mutations and mitotic recombinations, the heterozygous (mwh/+) strains possessing or lacking transgene poi 13 were used. The spontaneous frequency of small mwh spots, due to deletion or nondisjunction etc., in the non-transgenic w strain and the transgenic p[pol $\beta$]-130 strain was 0.351 and 0.606, respectively. The spontaneous frequency (0.063) of large mwh spots, arises mostly from somatic recombination between the centromere and the locus mwh, in the transgenic p[pol $\beta$]-130 strain was about three times higher than that (0.021) of the non-transgenic w strain. The mutant clone frequencies of small and large mwh spots induced by N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate in the transformant p[pol $\beta$]-130 were higher than those in the host strain w. The present results suggest that rat DNA polymerase $\beta$ participate at least in the somatic chromosome mutations and mitotic recombination processes.

• BMB Reports
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• 제35권3호
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• pp.320-329
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• 2002

A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2621 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39% to 47% identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the C-terminus. It was purified by heat treatment, followed by a $Ni^{2+}$-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'$\rightarrow$5' exonuclease activity.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.280-283
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• 1998

We have cloned the RNA polymerase sigma-like factors from a wide range of actinomycetes by using specific primers with the polymerase chain reaction (PCR). The specific oligonucleotide primers were designed on the basis of amino acid sequences of conserved regions from HrdA, B, D of Streptomyces griseus as well as from the rpoD box of many eubacteria. The consensus sequences were from the rpoD box and helix-turn-helix motif involved in -35 recognition. The designed primers were successfully applied to amplify the DNA fragments of the hrd homolog genes from 8 different strains of actinomycetes which produce a wide variety of important antibiotics. The 480 bp of the DNA fragment was amplified from all 8 strains, and it was identified as a part of hrdA and hrdB as we designed. The deduced amino acid sequence of PCR-amplified DNA fragments were highly homologous to those of other known RNA polymerase sigma factors of S. griseus and Streptomyces aureofaciens. Therefore, this study with specifically designed primers will support rapid cloning of the RNA polymerase sigma factors which recognize different classes of promoters from actinomycetes, and it will also be helpful in understanding the relationship of promoters and sigma factors leading to heterogeneity of RNA polymerases in actinomycetes.

• 생명과학회지
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• 제19권3호
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• pp.305-310
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• 2009

플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다.

• Journal of Microbiology
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• 제45권6호
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• pp.534-540
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• 2007

The Streptomyces coelicolor A3(2) genome contains six operons (rrnA to F) for ribosomal RNA synthesis. Transcription from rrnD occurs from four promoters (p1 to p4). We found that transcripts from the p1 and p3 promoters were most abundant in vivo in the early exponential phase. However, at later phases of exponential and stationary growth, transcripts from the p1 promoter decreased drastically, with the p3 and p4 transcripts constituting the major forms. Partially purified RNA polymerase supported transcription from the p3 and p4 promoters, whereas pure reconstituted RNA polymerase with core enzyme (E) and the major vegetative sigma factor ${\sigma}^{HrdB}$ ($E{\cdot}{\sigma}^{HrdB}$) did not. In order to assess any potential requirement for additional factor(s) that allow transcription from the p3 and p4 promoters, we fractionated a partially purified RNA polymerase preparation by denaturing gel filtration chromatography. We found that transcription from the p3 and p4 promoters required factor(s) of about 30-35 kDa in addition to RNAP holoenzyme ($E{\cdot}{\sigma}^{HrdB}$). Therefore, transcription from the p3 and p4 promoters, which contain a consensus -10 region but no -35 for ${\sigma}^{HrdB}$ recognition, are likely to be regulated by transcription factor(s) that modulate RNA polymerase holoenzyme activity in S. coelicolor.

• Journal of Magnetics
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• 제19권1호
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• pp.20-27
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• 2014

This study examined the relationships between protein expression and Poly ADP ribose polymerase in brain cell death in brains damaged by thrombotic stroke and treated with the Full Wave- Cockroft Walton (FWCW) method of Transcranial Magnetic Stimulation (TMS). The two-way switching element for TMS drove a half-bridge inverter of the current resonance of direct current voltage (+) and direct current voltage (-), and the experiment was conducted by stimulating the mice with thrombotic stroke through a range of pulses. Thrombotic stroke was caused of ligation of the common carotid artery of male SD mice, and blood reperfusion was conducted five minutes later. Protein expression was examined in immune reaction cells, which reacted to an antibody to Poly ADP ribose polymerase in the cerebrum cells, and western blotting. Observations of the PARP changes after thrombotic stroke showed that the number of Poly ADP ribose polymerase reactions were significantly lower (p < 0.05) in the group treated with TMS of the FWCW than the group with thrombotic stroke 24 hours after its onset. The application of FWCW-TMS helped prevent the necrosis of nerve cells and might prevent the brain damage that occurs as a result of thrombotic stroke, and improve the function recovery and disorder of brain cells.

• Journal of Microbiology
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• 제37권3호
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• 미생물학회지
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• 제24권3호
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• pp.276-287
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• 1986

ChIarella elliPsoidea를 rifampicin이 함유된 M4N 배지에 7일간 배양하였다. 배양기간 동안에 일정량 세포를 수확하여 생장율을 측정하였다. 이들 세포에서 분리된 엽록체로 부터 핵산과 RNA polymerase를 추출하여 함량을 염기별로, 분석, 활성도를 측정하여 핵산 합성에 비치는 rifampicin의 효과를 대조구와 비교하며 분석하였다. 생장 억제 효과를 나타내는rifampicin의 농도는 80ppm 이였다. Whole cell system과 분리한 엽록체에서의 DNA함량은 대조구에 비해 각각 70%, 60%의 감소를 나타내였다. Rifampicin은 RNA 염기 함양도 whole cell system에서는 46% 억제되었고 분리한 엽록체에서는 77% 저해효과가 관찰되었다. Rifampicin에 의한 RNA polymerase 활성도는 whole cell system에서는 10% 감소, 분리한 엽록체에서는 42% 억제를 나타내었다.

• BMB Reports
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• 제32권5호
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• pp.492-496
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• 1999

We have investigated the biochemical properties of the herpes simplex virus type 1 (HSV-1) DNA polymerase without the UL42 protein (Pol), purified from insect cells infected with a recombinant baculovirus containing the UL30 gene. BSA and DTT have inhibitory effects on dAMP incorporation. Pol showed a greater turnover rate of steady-state single nucleotide incorporation at 12 mM $MgCl_2$ than at 2 mM $MgCl_2$. However, it showed a greater processivity of DNA synthesis at lower $MgCl_2$ concentration (1 mM, 2 mM) than at a higher $MgCl_2$ concentration (12.5 mM). These results are consistent with a slow DNA dissociation at lower $MgCl_2$ concentrations. Pol does not incorporate a correct nucleotide into the primer with an incorrect nucleotide at the end; instead, it preferentially excises the incorrect nucleotide at the 3' end of the primer. Pol has DNA polymerase activity at pHs 6.5 and 7.5 but little at pHs 5.5, 8.5, and 9.5. It has exonuclease activity at pHs 6.5, 7.5, and 8.5 but little at pHs 4.5, 5.5, and 9.5. The finding that Pol has exonuclease activity but not DNA polymerase at pH 8.5 suggests that DNA binds to Pol, but deoxynucleotide binding or incorporation does not occur at pH 8.5.

• BMB Reports
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• 제39권5호
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• pp.571-577
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• 2006

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.