• 미생물학회지
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• 제47권3호
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• pp.268-274
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• 2011

남조세균 Anabaena (Cyanobacteria, Nostocales)는 담수 생태계에서 녹조 현상을 유발하거나 일부 종은 간독소(hepatotoxin)를 갖고 있어 수질관리 차원에서 주목 받아 왔다. 본 연구는 Anabaena RNA polymerase beta subunit (rpoB) 유전자 염기서열을 규명하였으며, 분류학적 분자 마커로 사용하기 위하여 이들 염기서열의 특성을 평가하였다. Anabaena rpoB 유전자는 16S rRNA 유전자와 비교하여 염기 유사도가 낮으며 유전자 변이가 큰 것으로 분석되었으며, 통계적으로 유의한 차이를 보였다(Student t-test, p<0.01). Parsimony 분석을 통해 rpoB 유전자가 4.8배의 속도로 빠르게 진화하는 것으로 파악되었다. 또한 rpoB 유전자 phylogeny 분석에서 16S rRNA tree보다 높은 해상도로 Anabaena 균주를 명확하게 구분해 주었다. 본 연구 결과는 Anabaena의 종 식별, 분자계통 분류, 분자적 검출을 위해 rpoB 유전자가 매우 효과적이라는 것을 제시해 준다.

• Journal of Microbiology and Biotechnology
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• 제4권3호
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• pp.183-190
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• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Applied Biological Chemistry
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• 제58권4호
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• pp.383-387
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• 2015

국내 유통 식품 수입 식품 중 토끼와 고양이 고기의 혼입 여부를 알아내고 불법 도축된 고양이 고기를 토끼 고기나 다른 고기로 속여 판매하는 것을 방지하기 위해 토끼와 고양이를 동시에 검출할 수 있는 polymerase chain reaction (PCR) 법을 개발하였다. 토끼와 고양이의 종 특이 프라이머는 미토콘드리아의 cytochrome b 유전자를 대상으로 하였고 개발된 프라이머를 가공식품에 활용하는 것을 고려하여 PCR 산물의 크기는 토끼 101 bp, 고양이 191 bp로 최소화 하였다. 프라이머의 특이성은 총 21종의 동물을 대상으로 검토하였다. 개발된 검출법의 검출 한계는 시료 DNA를 희석하여 PCR과 Bioanalyzer로 확인한 결과 토끼는 0.005 ng, 고양이는 0.0005 ng이었다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8177-8186
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• 2016

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1321-1324
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• 2012

Background: DNA polymerase beta ($pol{\beta}$) is a key enzyme in the base excision repair pathway. It is 39kDa protein, with two subunits, one large subunit of 31 kDa having catalytic activity between exon V to exon XIV, and an 8 kDa smaller subunit having single strand DNA binding activity. Exons V to VII have double strand DNA binding activity, whereas exons VIII to XI account for the nucleotidyl transferase activity and exons XII to XIV the dNTP selection activity. Aim: To examine the association between $pol{\beta}$ polymorphisms and the risk of ovarian cancer, the present case control study was performed using 152 cancer samples and non-metastatic normal samples from the same patients. In this study, mutational analysis of $pol{\beta}$ genomic DNA was undertaken using primers from exons IX to XIV - the portion having catalytic activity. Results: We detected alteration in DNA polymerase beta by SSCP. Two specific heterozygous point mutations of $pol{\beta}$ were identified in Exon 9:486, A->C (polymorphism 1; 11.18%) and in Exon 11:676, A->C (polymorphism 2; 9.86%). The correlation study involving polymorphism 1 and 4 types of tissue showed a significant correlation between mucinous type with a Pearson correlation value of 4.03 (p=0.04). The association among polymorphism 2 with serous type and stage IV together have shown Pearson ${\chi}^2$ value of 3.28 with likelihood ratio of 4.4 (p=0.07) with OR =2.08 (0.3-14.55). This indicates that there is a tendency of correlation among polymorphism 2, serous type and stage IV, indicating a risk factor for ovarian cancer. Conclusion: Hence, the results indicate that there is a tendency for $pol{\beta}$ polymorphisms being a risk factor for ovarian carcinogenesis in India.

