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Sample records for bax gene promoter

  1. A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death

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    Sheila J Semaan

    2010-03-01

    Full Text Available Pro-apoptotic Bax is essential for RGC (retinal ganglion cell death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2JBax+/− mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax−/− mice, but 129B6Bax+/− mice exhibited significant cell loss (similar to wild-type mice. The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T129B6 to CDBA/2J at position −515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53−/− cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA–protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli.

  2. Erythropoietin can promote survival of cerebral cells by downregulating Bax gene after traumatic brain injury in rats

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    Liao Z

    2009-01-01

    Full Text Available Background : Traumatic brain injury (TBI is an important cause of adult mortality and morbidity. Erythropoietin (Epo has been shown to promote the viability of cerebral cells by upregulating Bcl-2 gene; however, Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process. Aim : The present study examined the neuroprotective effect of Epo as a survival factor through the regulation of the Bax. Materials and Methods : Wistar rats were randomly divided into three groups: Recombinant human EPO treated (rhEPO TBI, vehicle-treated TBI, and sham-operated. Traumatic brain injury was induced by the Feeney free-falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR, western blotting and immunofluorescence. Results : Bax mRNA and protein levels were lower in the rhEPO-treated rat brains than in the vehicle-treated rat brains. Induction of Bax expression peaked at 24 h and remained stable for 72-120 h in vehicle-treated rat brains, whereas induction of Bax expression was only slightly elevated in rhEPO-treated rat brains. The number of TdT-mediated dUTP Nick-End Labeling(TUNEL-positive cells in the rhEPO-treated rat brains was far fewer than in the vehicle-treated rat brains. Conclusions : Epo exerts neuroprotective effect against traumatic brain injury via reducing Bax gene expression involved in inhibiting TBI-induced neuronal cell death.

  3. Study of regulatory promoter polymorphism (-248 G>A) of Bax gene in patients with gastric cancer in the northern provinces of Iran

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    Mirmajidi, Seyedeh Habibeh; Najafi, Mojtaba; Mirmajidi, Seyedeh Tahereh; Nasri Nasrabadi, Nafiseh

    2016-01-01

    Aim: The aim of this study is to evaluate the polymorphism in Bax gene and its association with some clinical pathology traits in gastric cancer. Background: Gastric cancer is considered as the fourth most common cancer in the north and northwest of Iran. Bcl2 family has a key role in regulation of apoptosis, and any changes in the expression of Bcl2 lead to cancer. Patients and methods: Blood samples were collected from 100 cases and 89 controls in the northern provinces of Iran to evaluate promoter polymorphism (-248GBax gene. Genotyping was carried out by PCR-RFLP method. Results: The result of this study demonstrated the existence of polymorphism in the above-mentioned region of Bax gene. Sixty-nine patients (%69) with genotype GG and 31 patients (%31) with genotype AG were observed in the case group. No mutant genotype was found among cases. Sixty-seven individuals (%75/28) with genotype GG, 21 individuals (%23/59) with genotype AG and only one mutant genotype (AA) were demonstrated in the control group. The bioinformatics analysis showed that this polymorphism removed the probable Sp1 motifs, which may affect its expression in the cells. Conclusion: Allele G was the most frequent between both patient and control samples. Polymorphism may be effective on Bax expression, but it requires further investigation. Our results showed significant effects between genotypes and features of gender and age, whereas no significant relation were observed between the genotypes and grade, stage as well as smoking traits. PMID:26744613

  4. BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

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    Boohaker, Rebecca J.; Zhang, Ge; Carlson, Adina Loosley; Nemec, Kathleen N.

    2011-01-01

    The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration. PMID:21289292

  5. PUMA promotes Bax translocation by competitive binding to Bcl-Xl during UV-induced apoptosis

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    Zhang, Yingjie; Xing, Da; Wu, Yinyuan; Liu, Lei

    2008-02-01

    Ultraviolet (UV) irradiation can induce apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways as DNA-damaging agents. PUMA, a BH3-only Bcl-2 family protein, plays an essential role in DNA damage-induced apoptosis. Bax, also a Bcl-2 family member, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis. However, the regulation of Bax activation induced by UV irradiation remains poorly understood. In this study, the FRET (fluorescence resonance energy transfer) technique was used to study the interactions of Bax, Bcl-Xl, and PUMA in ASTC-a-1 cells. The results show that Bax translocated from the cytosol to the mitochondrial membrane at about 7 h after UV irradiation, and the translocation can not be blocked completely when overexpressed Bcl-xl. Moreover, The interaction of Bax and Bcl-Xl weakened markedly. In addition, Co-immunoprecipitation shows that PUMA released Bax by directly binding to Bcl-XL after UV irradiation in ASTC-a-1 cells. Taken together, these results indicated that PUMA can promote Bax translocation by binding to Bcl-Xl during UV-induced apoptosis.

  6. THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun; YAO Zhen-xiang; ZHANG Jing

    1999-01-01

    Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer.Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues.Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05).Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.

  7. PUMA promotes Bax translocation in FOXO3a-dependent pathway during STS-induced apoptosis

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    Zhang, Yingjie; Chen, Qun

    2009-08-01

    PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently studies have been shown that Puma is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax translocation induced by STS were not blocked by Pifithrin-α (p53 inhibitor), which suggested that p53 was not involved in this signaling pathway. Taken together, these results indicate that PUMA promoted Bax translocation in a FOXO3a-dependment pathway during STS-induced apoptosis, while p53 was dispensable in this process.

  8. Effect of Exercise Training on Bcl-2 and Bax Gene Expression in the Rat Heart

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    Jafari; Pourrazi; Nikookheslat; Baradaran

    2015-01-01

    Background Apoptosis or programmed cell death plays an important role in the development of cardiovascular diseases, particularly heart failure. Current evidence suggests that exercise training may alter apoptosis-related signaling in sensitive somatic tissues such as the myocardium. Objectives The aim of this study was to assess the effect of exercise training on Bcl-2 and Bax genes expression as key molecules involved in intrins...

  9. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

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    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. PMID:26947183

  10. CO-EXPRESSIONS OF SURVIVIN GENE,BCL-2 AND BAX PROTEINS IN OVARIAN CARCINOMA

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    林蓓; 张淑兰; 赵长清

    2004-01-01

    Objective To characterize the cellular properties of ovarian cancer, we examined the correlation between the expression of apoptosis-related gene survivin and those of Bcl-2 and Bar proteins. Methods Expressions of survivin mRNA, and Bcl-2 and Bax proteins in 35 cases of ovarian carcinoma, 10 cases of borderline carcinoma, 10 cases of benign tumors and 10 cases of normal tissue were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry SABC method, respectively. Results Expression of survivin gene was detected in a significantly greater proportion in ovarian carcinoma and borderline carcinoma than those in benign tumors and normal tissues. Although there was no relationship between expression of survivin gene and FIGO stage, histologic grade, pathological type and lymphatic metastasis, expressions of Bcl-2 and Bar proteins were positively and negatively correlated with that of survivin gene, respectively. Conclusion Survivin may play an important role in pathogenesis of ovarian carcinoma, with a synergistic role of apoptosis-related gene Bcl-2protein and an antagonistic role of Bax protein in formation and progression of ovarian carcinoma.

  11. Low levels of Bax inhibitor-1 gene expression increase tunicamycin-induced apoptosis in human neuroblastoma SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    Dan Wu; Peirong Wang; Shiyao Wang

    2012-01-01

    A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment. In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.

