• International Journal of Oral Biology
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• 제46권3호
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• Archives of Pharmacal Research
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• 제24권1호
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• pp.55-63
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• 2001

Aucubin, an irdoid g1ucoside, was investigated to determine whether it has a stimulating effect on $\alpha$-amanitin excretion in $\alpha$-amanitin intoxicated rats, and whether there is binding activity to calf thymus DNA. High-performance liquid chromatography (HPLC) analysis of $\alpha$-amanitin in rat urine allowed quantitative measurement of the $\alpha$-amanitin concentration with a detection limit of 50${mu}g/ml$. In this system, a group treated with both $\alpha$-amanitin and aucubin showed that o(-amanitin was excreted about 1.4 times faster than in the $\alpha$-amanitin only treated group. Our previous results showed that the toxicity of $\alpha$-amanitin is due to specific inhibition of RNA polymerase activity and the resultant blockage of the synthesis of certain RNA species in the nucleus. However, no significant activity change on RNA polymerase from Hep G2 cells was observed when aucubin was treated with $\alpha$-amanitin at any concentration tested. Nevertheless, aucubigenin inhibited both DNA polymerase (IC50, 80.5${mu}g/ml$) and RNA polymerase (IC50, 135.0${mu}g/ml$) from the Hep G2 cells. The potential of both $\alpha$-amanitin and aucubin to interact with DNA were examined by spectrophotometric analysis. $\alpha$-Amanitin showed no significant binding capacity to calf thymus DNA, but aucubin was found to interact with DNA, and the apparent binding constant ($K_{app}$) and apparent number of binding sites per D7A phosphate ($B_{app}$) were 0.45$0.45{\times}$$10^4$ $M^{-1}$ and 1.25, respectively.

• 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.

• 자연과학논문집
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• 제14권1호
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• pp.39-50
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• 2004

박테리오 파아지 T7 RNA 중합효소는 다른 RNA 중합효소와 비교하여 볼 때 보조인자 없이 전사를 진행하는 하나의 subunit로 구성된 RNA 중합효소이다. 전사 진행 단계 중에서 T7 RNA 중합효소의 전사연장을 연구하기 위해 biotin이 결합된 DNA 주형을 streptavidin bead로 고정시킴으로서 T7 RNA 중합효소의 진행과정을 관찰할 수 있었고, 이러한 기작을 이용하여 일련의 활성을 가지는 가장 안정한 전사연장복합체들을 얻을 수 있었다. 전사 연장체들은 16번 염기 위치로부터 18번 염기의 위치까지 방사선 동위원소가 표지되어 있으며 이들 표지된 전사연장복합체들은 단계별로 합성하여 22-40개 핵산잔기들이 합성된 전사연장복합체들을 얻을 수 있었다. 이와 같은 전사연장복합체들을 PTH 전사종결 부위가 있는 주형으로 사용하여 야생형 및 R173C 돌연변이 RNA 중합효소를 이용하여 전사연장복합체를 제조하여 비교한 결과 PTH 전사종결에 둔감한 R173C 돌연변이 중합효소의 경우 야생형에 비해 PTH 전사종결부위를 지난 위치에서도 전사연장복합체가 생성되었다.

• 한국미생물·생명공학회지
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• 제37권1호
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• pp.17-23
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• 2009

PCR법을 이용하여 Thermus thermophilus HJ6 유래 DNA polymerase(Tod) 유전자를 클로닝하고 염기서열을 분석한 결과, ORF는 2,505개의 뉴클레오타이드로 구성되고 834개의 아미노산을 암호화하였다. 아마노산 서열을 바탕으로 상동성을 분석한 결과, Thermus thermophilus HB8 유래 DNA polymerase와 98%, Thermus aquaticus 유래 DNA polymorase와 86%의 identity를 나타내었다. 이 유전자를 박테리오파지 $\lambda$ 유래 온도감수성 프로모터(PR, PL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. 발현된 효소는 열처리, $HiTrap^{TM}$ Q column과 $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 columun으로 정제하여 94 kDa의 단백질을 얻을 수 있었다. 정제된 효소의 DNA 중합 활성에 대한 최적온도는 $75{\sim}80^{\circ}C$이고 최적 pH가 9.0이었다. $Mg^{2+}$ and $Mn^{2+}$에 대한 최적 농도는 각각 2.5mM과 1mM이었고 효소활성은 2가 양이온의 존재 하에서는 활성화 되지만 1가양이온에서는 저 해되었다. Tod DNA 중합효소를 이용한 PCR 실험결과, Tod DNA 중합효소는 DNA 증폭 및 PCR 관련 기술에 응용 가능할 것으로 생각된다.

• Bulletin of the Korean Chemical Society
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• 제30권6호
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• pp.1365-1367
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• 2009

• Bulletin of the Korean Chemical Society
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• 제32권10호
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• pp.3779-3782
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• 2011

• Journal of Microbiology
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• 제45권2호
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• pp.139-145
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• 2007

The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

• BMB Reports
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• 제43권4호
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• pp.284-290
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• 2010

Replication of positive-strand RNA virus is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Ectropis obliqua virus (EoV), a newly identified insect virus belonging to the family Iflaviradae, we expressed the RNA polymerase domain in Escherichia coli and purified it on a Ni-chelating HisTrap affinity column. It is demonstrated that EoV RdRp initiated RNA synthesis in a primer and poly (A)-dependent manner in vitro. Furthermore, the effect of primer concentration, temperature, metal ions ($Mg^{2+}$, $Mn^{2+}$, and $K^+$) on enzymatic activity were determined. Our study represented a first step towards understanding the mechanism of EoV replication.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2005년도 춘계학술대회
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• pp.5-14
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• 2005

Recently, data from several groups have raised the concept of 'checkpoint' in transcription. As capping of nascent RNA transcript is tightly coupled to RNA polymerase II transcription, we seek to obtain direct evidence that transcripiton checkpoint via capping enzyme functions in this early regulatory step. One of temperature sensitive (ts) alleles of ceg1, a guanylyltransferase subunit of the Saccharomyces cerevisiaecapping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongational defect. This ts allele, ceg1-63 also has an impaired ability to induce PUR5 in response to a 6AU treatment. However, this cellular and molecular defect is not due to the preferential degradation of the transcript attributed from a lack of guanylyltransferase activity. On the contrary, the data suggests that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation. First, in addition to the 6AU sensitivity, ceg1-63is synthetically lethal with elongation defective mutations of the largest subunit of RNA polymerase II. Secondly, it exhibited a lower GAL1 mRNA turn-over after glucoseshut off. Third, it decreased the transcription read through a tandem array of promoter proximal pause sites in an orientation dependent manner. Interestingly, this mutant also showed lower pass through a pause site located further downstream of the promoter. Taken together, these results suggest that the capping enzyme plays the role of an early transcription checkpoint possibly in the step of the reversion of repression by stimulating polymerase to escape from the promoter proximal arrest once RNA becomes appropriately capped.