• Title/Summary/Keyword: K11 RNA polymerase

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Cloning and Sequencing of the Gene Involved in Morphological Change of Zoogloea ramigera 115SLR

  • Lee, Sam-Pin;Kim, Tae-Rahk;Sinskey, Anthony-John
    • Journal of Microbiology and Biotechnology
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    • v.10 no.2
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    • pp.161-168
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    • 2000
  • Plasmid pLEX3 isolated from the recombinant cosmid library of Zoogloea ramigera 115 was found to be responsible for the restoration of the rugose colony phenotype. To confirm the essential region responsible for the complementation, subclones were constructed from plasmid pLEX3 and transformed into mutant strain Z. ramigera 115SLR. The recombinant plasmids pLEX10 and pLEX11 were shown to complement the slime-forming property of Z. ramigera 115SLR. In a compositional analysis of the exopolysaccharides from Z. ramigera 115, Z. ramigera 115SLR, and Z. ramigera 115SLR harboring plasmid pLEX11, the exopolysaccharides showed a similar composition with glucose, galactose, and side chain groups. The complete nucleotide sequence of the 3.25kb genocim DNA insert in plasmid pLEX11 was determined and its analysis identified two open reading frames which could encode two proteins. The gene products derived form the two open reading frames were confirmed by and in vivo transcription using a T7-RNA polymerase. The ORF1 produced a 30 kDa protein, whereas the ORF2 was found responsible for the complementation of the morphological mutation and produced a 14 kDa protein. An in vivo gene expression of plasmid pTEX10 showed another open reading frame encoding a 50 kDa protein. The gene products form ORF1 and ORF2 are regarded as novel proteins which do not show any homology with other proteins.

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The Clinical Assessment of Protease-Activated Receptor-2 Expression in Inflammatory Cells from Peripheral Blood and Bronchoalveolar Lavage Fluid in Idiopathic Pulmonary Fibrosis

  • Park, Young Sik;Yoo, Chul-Gyu
    • Tuberculosis and Respiratory Diseases
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    • v.74 no.6
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    • pp.264-268
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    • 2013
  • Background: Idiopathic pulmonary fibrosis (IPF) is a lethal pulmonary fibrotic disease. In general, the exaggerated activation of the coagulation cascade has been observed during initiation or maintenance of the fibrotic disease. In our recent study, immunohistochemical expression of protease-activated receptor-2 (PAR-2), which plays a key role in coagulation cascade, was observed in surgical specimen of IPF patients, and associated with poor clinical outcome. The aim of this study was to evaluate the overexpression of PAR-2 in inflammatory cells from peripheral blood and bronchoalveolar lavage fluid in IPF patients. Methods: From May 2011 to March 2012, IPF patients and controls were enrolled in Seoul National University Hospital. Peripheral blood and bronchoalveolar lavage fluid were collected for analysis of PAR-2 expression. Flow cytometry and reverse transcription polymerase chain reaction were used for PAR-2 receptor and mRNA assessment. Results: Twelve IPF patients and 14 controls were included in this study. Among them, flow cytometry analysis was conducted from 26 peripheral blood (patient group, 11; control group, 13) and 7 bronchoalveolar lavage fluid (patient group, 5; control group, 2). The expression of PAR-2 receptor was not different between patient and control groups (p=0.074). Among all 24 population, PAR-2 mRNA assessment was performed in 19 persons (patient group, 10; control group, 9). The mRNA expression of PAR-2 was not significant different (p=0.633). Conclusion: In IPF patients, PAR-2 receptor and mRNA expression were not different from control group.