  12. Effect of Exercise Training on Bcl-2 and Bax Gene Expression in the Rat Heart

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    Jafari

    2015-10-01

    Full Text Available Background Apoptosis or programmed cell death plays an important role in the development of cardiovascular diseases, particularly heart failure. Current evidence suggests that exercise training may alter apoptosis-related signaling in sensitive somatic tissues such as the myocardium. Objectives The aim of this study was to assess the effect of exercise training on Bcl-2 and Bax genes expression as key molecules involved in intrinsic pathway of apoptosis in the rat heart. Materials and Methods This study was conducted with a two-group experimental design (animal model and sixteen three-month-old male rats were selected and randomly divided to two groups of exercise training (n = 8 and control (n = 8. Rats in the trained group participated in an exercise training program for 12 weeks (10 – 60 m min-1, 24 – 33 min d-1, 15%. The rat hearts were removed forty-eight hours after the last training session. RNA extraction and synthesis of cDNA was done, and Bax and Bcl2 genes expression was analyzed through the Real Time-Polymerase Chain Reaction (RT-PCR. Kolmogorov-Smirnov and independent t-test were applied for statistical analysis of the data (P 0.05. However, Bcl2 expression was higher in the trained group compared to the control group (11%. Conclusions In general, it seems that three-month exercise training was effective in reducing cardiac mitochondrial pro-apoptotic protein. However, considering the results of the Bcl2 gene expression, more researches are needed to identify effects of exercise trainings on indices of myocardial apoptosis.

  13. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax

    Czech Academy of Sciences Publication Activity Database

    Lidman, M.; Pokorná, Šárka; Dingeldein, A. P. G.; Sparrman, T.; Wallgren, M.; Šachl, Radek; Hof, Martin; Gröbner, G.

    2016-01-01

    Roč. 1858, č. 6 (2016), s. 1288-1297. ISSN 0005-2736 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Apoptosis * Bax-protein * Calorimetry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.836, year: 2014

  14. Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer.

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    Ye, Zhiqiang; Hao, Rutian; Cai, Yefeng; Wang, Xiaobo; Huang, Guanli

    2016-04-01

    Accumulating evidence shows that microRNAs (miRNAs) have a critical role in the initiation and progression of types of human cancer, including breast cancer. Recent research indicated that miRNAs are also related with the chemotherapy on cancers. In this study, the expression of miR-221 in breast cancer (BC) patients' serum and cancer tissues was found to be significantly up-regulated. The results of in vitro MTT assay indicated that although the anti-miR-221 oligonucleotide alone did not influence the viability of BC cell lines markedly, it significantly promoted the cytotoxicity of cisplatin (DDP) to BC cells. Mechanistic studies demonstrated that the gene of BIM (Bcl-2 interacting mediator of cell death), a pro-apoptotic Bcl-2 family protein, was up-regulated by the knockdown of miR-221. We found that the synergetic effect of anti-miR-221 on increasing the sensitivity of MDA-MB-231 was BIM dependant. Furthermore, results of immunoprecipitation showed the up-regulated BIM directly combined with the Bax and Bak, leading to mitochondrial dysfunction. Our results suggest the anti-miR-221 could promote the cisplatin-inducing apoptosis by targeting the Bim-Bax/Bak axis in breast cancer. PMID:26503209

  15. The effect of pentoxifylline drug on bax/bcl2 gene dosage expression changes following ischemic reperfusion injury in kidney

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    Mehrdad Hashemi

    2015-11-01

    Full Text Available Global cerebral ischemia (GCI and reperfusion induced apoptosis that lead to cell injury and death. The bax and bcl-2 are pro-apoptotic and anti-apoptotic genes, respectively. These genes belong to The B-cell lymphoma-2 (bcl-2 family.In this study; we assessed the effect of pentoxifylline drug on bax/bcl2 gene dosage expression changes following ischemic reperfusion injury in kidney. In this experimental study, 20 male wistar rats were accidently divided them on two tenth group of control and treatment groups. In the control group, celiotomy was performed by ventral midline incision. The left kidney was isolated, and then both the renal artery and vein were obstructed. After 60 minutes of warm ischemia, vessel obstruction resolved and the right kidney was removed. 72 hours after reperfusion, tissue samples were taken from left kidney for histopathology. All these steps in treatment group were exactly repeated after administration of 45 mg/kg/PO pentoxifylline (3 hours before operation and in this group treatment was continued every 12h until 3 days. In this research quantitative real-time PCR is used for the detection expression Bcl2 and Bax genes in ischemia group and PNT drug group and compared to normal sample. The results showed the gene dosage ratio of bax/bcl2 in PNT group decline than to ischemia group. Therefore, the pentoxyfylline might have a role in control of apoptosis result from Ischemia- reperfusion

  16. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

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    Radogna, Flavia

    2015-03-01

    Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

  17. Effect of 147Pm β-irradiation on bcl-2 gene and bax gene expression in human leukemia cells

    International Nuclear Information System (INIS)

    Objective: To study the effect of exposure to a pure β radiator-147Pm on bcl-2 gene expression in human leukemia HL-60 cells. Methods: Immunohistochemical technique was used to identify the expression of bcl-2 and bax proteins in HL-60 cells, as well as RNA dot blot hybridization to show the expression of bcl-2 mRNA in HL-60 cells. Results: The expression of bcl-2 protein was as high as 88% in control human HL-60 cells. Down regulation of bcl-2 protein expression which lowered to 53% was noted after exposure to 147Pm. The expression of bax protein was low in control cells, and after irradiation it did not obviously change. An obvious down regulation of bcl-2 mRNA expression was found in irradiated HL-60 cells. With increasing time of 147Pm exposure, the down regulation of bcl-2 became more evident. Conclusion: The apoptotic action of 147Pm β-rays in human HL-60 cells is associated with the down regulation of the apoptosis-suppressing gene bcl-2

  18. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    International Nuclear Information System (INIS)

    Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the

  19. PUMA Promotes Bax Translocation by Both Directly Interacting with Bax and by Competitive Binding to Bcl-XL during UV-induced Apoptosis

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    Zhang, Yingjie; Xing, Da; Liu, Lei

    2009-01-01

    Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. p53 up-regulated modulator of apoptosis (PUMA) has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which PUMA regulates apoptosis, especially how PUMA activates Bcl-2-associated X protein (Bax) in response to UV irradiation is still controversial. In this study, by using real-time single-cell an...

  20. Effect of Bcl-2/Bax gene expression on apoptosis of spermatogenic cells of mouse testes induced by low dose radiation

    International Nuclear Information System (INIS)

    The different kinds of spermatogenic cells were separated using density gradient centrifugation and their apoptosis and Bcl-2 and Bax protein expression were measured with flow cytometry and immunohistochemical method, respectively. The results showed the apoptosis in all kinds of spermatogenic cells induced by low dose radiation (LDR) had a obvious regularity. When the doses were 0.025 and 0.05 Gy, spermatogonia apoptosis was dominant. With the increase of irradiation dose (0.075-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic percentage of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarcely occurred after LDR. Bax protein was primarily expressed in spermatogonia and spermatocytes, and the former was significantly higher than that of the latter after LDR. With the increase of irradiation dose, Bax protein expression showed a upgrading tendency, but that of spermatids and spermatozoa scarcely occurred. Bcl-2 protein was primarily expressed in spermatids and spermatozoa, but the Bcl-2 protein expressions of spermatogonia and spermatocytes scarcely occurred after LDR. These results imply that the interacting regulation of Bcl-2 and Bax gene expression might be involved in selective apoptosis of spermatogenic cells induced by LDR, which provided an experimental evidence for further exploring the apoptotic mechanism of adaptive response of spermatogenic cells by LDR

  1. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

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    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival. PMID:24305165

  2. Identification of Novel Pepper Genes Involved in Bax- or INF1-Mediated Cell Death Responses by High-Throughput Virus-Induced Gene Silencing

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    Jeong Hee Lee

    2013-11-01

    Full Text Available Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7% pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.