• 대한바이러스학회지
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• 제27권2호
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• pp.185-195
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• 1997

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8FIIa/68/US, with exceptions of HuCV 185/87/Korea and HuCV 1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV 185/87/Korea and HuCV 1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV 185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.

• Asian-Australasian Journal of Animal Sciences
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• 제25권6호
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• pp.789-793
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• 2012

Fertilization of the oocyte commences embryogenesis during which maternally inherited mRNAs are degraded and the embryonic genome is activated. Transcription of embryonic mRNA is initiated by embryonic genome activation (EGA). RNA polymerase II (RNA Pol II) is responsible for the synthesis of mRNAs and most small nuclear RNAs, and consists of 12 subunits, the largest of which characteristically harbors a unique C-terminal domain (CTD). Transcriptional activity of RNA Pol II is highly regulated, in particular, by phosphorylation of serine residues in the CTD. Here, we have shown the presence of RNA Pol II CTD phosphoisoforms in porcine oocytes and preimplantation embryos. The distribution pattern as well as phosphorylation dynamics in germinal vesicles and during embryogenesis differed in developmental stages with these isoforms, indicating a role of RNA Pol II CTD phosphorylation at the serine residue in transcriptional activation during both oocyte growth and embryonic genome activation. We additionally examined the effects of the RNA Pol II inhibitor, ${\alpha}$-amanitin, on embryo development. Our results show that inhibition of polymerase, even at very early stages and for a short period of time, dramatically impaired blastocyst formation. These findings collectively suggest that the functionality of maternal RNA Pol II, and consequently, expression of early genes regulated by this enzyme are essential for proper embryo development.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1999-2002
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• 2012

Background: DNA polymerase is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. The encoded enzyme is a key to the base excision repair (BER) pathway. It is evident that pol beta has mutations in various cancer samples, but little is known about ovarian cancer. Aim: Identification of any variant form of $pol{\beta}$ cDNA in ovarian carcinoma and determination of association between the polymorphism and ovarian cancer risk in Indian patients. We used 152 samples to isolate and perform RT-PCR and sequencing. Results: A variant of polymerase beta (deletion of exon 4-6 and 11-13, comprising of amino acid 63-123, and 208-304) is detected in heterozygous condition. The product size of this variant is 532 bp while wild type pol beta is 1 kb. Our study of association between the variant and the endometrioid type shows that it is a statistically significant factor for ovarian cancer [OR=31.9 (4.12-246.25) with p<0.001]. The association between variant and stage IV patients further indicated risk (${\chi}^2$ value of 29.7, and OR value 6.77 with 95% CI values 3.3-13.86). The correlation study also confirms the association data (Pearson correlation values for variant/stage IV and variant/endometrioid of 0.44 and 0.39). Conclusion: Individuals from this part of India with this type of variant may be at risk of stage IV, endometrioid type ovarian carcinoma.

• 한국정보통신학회논문지
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• 제17권4호
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• pp.1005-1009
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• 2013

마산병원과 결핵연구원에서 rifampin 내성균주를 확보하여 기존의 전통방법으로 검사된 항결핵제 내성균의 rifampin 내성관련 유전자인 rpoB (RNA polymerase beta subunit)의 변이를 DNA 염기서열 분석방법에 의하여 분석하였다. 그 결과 국내에서 분리되는 결핵균에서는 기존에 보고된 rpoB 변이부위와는 다른 위치의 DNA 염기 변이가 확인되었고 국내에서 여러 내성 결핵균주에서 변이가 발견되고 있지만 이러한 변이가 리팜핀 내성을 실제로 유발하는지 실험적으로 검증된 바는 없었다. 따라서 이러한 특이 부위의 rpoB변이들이 실제로 리팜핀 내성을 유발하는가를 확인하기 위하여 내성 결핵균주들의 rpoB 변이유전자를 polymerase chain reaction(PCR)으로 증폭하고 이것을 리팜핀 감수성 결핵균주에 cloning(클로닝)하고 발현시켜 rpoB 변이가 리팜핀에 대한 내성을 발생시켰음을 실험적으로 확인하였다.