• Title/Summary/Keyword: T7 promoter

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Neoplastic Transformation of Immortalized Human Keratinocytes by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin

  • Kang, Mi-Kyung;Kang, Ho-Il;Park, Young-Sill;Ryeom, Tai-Kyung;Eom, Mi-Ok;Park, Mi-Sun;Jee, Seung-Wan;Kim, Ok-Hee
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2003.10b
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    • pp.178-178
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    • 2003
  • 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototype of many halogenated aromatic hydrocarbons, is a ubiquitous, persistent environmental contaminant and the most powerful carcinogen categorized by IARC. It is display high toxicity in animals and is associated with several cancers in human. Although the mechanism of carcinogenesis by TCDD is unclear, it is considered to be a non-genotoxic and rumor promoter.(omitted)

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Bacillus cellulyticus K-12 Crystalline Cellulose-Degrading Avicelase Gene and Expression in Eschterichia coli

  • Cheorl-Ho Kim;Woo
    • The Korean Journal of Food And Nutrition
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    • v.6 no.4
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    • pp.314-321
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    • 1993
  • We have cloned the Bacillus cellulyticus K-12 avicelase (Avi, E.C.3.2.1.4) gene (ace A) In E. coli. This was accompanied by using the vector PT7T3U 19 and Hind W -Hind m libraries of Bacillus cellulyticus K-12 chromosomal inserts created in 5.cofi. The Libraries were screened for the expression of avicelase by monitoring the immunoreaction of the anti-avicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5kb Hind III fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constituvely using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide which showed a CMCase activity with an Mr of 54000 was detected.

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Association between Maternal and Cord Blood Interleukin-10 (-819T/C and -592A/C) Gene Polymorphisms and Respiratory Distress Syndrome in Preterm Korean Infants (한국 미숙아 산모와 제대혈의 IL-10 유전자형 빈도와 신생아 호흡 곤란증 발생과의 연관성)

  • Park Eun-Ae;Cho Su-Jin;Kim Young-Ju;Park Hye-Sook;Ha Eun-Hee;Suh Young-Ju
    • Neonatal Medicine
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    • v.16 no.2
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    • pp.137-145
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    • 2009
  • Purpose: The aim of this study was to determine the genotype frequencies of interleukin 10 (IL-10) gene polymorphisms and to investigate their association with the risk of respiratory distress syndrome (RDS) in preterm Korean infants. Methods: Two hundred fourteen preterm infants born at Ewha Womans University Mok Dong Hospital between November 2003 and July 2008 were studied. The cord blood of preterm neonates and the corresponding maternal blood were analyzed by PCR for IL-10 gene (IL-10 -1082A/G, -819T/C, and -592A/C) polymorphisms. The clinical data of patients were collected retrospectively by chart review. Results: The genotype frequencies of IL-10 genes in Korean mothers with preterm infants differ from other reports. The prevalence of two promoter SNPs of the IL-10 cytokine gene was similar but none had the IL-10-1082GG homozygote. Multiple logistic regression analysis demonstrated the risk of RDS to be significantly lower in the infants of the mothers with an IL-10-592AC/CC genotype than in those with an AA genotype (P=0.033). The risk of RDS was significantly lower in the mother with an IL-10-819TC/CC genotype than in those with a TT genotype (P=0.030). However, IL-10 polymorphisms in the cord blood were not significantly different in preterm infants with RDS compared with the preterm infants without RDS. When we compared the incidence of RDS and each IL-10A-1082G/T-819C/A-592C haplotype, the ACC haplotype had a protective effect on RDS (P=0.007). Conclusion: We conclude that the maternal IL-10-592A/C and IL-10-819T/C polymorphisms may have a role in the development of the RDS in preterm infants.