  3. bax和p53基因共转染对人舌癌细胞增 殖和凋亡的影晌%Effect on Proliferation and Apoptosis of Human Lingual Carc inoma Cells Cotransfected by bax and p53 Genes

    Institute of Scientific and Technical Information of China (English)

    马英红; 杨绍华; 陈俊杰; 高家让; 彭文珍; 王若菡

    2001-01-01

    Objective To investigate th e effects on prolife ration andapoptosis of human cancerous cells cotransfected by bax apoptosis-in ducing gene and p53 tumor suppressor gene. Methods The chi meric gene pSV-CIP-bax -CAT was constructed in which bax gene was flanked upstream by a 217bp fragment (+822~+1093) of the first intron and a 317bp promoter fragment of human a 1(Ⅰ) colla gen gene . Human lingual carcinoma cell line Tca 8113 (LCC) in culture was respe ctively transfected with pSV-CIP-bax-CAT, and cotransfected with pSV-p53-CA T and pSV-CIP-bax-CAT by using the transfection reagent DOSPER. Res ults Immuno- slot blot and ELISA demonstrated that the expression of bax and p53 genes incr eased remarkably in the transfected and cotransfected LCC, compared with the con trols(LCC transfected with pSV-CIP-CAT and LCC).MTT colorimetric assay,TUNE L fluorecence microscopy and flow cytometry showed that foreign bax or p53 gene inhibited the LCC growth and induced apoptosis(23.9% and 26.1% of inhibitory ra te by bax gene and p53 plus bax genes respectively). Conclusion The ectopic expression of the bax gene in the LCC promoted by the cis-act ing elements of human a1(Ⅰ)collagen gene has obviously synergistic effect on th e proliferation and apoptosis of the LCC cotransfected with p53 gene plus bax gene for 48 hours.%目的 探讨诱导凋亡的bax基因和p53抑癌基因共转染对人癌细胞增殖和凋亡的作用。方法 构建pSV-CIP-bax-CAT嵌合基因。在其bax基因上游含人a1(Ⅰ)型胶原基因的第一内含子217bp片段(+822~+1093)和317bp启动子片段。经阳离子转染试剂DOSPER介导,分别用pSV-CIP-bax-CAT转染、pSV-CIP-bax-CAT和pSV-p53-CAT共转染培养的人舌癌Tca8113细胞系(LCC)。结果 免疫狭缝印迹和ELISA检测证实,转染组和共转染组比对照的bax和p53基因表达量明显增加。MTT显色分析、TUNEL荧光显微镜和流式细胞仪监测结果显示,bax基因或p53基因均具抑

  4. Effects of Chronic Mild Stress in Female Bax Inhibitor-1-Gene Knockout Mice

    OpenAIRE

    Sui, Zhi-Yan; Chae, Han-Jung; Huang, Guang-Biao; Zhao, Tong; Shrestha Muna, Sushma; Chung, Young-Chul

    2012-01-01

    Objective The anti-apoptotic protein Bax inhibitor-1 (BI-1) is a regulator of apoptosis linked to endoplasmic reticulum (ER) stress, and BI-1-/- mice exhibit increased sensitivity to tissue damage. The purpose of this study was to investigate the role of BI-1 in the pathogenesis of chronic mild stress (CMS)-induced depression-like behaviors in BI-1-/- mice. Methods We delivered CMS for 2 or 6 weeks in BI-1-knockout and wild-type mice. Control groups of BI-1-knockout and wild-type mice were le...

  5. Quantitative Expression of BAG1, BAX and BCL-2 genes in Human Embryos with Different Fragmentation Grades Derived from ART

    Directory of Open Access Journals (Sweden)

    Poopak Eftekhari Yazdi

    2010-01-01

    Full Text Available Objective: The purpose of this study was to evaluate the quantitative expressions ofBAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derivedfrom assisted reproduction technology (ART.Materials and Methods: Fragmented and normal human 8-cell embryos were scoredaccording to the degree of fragmentation with an inverted microscope and divided intofour grades (grade І: no or minimal fragmentation (25% fragmentation and grade ІV: apoptotic inducedembryos with actinomycin D. In this study, TUNEL labeling was initially used todetect apoptosis, and then revers transcription polymerase chain reaction (RT-PCR andquantitative PCR were used to define the quantitative expressions of experimental genesin human embryos with different fragmentation grades.Results: The results of TUNEL labeling showed that embryos with higher fragmentationhad a high number of apoptotic bodies. The results of RT-PCR and q-PCR analysesshowed a significantly decreased amount of BAGI transcript expression from group I togroup IV. The highest expression of BAX gene was observed in group II, however, thetranscript of BCL-2 gene was not observed in any of the experimental groups. The effectof actinomycin D on transcript expression amounts of experimental genes in apoptoticinduced embryos (group IV compared to control embryos (group I showed a significantdecrease.Conclusion: mRNA expression of BAG1 gene can be used as a good marker to detectapoptosis in human embryos. However, the transcript of BCL-2 gene does not play a rolein the detection of apoptosis in human embryos at the 8-cell stage.

  6. bax, but not bcl-2, influences the prognosis of human pancreatic cancer

    OpenAIRE

    Friess, H; Lu, Z; H. Graber; Zimmermann, A.; Adler, G.; Korc, M.; Schmid, R; Buchler, M

    1998-01-01

    Background—bcl-2 and bax belong to the bcl-2-related gene family, which marks a new class of genes that influence apoptosis. The bcl-2 oncogene acts as a broad antiapoptotic factor and extends both normal and tumour cell survival. In contrast, the bax gene is a promoter of apoptosis. 
Aims—To analyse the expression of bcl-2 and bax in pancreatic cancer and correlate the results with clinical parameters. 
Patients—Pancreatic cancer tissue samples were obtained fro...

  7. The Impact of Adenosine Fast Induction of Myocardial Arrest during CABG on Myocardial Expression of Apoptosis-Regulating Genes Bax and Bcl-2

    Directory of Open Access Journals (Sweden)

    Ahmed Shalaby

    2009-01-01

    Full Text Available Background. We studied the effect of fast induction of cardiac arrest with denosine on myocardial bax and bcl-2 expression. Methods and Results. 40 elective CABG patients were allocated into two groups. The adenosine group (n=20 received 250 μg/kg adenosine into the aortic root followed by blood potassium cardioplegia. The control group received potassium cardioplegia in blood. Bcl-2 and bax were measured. Bax was reduced in the postoperative biopsies (1.38 versus 0.47, P=.002 in the control group. Bcl-2 showed a reducing tendency (0.14 versus 0.085, P=.07. After the adenosine treatment, the expression of both bax (0.52 versus 0.59, P=.4 and bcl-2 (0.104 versus 0.107, P=.4 remained unaltered after the operation. Conclusion. Open heart surgery is associated with rapid reduction in the expression of apoptosis regulating genes bax and bcl-2. Fast Adenosine induction abolished changes in their expression.

  8. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells.