Expression of the Pokemon Proto-oncogene in Nasopharyngeal Carcinoma Cell Lines and Tissues

  • Jiao, Wei;Liu, Fei;Tang, Feng-Zhu;Lan, Jiao;Xiao, Rui-Ping;Chen, Xing-Zhou;Ye, Hui-Lan;Cai, Yong-Lin
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.11
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    • pp.6315-6319
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    • 2013
  • To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

Regulation of Histone Acetylation and Methylation of the p11 Gene in the Hippocampus of Chronic Unpredictable Stress-induced Depressive Mice (장기간 예측 불가능한 스트레스를 받은 마우스 해마에서 p11 유전자의 히스톤 아세틸화 및 메틸화의 조절)

  • Seo, Mi Kyoung;Seog, Dae-Hyun;Park, Sung Woo
    • Journal of Life Science
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    • v.31 no.11
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    • pp.995-1003
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    • 2021
  • Chromatin remodeling regulates gene expression through epigenetic mechanisms. Aberrations in histone modification have been associated with depression-like behaviors in animal models. Additionally, growing evidence also indicates that epigenetic modification is associated with depression. p11 (S100A10) has been implicated in the pathophysiology of depression both in human and rodent models. In the present study, we investigated alterations in histone acetylation and methylation at the promoter of the p11 gene in the hippocampus of mice subjected to chronic unpredictable stress (CUS). C57BL/6 mice were exposed to CUS daily for 3 weeks. Depression-like behaviors were measured with the forced swimming test (FST). The levels of hippocampal p11 expression were analyzed by quantitative real-time polymerase chain reaction (PCR) and Western blotting. The levels of acetylated and methylated histone H3 at the promoter of p11 were measured by chromatin immunoprecipitation followed by real-time PCR. CUS-exposed mice displayed depression-like behaviors with prolonged immobility in FST. CUS led to significant decreases in the expression of p11 at both protein and mRNA levels. Meanwhile, there was a decrease in histone H3 acetylation (Ac-H3) and H3-K4 trimethylation (H3K4met3) and an increase in H3-K27 trimethylation (H3K27met3) at the p11 promoter. These results indicate that chronic stress causes the epigenetic suppression of p11 expression in the hippocampus.

Bi-functional Activities of Chimeric Lysozymes Constructed by Domain Swapping between Bacteriophage T7 and K11 Lysozymes

  • Alcantara, Ethel H.;Kim, Dong-Hee;Do, Su-Il;Lee, Sang-Soo
    • BMB Reports
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    • v.40 no.4
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    • pp.539-546
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    • 2007
  • The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.

Monitoring of Chicken RNA Integrity as a Function of Prolonged Postmortem Duration

  • Malila, Yuwares;Srimarut, Yanee;U-chupaj, Juthawut;Strasburg, Gale;Visessanguan, Wonnop
    • Asian-Australasian Journal of Animal Sciences
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    • v.28 no.11
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    • pp.1649-1656
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    • 2015
  • Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

Analysis of rpoB Gene in Rifampin-Resistant M. Tuberculosis by Direct Sequencing and Line Probe Assay (염기서열결정과 Line Probe 분석법에 의한 Rifampin내성 결핵균의 rpoB 유전자 분석)

  • Lee, Min-Ki;Kim, Yun-Seong;Lee, Hyo-Jin;Cheon, Du-Su;Yun, Sang-Myung;Park, Sam-Seok;Kim, Cheol-Min;Park, Soon-Kew
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.2
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    • pp.251-263
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    • 1997
  • Background : The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture. Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in $\beta$subunit of RNA polymerase is main mechanism of resistance. Method : In this study, rpoB gene for the $\beta$subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. Results : All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S531L) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not confirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513~515 were new identified mutations for the first time. Conclusion : Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.

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A Korean case of neurofibromatosis type 1 with an exonic splicing enhancer site mutation

  • Park, Sangwook;Sohn, Young Bae;Chung, In-Soon;Hong, Ji-Hee;Jung, Eun-Jung;Jeong, Seon-Yong;Jin, Hyun-Seok
    • Journal of Genetic Medicine
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    • v.11 no.1
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    • pp.40-42
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    • 2014
  • Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by neurological, cutaneous, and ophthalmological manifestations. A 33-year-old woman with typical symptoms of NF1 visited Ajou University Hospital. Screening of the whole-messenger RNA region of NF1 at the complementary DNA level by polymerase chain reaction-direct sequencing confirmed the presence of an NF1 mutation at the genomic level. The mutation analysis revealed an in-frame skipping of exon 46 (c.6757_6858del) caused by a point mutation (c. 6792C>A) in exon 46. In this report, we have described the first Korean case of a proband with NF1 that carries an allele with an exon 46 deletion caused by an exonic splicing enhancer site mutation, leading to the skipping of the whole of exon 46 (c.6757_6858del).