The MMP-2 -735 C Allele is a Risk Factor for Susceptibility to Breast Cancer

  • Yari, Kheirollah;Rahimi, Ziba;Moradi, Mohamad Taher;Rahimi, Zohreh
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6199-6203
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    • 2014
  • Background: The expression of MMP genes has been demonstrated to be associated with tumor invasion, metastasis and survival rate for a variety of cancers. The functional promoter polymorphism MMP-2 C-735T is associated with decreased expression of the MMP-2 gene. The aim of present study was to detect any association between MMP-2 C-735T and susceptibility to breast cancer. Materials and Methods: The MMP-2 C-735T polymorphism was studied in 233 women (98 with breast cancer and 135 healthy controls). All studied women were from Kermanshah and Ilam provinces of Western Iran. The MMP-2 C-735T polymorphism was detected using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: The frequencies of MMP-2 CC, CT and TT genotypes in healthy individuals were 59.3, 38.5 and 2.2%, respectively. However, in breast cancer patients, only CC (71.4%) and CT (28.6%) genotypes were observed (p=0.077). In patients the frequency of the MMP-2 C allele was significantly higher (85.7%) compared to that in controls (78.5 %, p=0.048). The presence of C allele of MMP-2 increased the risk of breast cancer by 1.64-fold [OR=1.64 (95%CI 1.01-2.7, p=0.049)]. The frequency of MMP-2 C allele was also higher in patients ${\leq}40$ years (88.9%) than those aged ${\geq}41$ years (67.5%, p=0.07). In addition, the frequency of MMP-2 C allele tended to be higher in patients with a family history of cancer in first-degree relatives (76.6%) compared to that without a family history of cancer (67.3%, p=0.31). Conclusions: Our findings indicate that the C allele of MMP-2 C-735T polymorphism is associated with increased risk of breast cancer. Also, the MMP-2 C allele might increase the risk of young onset breast cancer in our population.

Construction and Characterization of Escherichia coli-Corynebacterium nephridii Hybrid Thioredoxins

  • Sa, Jae-Hoon;Kim, Kyung-Hoon;Lim, Chang-Jin
    • BMB Reports
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    • v.28 no.1
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    • pp.51-56
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    • 1995
  • Thioredoxin is a small redox protein with an active-site disulfide/dithiol, and is ubiquitous in bacteria, plants, and animals. To investigate the structure-function relationship of thioredoxin, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3(N to C-terminal) and C3-E. The hybrid thioredoxin genes were put under the T7 promoter and their productions were confirmed. The two hybrid thioredoxins complemented phenotypes of a thioredoxin-deficient E. coli strain. A strain containing the C3-E hybrid thioredoxin supported growth of the T7 phage, whereas a strain expressing the E-C3 hybrid thioredoxin did not. However, both hybrids supported growth of M13 phages. The two hybrid thioredoxins were also characterized in other aspects. Differences in activity between the hybrid thioredoxins were attributed to altered interactions of the N- and C-terminal domains of the molecule, which produced changes in the three-dimensional structure of the active site region.

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Agroinfiltration-based Potato Virus X Replicons to Dissect the Requirements of Viral Infection

  • Park, Sang-Ho;Kim, Kook-Hyung
    • The Plant Pathology Journal
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    • v.22 no.4
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    • pp.386-390
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    • 2006
  • Extensive research of the Potato virus X(PVX) has been performed in in vitro transcription system using the bacteriophage T7 promoter. We constructed an efficient T-DNA based binary vector, pSNU1, and modified vectors carrying PVX replicons. The suitability of the construct to transiently express PVX RNA using Agrobacterium tumefaciens was tested by analysis of infectivity in plants. The expressed PVX RNA was infectous and systemically spread in three plant species including Nicotiana benthamiana, N. tabacum cv. Xanthi-nc, and Capsicum annuum cv. Chilsungcho. The PVX full length construct, pSPVXp31, was caused severe mosaic symptoms on N. benthamiana, severe necrotic lesions on C. annuum while milder symptoms and delayed mosaic symptoms were appeared on the systemic leaves on N. tabaccum. RT-PCR analysis confirmed the presence of PVX RNAs on both inoculated and systemic leaves in all three plant species tested. Our results indicated that PVX replicons were efficiently expressed PVX RNA in at least three tested species. Further investigation win be needed to elucidate the mechanism of PVX replication, translation, movement and assembly/disassembly processes.

Production of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in E. coli by Control of Growth Rate. (대장균에서 증식속도 조절에 의한 수용성 재조합 인간 과립구 콜로니 촉진인자의 생산)

  • 박세철;고인영;강희일
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.135-141
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    • 2004
  • Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, recombinant hG-CSF is used successfully in the treatment of chemotheraphy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In this study, we have constructed rhG-CSF secretion plasmid pYRC1 in which OmpA signal sequence/hG-CSF gene was expressed under the control of the T7 promoter. rhG-CSF produced in E. coli BL21 (pYRC1) grown at $37{\circ}C$ was found in aggregates. However, 15% of the periplasmic protein was soluble rhG-CSF when the E. coli BL21 (pYRC1) was cultured at $25^{\circ}C$ for 7 h in the modified MBL medium containing 10 g/$\ell$ glucose with 10 $\mu$M IPTG induction. The production of soluble rhG-CSF in E. coli BL21 (pYRC1) using fed batch culture was also studied. In the fed batch culture system, the final yield of rhG-CSF produced from E. coli BL21 (pYRC1) was increased from 4.4 mg/$\ell$to 24 mg/$\ell$by controlling the specific growth rate from $0.43 h^{-1}$ to $0.14 h^{-1}$, and optimizing the time of induction.