    Science.gov (United States)

    Lowe, S L; Rubinchik, S; Honda, T; McDonnell, T J; Dong, J Y; Norris, J S

    2001-09-01

    In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude

  9. The Expression of Apoptosis-Related Genes Bcl-2 and Bax Protein and Apoptosis Positivity in Cervical Carcinoma during Irradiation

    Institute of Scientific and Technical Information of China (English)

    ZHAODongli; SHIJingsen; LIMingzhong; SONGLiping; WANGShuwen

    2005-01-01

    Objective: To evaluate the apoptosis positivity, the expression of Bcl-2. bax proteins in 30 patients with squamous cell cervix carcinoma before and after radiotherapy. Methods: By using immunohistochemical and TDT-dUTP nick end labelling techniques. 30 cases of squamous cell cervical carcinoma were analyzed. Results: The apoptosis positivity before and after irradiation was 76.7%, and 100% respectively, with the difference being significant (P<0.05); The positive rates of Bcl-2 protein before and after irradiation were 73.3% and 46.7% respectively, with the difference being significant (P<0.05): The positive rates of bax protein before and after irradiation were 86% and 100 respectively, with the difference being significant (P<0.05). Conclusion: bax and Bcl-2 protein play an important role in apoptosis induced by fractionated radiation therapy. Apoptosis induced by irradiation is contributed to upregulation of bax protein or downregulation of Bcl-2 protein.

  10. Expression of bax and bcl2 Genes in MDMA-induced Hepatotoxicity on Rat Liver Using Quantitative Real-Time PCR Method through Triggering Programmed Cell Death

    OpenAIRE

    2015-01-01

    Background: 3-4methylenedioxymethamphetamine (MDMA) is a synthetic and psychoactive drug, which is known popularly as Ecstasy and has toxic effects on human organs. Objectives: Considering the potential toxic interaction, this study was performed to quantify the expression of bax and bcl2 genes in MDMA-induced hepatotoxicity on rat liver. Subsequently, we evaluated pentoxifylline as a possible protective drug on hepatotoxicity. Materials and Methods: Adult male Wistar rats weighting 250 - 300...

  11. Effect of Helicobacter pylori infection on Bax protein expression in patients with gastric precancerous lesions

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Liu; Wei-Wen Liu; Guo-An Wang; Xiao-Chun Teng

    2005-01-01

    AIM: To investigate the effect of Helicobacter pylori (H pylori) infection on Bax protein expression, and explore the role of H pylori in gastric carcinogenesis.METHODS: H pylori was assessed by rapid urease test and Warthin-Starry method, and expression of Bax protein was examined immunohistochemically in 72 patients with pre-malignant lesions.RESULTS: Bax protein was differently expressed in intestinal metaplasia and gastric dysplasia, and showed 63.99% positivity. The positivity of Bax protein expression in H pylori-positive gastric precancerous lesions (72.3%) was significantly higher than that in H pylori-negative gastric precancerous lesions (48.0%, χ2 = 4.191, P<0.05).H pylori infection was well correlated with the expression of Bax protein in gastric precancerous lesions (r = 0.978,P<0.01). After eradication of H pylori, the positivity of Bax protein expression significantly decreased in H pylori-positive gastric precancerous lesions (χ2= 5.506,P<0.05). In the persisting H pylori-infected patients,the positivity of Bax protein expression was not changed.CONCLUSION: H pylori infection may be involved in the upregulation of Bax gene, which might be one of the mechanisms of H pylori infection-induced gastric epithelial cell apoptosis. H pylori might act as a tumor promoter in the genesis of gastric carcinoma and eradication of H pylori could inhibit gastric carcinogenesis.

  12. The bcl-2, bax gene expression and apoptosis of continuous low-dose-rate irradiation on PC-3 transplanting tumor

    International Nuclear Information System (INIS)

    Objective: The aim of this study was to investigate bcl-2, bax expression and apoptosis of continuous low-dose-rate irradiation on prostate cancer (PC)-3 transplanting tumor. Methods: The expression of bcl-2 and bax associated with apoptosis between experiment and control groups were analyzed using immunohistochemistry at 48, 96 and 192 h after two 125I seed sources implanting model. The correlation between apoptosis and the ratio of bax/bcl-2 was analyzed using Bi-variable linear correlation. SPSS 11.0 was used to analyse the data. Results: The bcl-2 expression in experiment group began to down-regulated significantly after 125I seed irradiation for 48 h as compared with control(t=2.500, P=0.067), though it was not reached to statistical significance. At 96 and 192 h after irradiation, significantly low expression of bcl- 2 were noted (t=4.950, 3.464; P=0.008 and 0.026). In contrast, significantly over expression of bax was noted at 48, 96 and 192 h after 12si irradiation (t=3.334,4.025,5.292;P=0.029, 0.016 and 0.006). The apoptotic index (AI) for PC-3 at 48, 96 and 192 h after 125I irradiation were 22.3%, 21.7% and 30.7%, which was significantly higher than controls when at 96 and 192 h after 125I irradiation (P= 0.016 and 0.036). Moreover, positive correlation was noted between AI and bax/bcl-2 ratio (r=0.784, P= 0.012). Conclusion: Low-dose-rate irradiation could down-regulate the expression of bcl-2, up-regulate the expression of bax and induced PC-3 cells apoptosis. (authors)

  13. Driving p53 Response to Bax Activation Greatly Enhances Sensitivity to Taxol by Inducing Massive Apoptosis

    Directory of Open Access Journals (Sweden)

    Paola De Feudis

    2000-05-01

    Full Text Available The proapoptotic gene bax is one of the downstream effectors of p53. The p53 binding site in the bax promoter is less responsive to p53 than the one in the growth arrest mediating gene p21. We introduced the bax gene under the control of 13 copies of a strong p53 responsive element into two ovarian cancer cell lines. The clones expressing bax under the control of p53 obtained from the wild-type (wt p53-expressing cell line A2780 were much more sensitive (500- to 1000-fold to the anticancer agent taxol than the parent cell line, with a higher percentage of cells undergoing apoptosis after drug treatment that was clearly p53-dependent and bax-mediated. Xenografts established in nude mice from one selected clone (A2780/C3 were more responsive to taxol than the parental line and the apoptotic response of A2780/C3 tumors was also increased after treatment. Introduction of the same plasmid into the p53 null SKOV3 cell line did not alter the sensitivity to taxol or the induction of apoptosis. In conclusion, driving the p53 response (after taxol treatment by activating the bax gene rather than the p21 gene results in induction of massive apoptosis, in vitro and in vivo, and greatly enhances sensitivity to the drug.

  14. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0- and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  15. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  16. A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Fabiola Méndez-Catalá

    2013-08-01

    Full Text Available We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF, in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.

  17. Quantitative Expression of BAG1, BAX and BCL-2 genes in Human Embryos with Different Fragmentation Grades Derived from ART

    OpenAIRE

    Poopak Eftekhari Yazdi; Azam Piltan; Mohamadreza Baghaban Eslaminejad; Leila Karimian; Hamid Gourabi; Mojtaba Rezazadeh; Mehdi Totonchi

    2010-01-01

    Objective: The purpose of this study was to evaluate the quantitative expressions ofBAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derivedfrom assisted reproduction technology (ART).Materials and Methods: Fragmented and normal human 8-cell embryos were scoredaccording to the degree of fragmentation with an inverted microscope and divided intofour grades (grade І: no or minimal fragmentation (

  18. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...... knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can...... be met by using promoter libraries. This approach generally consists of inserting a library of promoters in front of the gene to be studied, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter...