Gene Expression of Smad3 and Estrogen Receptor-related $Receptor\;{\beta}$ like 1 in Sea Urchin, Strongylocentrotus nudus (둥근성게(Strongylocentrotus nudus)의 Smad3와 Estrogen Receptor-related $Receptor\;{\beta}$ like 1 유전자 발현)

  • Jun, Yu-Jung;Sohn, Young-Chang
    • Development and Reproduction
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    • v.11 no.1
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    • pp.43-47
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    • 2007
  • Smad proteins mediate transforming growth $factor(TGF)-{\beta}$ signaling and play a pivotal role in embryonic development. The estrogen receptor-related receptors(ERRs), which are structurally similar to estrogen receptors, are members of orphan nuclear receptor in the nuclear receptor superfamily and their functions are known to be involved in the formation of extra-embryonic ectoderm. To investigate the involvement of Smad3 and $ERR{\beta}$ like 1 in reproductive activities and embryogenesis in marine invertebrate, we examined gene expression of Smad3 and $ERR{\beta}$ like 1 in Strongylocentrotus nudus during their seasonal changes and embryonic development using real-time polymerase chain reaction. The Smad3 mRNA levels in gonad showed an increasing pattern from February to June 2004 but decreased at August(spawning season) followed by an elevation of the levels at October and December 2004. The mRNA levels of the $ERR{\beta}$ like 1 significantly elevated during the spawning season. During embryonic development, Smad3 mRNA levels at $8{\sim}16$ cell stages were significantly higher than those of other stages, whereas the mRNA of the $ERR{\beta}$ like 1 was significantly high levels at late development stages, i.e., blastular, gastrula and plutei stages. These results suggest that the Smad3 could be involved at least in part in the early cleavage stages and the $ERR{\beta}$ like 1 may play an important role in the spawning season and late developmental stage in the sea urchin.

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Anti-inflammatory effect of lycopene in SW480 human colorectal cancer cells

  • Cha, Jae Hoon;Kim, Woo Kyoung;Ha, Ae Wha;Kim, Myung Hwan;Chang, Moon Jeong
    • Nutrition Research and Practice
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    • v.11 no.2
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    • pp.90-96
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    • 2017
  • BACKGROUND/OBJECTIVES: Although the antioxidative effects of lycopene are generally known, the molecular mechanisms underlying the anti-inflammatory properties of lycopene are not fully elucidated. This study aimed to examine the role and mechanism of lycopene as an inhibitor of inflammation. METHODS/MATERIALS: Lipopolysaccharide (LPS)-stimulated SW 480 human colorectal cancer cells were treated with 0, 10, 20, and $30{\mu}M$ lycopene. The MTT assay was performed to determine the effects of lycopene on cell proliferation. Western blotting was performed to observe the expression of inflammation-related proteins, including nuclear factor-kappa B ($NF-{\kappa}B$), inhibitor kappa B ($I{\kappa}B$), mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 (p38 MAP kinase). Real-time polymerase chain reaction was performed to investigate the mRNA expression of tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), interleukin-1 beta ($IL-1{\beta}$), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Concentrations of nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) were determined via enzyme-linked immunosorbent assays. RESULTS: In cells treated with lycopene and LPS, the mRNA expression of $TNF-{\alpha}$, $IL-1{\beta}$, IL-6, iNOS, and COX-2 were decreased significantly in a dose-dependent manner (P < 0.05). The concentrations of $PGE_2$ and NO decreased according to the lycopene concentration (P < 0.05). The protein expressions of $NF-{\kappa}B$ and JNK were decreased significantly according to lycopene concertation (P < 0.05). CONCLUSIONS: Lycopene restrains $NF-{\kappa}B$ and JNK activation, which causes inflammation, and suppresses the expression of $TNF-{\alpha}$, $IL-1{\beta}$, IL-6, COX-2, and iNOS in SW480 human colorectal cancer cells.