Expression of the EPO-like Domains of Human Thrombopoietin in Escherichia coli

  • Koh, Yeo-Wook;Koo, Tai-Young;Ju, Sang-Myoung;Kwon, Chang-Hyuk;Chung, Joo-Young;Park, Myung-Hwan;Yang, Jai-Myung;Park, Seung-Kook
    • Journal of Microbiology and Biotechnology
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    • v.8 no.6
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    • pp.553-559
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    • 1998
  • cDNA of human thrombopoietin (hTPO) amplified by polymerase chain reaction from a cDNA library of human fetal liver was cloned. EPO-like domains ($hTPO_{153} \;or\; hTPO_{l63})\; of\; hTPO(hTPO_{332}$) were expressed in Escherichin coli using several kinds of expression systems, such as ompA secretion, thioredoxin fusion, and the $P_L$ and T7 expression systems. To obtain $hTPO_{153}$ in soluble form, $hTPO_{153}$ cDNA was fused in-frame behind the gene encoding ompA signal sequence and thioredoxin protein. When fused with either of the genes, $hTPO_{153}$ was not expressed to the detectable level. However, a high level expression of the EPO-like domain of hTPO was obtained using the PL and T7 expression system. $hTPO_{153} \;or\; hTPO_{l63} cDNA were subcloned into the pLex and pET-28a(+) vectors under the control of the inducible$ P_L\;T_7$ promoter, respectively. Proteins expressed using pl.ex vector and pET-28a(+) detected in insoluble forms with an expression level of about 14% and 9% of total cellular proteins, respectively, and the level of expression was rapidly diminished in 2 h after the maximum level of expression was reached.

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Association of the CYP17-34T/C Polymorphism with Pancreatic Cancer Risk

  • Hussain, Shahid;Bano, Raisa;Khan, Muhammad Tahir;Khan, Mohammad Haroon
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.sup3
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    • pp.71-75
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    • 2016
  • Pancreatic cancer is a leading cause of fatality worldwide. Several population studies have been conducted on genetic diagnosis of pancreatic cancer but the results from epidemiologic studies are very limited. CYP17A gene has a role in disease formation but its influence on pancreatic cancer is unclear. A polymorphism in the 5'UTR promoter region of CYP17A1-34T/C (A1/A2) has been associated with multiple cancers. The aim of the current study was to assess associations of this polymorphism and socio-demographic risk factors with pancreatic cancer. A total of 255 and 320 controls were enrolled in the study, and were genetically analyzed through PCR-RFLP. Statistical analysis was conducted with observed genotype frequencies and odds ratios (ORs) and 95% CIs were estimated using unconditional logistic regression. The impact of socio-demographic factors was accessed through Kaplen-Meir analysis. According to our results, the A2/A2 genotype was significantly associated with pancreatic cancer (OR=2.1, 95%CI = 1.3-3.5). Gender female (OR=2.6, 95%CI=1.8-3.7), age group 80s/80+ years (OR=2.2, 95% CI=1.2-4), smoking both former (OR=4.6, 95% CIs=2.5-8.8) and current (OR=3.6, 95% CI=2-6.7), and family history (OR=7.1; 95%CI = 4.6-11.4) were also found associated with increased risk. Current study suggests that along with established risk factors for pancreatic cancer CYP17A1-34T/C may play a role. However, on the basis of small sample size the argument cannot be fully endorsed and larger scale studies are recommended.

The Optimization of Expression System for Recombinant Protein Production by Pichia pastoris and Hansenula polymorphs (유전자 재조합 단백질 생산에 있어서 Pichia pastoris와 Hansenula polymorpha를 이용한 최적 발현 방법 개발)

  • 강환구;전희진;김재호
    • KSBB Journal
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    • v.15 no.2
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    • pp.174-180
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    • 2000
  • Pichia pastoris and Hansenula polymorpha, the methylotrophic yeasts have been widely used as a host for the production of e eudaryotic proteins due to the advantages related to their inherited characters. This paper describes the method to enhance t the productivity of recombinant proteins by P. pastoris and H. po$\psi$morpha. In the production of recombinant proteins using a f fed batch fermentation system, the effects of specific growth rate on the specific expression rate of re$\infty$mbinant proteins w were studied. In both species, the expression system of recombinant proteins using the fed batch fermentation was optimezed.

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