  19. Bax-induced cell death in Candida albicans.

    Science.gov (United States)

    De Smet, Kris; Eberhardt, Ines; Reekmans, Rieka; Contreras, Roland

    2004-12-01

    Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans. Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans. Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans, Bax co-localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region. PMID:15565645

  20. Expression of protein encoded by apoptosis-associated gene p53, bcl-2, and bax in adaptive response of thymocyte apoptosis in mice induced by low dose radiation with X-rays

    International Nuclear Information System (INIS)

    Objective: To explore the regulative mechanism of apoptosis-associated gene proteins on the adaptive response of thymocyte apoptosis in mice induced by low dose radiation with X-rays. Methods: Kunming male mice were irradiated with the inductive doses (D1: 25, 50, 75, 100 and 200 mGy; dose rate: 12.5 mGy ·min-1) and the challenging dose (D2: 1.5 Gy; dose rate: 287 mGy·min-1). The time interval between D1 and D2 was 6 h. The expressive levels of thymocyte apoptosis-associated gene proteins were measured with flow cytometry. Results: As compared with the sham-irradiation, the positive percentage of thymocyte Bcl-2 protein expression decreased significantly in D2 group (P<0.05), Bax increased significantly (P<0.05), and Bcl-2/Bax decreased significantly (P<0.001); p 53 increased significantly (P<0.001). As compared with D2 group, the positive percentage of thymocyte Bcl-2 protein expression increased in varying degree in D1+ D2 group of 25-75 mGy D1, Bax decreased in varying degree, and Bcl-2/Bax increased significantly (P<0.01); p53 decreased significantly (P<0.001 or P<0.05). Conclusion: The apoptotic thymocytes in the adaptive response of thymocyte apoptosis in mice induced by irradiation with 25-75 mGy decrease significantly due to the increase of apoptosis-associated gene Bcl-2 protein expression and Bcl-2/Bax, the decrease of Bax and p53 protein expressions. (authors)

  1. Small molecules reveal an alternative mechanism of Bax activation.

    Science.gov (United States)

    Brahmbhatt, Hetal; Uehling, David; Al-Awar, Rima; Leber, Brian; Andrews, David

    2016-04-15

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  2. Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

    Science.gov (United States)

    Luo, Sheng-Wei; Wang, Wei-Na; Sun, Zuo-Ming; Xie, Fu-Xing; Kong, Jing-Rong; Liu, Yuan; Cheng, Chang-Hong

    2016-07-01

    Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process. PMID:26905633

  3. Lycopene modulates cholinergic dysfunction, Bcl-2/Bax balance, and antioxidant enzymes gene transcripts in monosodium glutamate (E621) induced neurotoxicity in a rat model.

    Science.gov (United States)

    Sadek, Kadry; Abouzed, Tarek; Nasr, Sherif

    2016-04-01

    The effect of monosodium glutamate (MSG) on brain tissue and the relative ability of lycopene to avert these neurotoxic effects were investigated. Thirty-two male Wistar rats were distributed into 4 groups: group I, untreated (placebo); group II, injected with MSG (5 mg·kg(-1)) s.c.; group III, gastrogavaged with lycopene (10 mg·kg(-1)) p.o.; and group IV received MSG with lycopene with the same mentioned doses for 30 days. The results showed that MSG induced elevation in lipid peroxidation marker and perturbation in the antioxidant homeostasis and increased the levels of brain and serum cholinesterase (ChE), total creatine phosphokinase (CPK), creatine phosphokinase isoenzymes BB (CPK-BB), and lactate dehydrogenase (LDH). Glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) activities and gene expression were increased and glutathione content was reduced in the MSG-challenged rats, and these effects were ameliorated by lycopene. Furthermore, MSG induced apoptosis in brain tissues reflected in upregulation of pro-apoptotic Bax while lycopene upregulated the anti-apoptotic Bcl-2. Our results indicate that lycopene appears to be highly effective in relieving the toxic effects of MSG by inhibiting lipid peroxidation and inducing modifications in the activity of cholinesterase and antioxidant pathways. Interestingly, lycopene protects brain tissue by inhibiting apoptosis signaling induced by MSG. PMID:26900785

  4. Promoter architectures and developmental gene regulation.

    Science.gov (United States)

    Haberle, Vanja; Lenhard, Boris

    2016-09-01

    Core promoters are minimal regions sufficient to direct accurate initiation of transcription and are crucial for regulation of gene expression. They are highly diverse in terms of associated core promoter motifs, underlying sequence composition and patterns of transcription initiation. Distinctive features of promoters are also seen at the chromatin level, including nucleosome positioning patterns and presence of specific histone modifications. Recent advances in identifying and characterizing promoters using next-generation sequencing-based technologies have provided the basis for their classification into functional groups and have shed light on their modes of regulation, with important implications for transcriptional regulation in development. This review discusses the methodology and the results of genome-wide studies that provided insight into the diversity of RNA polymerase II promoter architectures in vertebrates and other Metazoa, and the association of these architectures with distinct modes of regulation in embryonic development and differentiation. PMID:26783721

  5. Expression and significance of Bcl-2 gene and Bax gene in lung cancer%凋亡相关基因bcl-2和bax在肺癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    郭雷; 王福昌; 杨五计

    2001-01-01

    To study the relationship between expression of bcl-2 and baxgene and lung cancer.The bcl-2 and bax gene expression in 51 lung cancer tissues and tissues near cancer were deteeted by immunohistochemical SP method.The positive degree of bcl-2 gene expression in lung cancer was higher than that in tissues near cancer.The positive degree of bax gene expression in lung cancer was lower than that in tissues near cancer.Both differences were very significant(P<0.01).There was no correlations between the immunoreactivity of bcl-2 and bax gene and histology,gender,smoking history,clinical subtypes,stage and lymphatic metastasis(P>0.05).It suggests that bcl-2 and bax gene may play an important role in tumorgenesis and tumor development.The detection and determination of bcl-2 and bax gene in preinvasive lesions may contribute to the early diagnosis of lung cancer.Some drugs can down-regulate bcl-2 expression and speed apoptosis so as to improve the sensitivity to chemical therapy and radiation.%为探讨B细胞淋巴瘤/白血病-2(bcl-2)基因及其同源类似物bax基因的表达与肺癌发生的关系,应用免疫组化SP法检测了51例肺癌患者的肺癌标本及其癌旁组织中bcl-2、bax的表达。结果显示,bcl-2在肺癌中染色比在癌旁组织中深,bax在肺癌中染色比在癌旁组织中浅,两者之间均有极显著差异(P<0.01);bcl-2、bax表达与肺癌的组织分型、患者的性别、吸烟史、临床分型、分期及淋巴结转移无关(P>0.05)。提示:①bcl-2、bax在肺癌发生中具有重要作用。②在癌前病变组织中检测并比较bcl-2和bax的表达,有利于肺癌的早期诊断。③可通过药物使bcl-2表达降低,促进凋亡,提高患者对放、化疗的敏感性。

  6. Silver Nanoparticles Biosynthesized Using Achillea biebersteinii Flower Extract: Apoptosis Induction in MCF-7 Cells via Caspase Activation and Regulation of Bax and Bcl-2 Gene Expression

    Directory of Open Access Journals (Sweden)

    Javad Baharara

    2015-02-01

    Full Text Available Silver nanoparticles (Ag-NPs, the most popular nanoparticles, possess unique properties. Achillea biebersteinii is a plant of the Asteraceae family rich in active antitumor components. The aim of this research was the characterization and investigation of the cytotoxic properties of Ag-NPs synthesized using A. biebersteinii flower extract, on a human breast cancer cell line. The Ag-NPs were synthesized after approximately 180 min of reaction at 40 °C, then they were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR, transmission electron microscopy (TEM and dynamic light scattering (DLS. The anti-apoptosis effect of Ag-NPs on the MCF-7 cell line was investigated by MTT assay, DAPI and acridine orange staining and caspase activity. The transcriptional expression of bax, bcl-2, caspase-3, -8 and -9 were also evaluated by RT-PCR. The TEM images revealed that the Ag-NPs morphology had a different shape. The DLS indicated that the average hydrodynamic diameter of the biosynthesized Ag-NPs was around 12 nm. By UV-visible spectroscopy the strongest absorbance peak was observed at 460 nm. The FTIR results also showed interaction between the plant extract and Ag-NPs due to the similarity in the peak patterns. The EDS results showed that Ag-NPs display an absorption peak at 3 keV, indicating the presence of the element silver. The Ag-NPs caused a dose-dependent decrease in cell viability, fragmentation in nucleic acid, inhibited the proliferation and induction of apoptosis on MCF-7 by suppressing specific cell cycle genes, and simulation programmed cell dead genes. Further investigation is required to establish the potential of this novel and promising approach in cancer therapy.

  7. Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

    Directory of Open Access Journals (Sweden)

    Elainy Patricia Lino Gasparotto

    2011-12-01

    Full Text Available Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV. This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005. CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020; while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19 compared with controls (1.39 and 2.01, p=0.006 and p=0.020. CD34+ cells in PV patients showed an elevated Bid expression (14.4 in comparison with healthy subjects (1.0; p=0.002. Patients' leukocytes showed an A1 augmentation (7.41, p=0.001 and a reduced expression of Bax (0.19; p=0.040 and Bad (0.2; p=0.030. There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV. Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005. Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020 e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19 em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0

  8. Precise regulation of gene expression dynamics favors complex promoter architectures.

    Directory of Open Access Journals (Sweden)

    Dirk Müller

    2009-01-01

    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  9. Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells

    Institute of Scientific and Technical Information of China (English)

    Jian-Yong Zheng; Guang-Shun Yang; Wei-Zhong Wang; Jiang Li; Kai-Zong Li; Wen-Xian Guan; Wen-Liang Wang

    2005-01-01

    AIM: To investigate the role of overexpression of Bax in apoptotic pathways and the response of human hepatocellular cancer (HCC)-9204 cells to cell death induced by adriamycin.METHODS: The whole length of Bax cDNA was transfectrd into human HCC-9204 cells by the method of lipofectamine transfection. An inducible MT-Ⅱ regulatory system was constructed, which allowed controlled expression of protein upon addition of ZnSO4 (100 μmol/L) as an external inducer. Stable transfecting inducible expression vector containing Bax gene was performed. Expression of Bax in protein was analyzed by immunohistochemistry and Western blotting. TUNEL and flow cytometry were used to assess the effect of Bax on apoptosis. Colony assay and tetrazolium blue (MTT) assay were used to evaluate the difference in drug sensitivity of HCC-9204 cells after Bax-transfection.RESULTS: Immunohistochemistry and Western blotting demonstrated that the expression of Bax protein markedly increased in Bax-transfected cells 4 h after the addition cytoplasm and perinuclear region of HCC-9404 cells, and there was ectopic expression in cells with marked condensation of chromatin and cytoplasm (apoptotic cells). Apoptotic index significantly increased in Bax-transfected HCC-9204/Bax cells (3.6 vs 27.2, 4.2 vs 32.3, P<0.05).Flow cytometry analysis showed a significant sub-G1 peak and apoptosis in 15.4% HCC-9204/Bax cells 24 h after treatment. Furthermore, colony survival rate decreased from 66% (HCC-9204/pMD) to 45% (HCC-9204/Bax) 2 dafter ADR withdrawal. MTT assay result showed that the effects of Bax on cell viability following ADR exposure were significant as compared to the vehicle-transfected HCC-9204/pMD cells (21% vs 44%, P<0.01).CONCLUSION: Overexpression of Bax not only induces apoptosis, but also sensitizes HCC-9204 cells to cell death induced by adriamycin.

  10. Clinicopathological significance of Bcl-2 and Bax protein expression in human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Ming Dong; Jian-Ping Zhou; Hao Zhang; Ke-Jian Guo; Yu-Lin Tian; Yu-Ting Dong

    2005-01-01

    AIM: To assess the clinicopathological significance of the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. METHODS: Fifty-nine surgical specimens of IDCs of the pancreas were stained immunohistochemically to detectpBcl-2 and pBax expressions whose correlation to tumor classification, staging, and prognosis was analyzed by univariate and multivariate analyses. RESULTS: The expression of pBcl-2 and pBax was detected in 21 of 59 (35.6%) and in 29 of 59 (49.2%) patients with IDCs of the pancreas, respectively. Neither pBcl-2 nor pBax alone was correlated to TNM staging and differentiation degree of IDCs of the pancreas according to univariate analysis. By Mantel-Cox test, the median survival time after surgery for pBcl-2(+) and pBcl-2(-) groups were 14.3 and 7.3 mo, respectively (χ2= 9.357, P = 0.002) and that for pBax(+) and pBax(-) groups were 12.9 and 10.2 mo, respectively (χ2= 0.285, P>0.05).Contingency coefficient between pBd-2 and pBax expression was 0.298, indicating that there was correlation between them (χ2= 5.74, P<0.05). The median survival time after surgery for pBd-2(+)pBax(+) and pBcl-2(+)pBax(-) groups were 14.3 and 14.1 mo, respectively, and that for pBcl-2 (-)pBax(+) and pBcl-2(-)pBax(-) groups were 5.9 and 9.9 mo, respectively. There was a significant difference between pBcl-2(+)pBax(+) and pBcl-2(-)pBax(+) (χ2 = 5.06,P<0.05), such was the case for pBcl-2(+)pBax(+) andpBcl-2(-)pBax(-) (χ2= 7.18, P<0.01). Cox proportional hazards model for multivariate analysis was applied, indicating that pBcl-2, TNM staging, age and pBax were high risk factors of post-surgical survival time. CONCLUSION: Both pBcl-2 and pBax have high expression in IDCs of the pancreas, indicating that co-expression of pBcl-2 and pBax is a good indicator of favorable prognosis in IDCs of the pancreas.

  11. The rat androgen receptor gene promoter

    NARCIS (Netherlands)

    W.M. Baarends (Willy); A.P.N. Themmen (Axel); L.J. Blok (Leen); P. Mackenbach (Petra); A.O. Brinkmann (Albert); D.N. Meijer (Dies); P.W. Faber; J. Trapman (Jan); J.A. Grootegoed (Anton)

    1990-01-01

    markdownabstractAbstract The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was clon

  12. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang;

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory...... mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression....

  13. Epigenetic regulation of transposable element derived human gene promoters.

    Science.gov (United States)

    Huda, Ahsan; Bowen, Nathan J; Conley, Andrew B; Jordan, I King

    2011-04-01

    It was previously thought that epigenetic histone modifications of mammalian transposable elements (TEs) serve primarily to defend the genome against deleterious effects associated with their activity. However, we recently showed that, genome-wide, human TEs can also be epigenetically modified in a manner consistent with their ability to regulate host genes. Here, we explore the ability of TE sequences to epigenetically regulate individual human genes by focusing on the histone modifications of promoter sequences derived from TEs. We found 1520 human genes that initiate transcription from within TE-derived promoter sequences. We evaluated the distributions of eight histone modifications across these TE-promoters, within and between the GM12878 and K562 cell lines, and related their modification status with the cell-type specific expression patterns of the genes that they regulate. TE-derived promoters are significantly enriched for active histone modifications, and depleted for repressive modifications, relative to the genomic background. Active histone modifications of TE-promoters peak at transcription start sites and are positively correlated with increasing expression within cell lines. Furthermore, differential modification of TE-derived promoters between cell lines is significantly correlated with differential gene expression. LTR-retrotransposon derived promoters in particular play a prominent role in mediating cell-type specific gene regulation, and a number of these LTR-promoter genes are implicated in lineage-specific cellular functions. The regulation of human genes mediated by histone modifications targeted to TE-derived promoters is consistent with the ability of TEs to contribute to the epigenomic landscape in a way that provides functional utility to the host genome. PMID:21215797

  14. Archaeal promoter architecture and mechanism of gene activation.

    Science.gov (United States)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang; She, Qunxin

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression. PMID:21265754

  15. Evolution of Drosophila ribosomal protein gene core promoters

    OpenAIRE

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2008-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, a...

  16. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

    Institute of Scientific and Technical Information of China (English)

    Ti Liu; Tianhong Wang; Xian Li; Xuan Liu

    2008-01-01

    To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from-677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from-620 to-820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4,and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

  17. PROMOTER HYPERMETHYLATION OF p16 GENE AND DAPK GENE IN SERA FROM HEPATOCELLULAR CARCINOMA (HCC) PATIENTS

    Institute of Scientific and Technical Information of China (English)

    LIN Qing; CHEN Long-bang; TANG Yong-ming; WANG Jing

    2005-01-01

    Objective: To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods: A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results: 76.6%(49/64) of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% (26/64) for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage,metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion: Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

  18. Limited specificity of promoter constructs for gene therapy in osteosarcoma.

    Science.gov (United States)

    Pollmann, Annika; Kabisch, Hartmut; Block, Andreas; Müller, Jürgen; Hellwinkel, Olaf J C

    2004-10-01

    Osteosarcoma (OS), a malignant bone neoplasia in childhood, has poor prognosis if metastases appear in the lung. A novel therapeutic approach could consist in a gene therapeutic treatment of OS metastases. However, if promiscuous viral vectors are applied for the delivery of potentially toxic transgenes, their misdelivery into normal tissues could cause severe complications. This problem could be circumvented by application of OS-specific promoters for transgene expression control. We analysed the function of promoters described to be tumour-, osteosarcoma- or osteoblast-specific. Expression rates driven by osteoblast- specific fragments from the collagen1A1-promoter, the human Osteocalcin-promoter, the bone-sialoprotein promoter and the beta-catenin promoter depending on vitamin supplementation were analysed in five OS cell lines, in normal lung fibroblasts and in a non-osteoblastic prostate cancer cell line (LNCaP) by dual luciferase assays. In addition, an unspecific but doxycyclin-repressible promoter construct (pAd.3r-luc) was examined. We found that all constructs were active in OS cell lines to varying extents. The complete human Osteocalcin promoter and the bone-sialoprotein promoter were partially induced by vitamin D3 or C respectively while the pAd.3r-luc activity could be shut down by doxycyclin. In contrast, the human Osteocalcin-promoter was not activated by vitamin D3 in LNCaP cells; its action remained relatively low. Interestingly, excepting the beta-catenin promoter, we measured strong activities of all promoters in lung fibroblast cells. Our study demonstrates that promoter activity should be evaluated not only for the target cells of the gene therapeutic approaches, but also for neighbouring normal tissues. Unspecific but repressible promoters could represent an alternative. PMID:15375610

  19. Firefly luciferase gene contains a cryptic promoter

    Czech Academy of Sciences Publication Activity Database

    Vopálenský, V.; Mašek, T.; Horváth, Ondřej; Vicenová, B.; Mokrejš, M.; Pospíšek, M.

    2008-01-01

    Roč. 14, č. 9 (2008), s. 1720-1729. ISSN 1355-8382 Grant ostatní: GAČR(CZ) GA204/03/1487; GAČR(CZ) GA301/07/0607; Mšk(CZ) LC06066 Institutional research plan: CEZ:AV0Z50520514 Keywords : luciferase * cryptic promoter * hepatitis C virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.018, year: 2008

  20. Evidence for an Induced-Fit Process Underlying the Activation of Apoptotic BAX by an Intrinsically Disordered BimBH3 Peptide.

    Science.gov (United States)

    Jhong, Siao-Ru; Li, Ching-Yu; Sung, Tai-Ching; Lan, Yu-Jing; Chang, Kuo-Jung; Chiang, Yun-Wei

    2016-03-17

    Apoptotic BAX protein functions as a critical gateway to mitochondria-mediated apoptosis. A diversity of stimuli has been implicated in initiating BAX activation, but the triggering mechanism remains elusive. Here we study the interaction of BAX with an intrinsically disordered BH3 motif of Bim protein (BimBH3) using ESR techniques. Upon incubation with BAX, BimBH3 binds to BAX at helices 1/6 trigger site to initiate conformational changes of BAX, which in turn promotes the formation of BAX oligomers. The study strategy is twofold: while BAX oligomerization was monitored through spectral changes of spin-labeled BAX, the binding kinetics was studied by observing time-dependent changes of spin-labeled BimBH3. Meanwhile, conformational transition between the unstructured and structured BimBH3 was measured. We show that helical propensity of the BimBH3 is increased upon binding to BAX but is then reduced after being released from the activated BAX; the release is due to the BimBH3-induced conformational change of BAX that is a prerequisite for the oligomer assembling. Intermediate states are identified, offering a key snapshot of the coupled folding and binding process. Our results provide a quantitative mechanistic description of the BAX activation and reveal new insights into the mechanism underlying the interactions between BAX and BH3-mimetic peptide. PMID:26913490

  1. Functional analysis of the Myostatin gene promoter in sheep

    Institute of Scientific and Technical Information of China (English)

    DU; Rong; AN; XiaoRong; CHEN; YongFu; QIN; Jian

    2007-01-01

    Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNProW-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNProE(3+5+7)M-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3―1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin

  2. Common promoter elements in odorant and vomeronasal receptor genes.

    Directory of Open Access Journals (Sweden)

    Jussara S Michaloski

    Full Text Available In mammals, odorants and pheromones are detected by hundreds of odorant receptors (ORs and vomeronasal receptors (V1Rs and V2Rs expressed by sensory neurons that are respectively located in the main olfactory epithelium and in the vomeronasal organ. Even though these two olfactory systems are functionally and anatomically separate, their sensory neurons show a common mechanism of receptor gene regulation: each neuron expresses a single receptor gene from a single allele. The mechanisms underlying OR and VR gene expression remain unclear. Here we investigated if OR and V1R genes share common sequences in their promoter regions.We conducted a comparative analysis of promoter regions of 39 mouse V1R genes and found motifs that are common to a large number of promoters. We then searched mouse OR promoter regions for motifs that resemble the ones found in the V1R promoters. We identified motifs that are present in both the V1R and OR promoter regions. Some of these motifs correspond to the known O/E like binding sites while others resemble binding sites for transcriptional repressors. We show that one of these motifs specifically interacts with proteins extracted from both nuclei from olfactory and vomeronasal neurons. Our study is the first to identify motifs that resemble binding sites for repressors in the promoters of OR and V1R genes. Analysis of these motifs and of the proteins that bind to these motifs should reveal important aspects of the mechanisms of OR/V1R gene regulation.

  3. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  4. Melatonin restores normal Bax and Bcl-2 protein expression in the subgranular zone of the dentate gyrus in pinealectomized rats

    Institute of Scientific and Technical Information of China (English)

    Shengchang Zhang; Shuang Zhao; Lu Bai; Mingming Guan; Jielin Mo; Ling Lan

    2011-01-01

    In this study, we sought to elucidate the effects of melatonin on learning and memory as well as apoptosis and expression of the Bax or Bcl-2 proteins in the subgranular zone of the dentate gyrus in pinealectomized rats. Using the Morris water maze and the olfactory memory tests, we found that the average escape latency in pinealectomized rats was clearly increased compared with sham-operated rats. Moreover, the average escape latency in the melatonin-treated and pinealectomized rats was longer than that in the sham-operated rats and shorter than that in the pinealectomized and untreated rats. Immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) showed that there were fewer Bax immunoreactive cells and TUNEL-positive (apoptotic) cells but more Bcl-2 immunoreactive cells in the melatonin-treated rats compared with the pinealectomized rats. The sham-operated rats showed numbers of these cells similar to the melatonin-treated rats. These experimental findings demonstrate that melatonin treatment may reduce abnormal apoptosis by promoting gene expression of Bax and suppressing gene expression of Bcl-2 in the subgranular zone of the dentate gyrus in pinealectomized rats. These effects appear to result in the inhibition of cellular apoptosis and the improvement of spatial learning and memory in pinealectomized rats.

  5. Cloning and Characterization of Gene Promoters from Bacillus pumilus

    Institute of Scientific and Technical Information of China (English)

    Pan Jiao(潘皎); Zhang Yizheng

    2004-01-01

    DNA fragments obtained from Sau3AI partially digested total DNA of Bacillus pumilus UN31-C-42 are first inserted into BamHI site of pSUPV4, a promoter-probe vector. The recombinant DNA molecules are transformed into Escherichia coli cells and eight-three Kanr clones (named pSUBp1- pSUBp83) are obtained. The inserted fragments in pSUBp53, pSUBp57, pSUBp21, which showed high level of kanamycin - resistance, are sequenced and analyzed, respectively. These fragments contain some conserved sequences of prokaryotic gene promoters, such as TATAAT and TTGACA box. The promoter fragment Bp53 could efficiently promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells.

  6. Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

    OpenAIRE

    Victoria Gonzalo; Juan José Lozano; Jenifer Muñoz; Francesc Balaguer; Maria Pellisé; Cristina Rodríguez de Miguel; Montserrat Andreu; Rodrigo Jover; Xavier Llor; M Dolores Giráldez; Teresa Ocaña; Anna Serradesanferm; Virginia Alonso-Espinaco; Mireya Jimeno; Miriam Cuatrecasas

    2010-01-01

    BACKGROUND: Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-...

  7. The long-range interaction landscape of gene promoters.

    Science.gov (United States)

    Sanyal, Amartya; Lajoie, Bryan R; Jain, Gaurav; Dekker, Job

    2012-09-01

    The vast non-coding portion of the human genome is full of functional elements and disease-causing regulatory variants. The principles defining the relationships between these elements and distal target genes remain unknown. Promoters and distal elements can engage in looping interactions that have been implicated in gene regulation. Here we have applied chromosome conformation capture carbon copy (5C) to interrogate comprehensively interactions between transcription start sites (TSSs) and distal elements in 1% of the human genome representing the ENCODE pilot project regions. 5C maps were generated for GM12878, K562 and HeLa-S3 cells and results were integrated with data from the ENCODE consortium. In each cell line we discovered >1,000 long-range interactions between promoters and distal sites that include elements resembling enhancers, promoters and CTCF-bound sites. We observed significant correlations between gene expression, promoter-enhancer interactions and the presence of enhancer RNAs. Long-range interactions show marked asymmetry with a bias for interactions with elements located ∼120 kilobases upstream of the TSS. Long-range interactions are often not blocked by sites bound by CTCF and cohesin, indicating that many of these sites do not demarcate physically insulated gene domains. Furthermore, only ∼7% of looping interactions are with the nearest gene, indicating that genomic proximity is not a simple predictor for long-range interactions. Finally, promoters and distal elements are engaged in multiple long-range interactions to form complex networks. Our results start to place genes and regulatory elements in three-dimensional context, revealing their functional relationships. PMID:22955621

  8. IGFBP-2 enhances VEGF gene promoter activity and consequent promotion of angiogenesis by neuroblastoma cells.

    Science.gov (United States)

    Azar, Walid J; Azar, Sheena H X; Higgins, Sandra; Hu, Ji-Fan; Hoffman, Andrew R; Newgreen, Donald F; Werther, George A; Russo, Vincenzo C

    2011-09-01

    IGF binding protein (IGFBP)-2 is one of the most significant genes in the signature of major aggressive cancers. Previously, we have shown that IGFBP-2 enhances proliferation and invasion of neuroblastoma cells, suggesting that IGFBP-2 activates a protumorigenic gene expression program in these cells. Gene expression profiling in human neuroblastoma SK-N-SHEP (SHEP)-BP-2 cells indicated that IGFBP-2 overexpression activated a gene expression program consistent with enhancement of tumorigenesis. Regulation was significant for genes involved in proliferation/survival, migration/adhesion, and angiogenesis, including the up-regulation of vascular endothelial growth factor (VEGF) mRNA (>2-fold). Specific transcriptional activation of the VEGF gene by IGFBP-2 overexpression was demonstrated via cotransfection of a VEGF promoter Luciferase construct in SHEP-BP-2. Cotransfection of VEGF promoter Luciferase construct with IGFBP-2 protein in wild-type SHEP cells indicated that transactivation of VEGF promoter only occurs in the presence of intracellular IGFBP-2. Cell fractionation and immunofluorescence in SHEP-BP-2 cells demonstrated nuclear localization of IGFBP-2. These findings suggest that transcriptional activation of VEGF promoter is likely to be mediated by nuclear IGFBP-2. The levels of secreted VEGF (up to 400 pg/10(6) cells) suggested that VEGF might elicit angiogenic activity. Hence, SHEP-BP-2 cells and control clones cultured in collagen sponge were xenografted onto chick embryo chorioallantoic membrane. Neomicrovascularization was observed by 72 h, solely in the SHEP-BP-2 cell xenografts. In conclusion, our data indicate that IGFBP-2 is an activator of aggressive behavior in cancer cells, involving nuclear entry and activation of a protumorigenic gene expression program, including transcriptional regulation of the VEGF gene and consequent proangiogenic activity of NB cell xenografts in vivo. PMID:21750048

  9. Isolation of the promoters of Atlantic salmon MHCII genes.

    Science.gov (United States)

    Syed, Mohasina; Vestrheim, Olav; Mikkelsen, Birthe; Lundin, Maria

    2003-01-01

    The major histocompatibility complex class II (MHCII) has a central role in the immune response of vertebrates with its function of presenting antigenic peptides to the T-cell receptors. We have isolated the promoters and intron 1 of MHCIIalpha and MHCIIbeta genes of Atlantic salmon. To isolate these promoters, we constructed an Atlantic salmon ( Salmo salar) promoter finder kit (analogous to the commercially available "human promoter finder kit"). By nucleotide sequence alignment of known MHCII promoter regions, we identified the 3 conserved regulatory X, X2, and Y boxes in the salmon promoters. The W box was not found. In contrast, a salmon-specific putative W box was identified. Both of the isolated Atlantic salmon MHCIIalpha and beta promoters (included in patent applications by Genomar A/S, Oslo, Norway) were found to be functional since they both gave positive yellow fluorescence protein signal when inserted as promoters in the pEYFP-1 reporter plasmid and transfected into the salmon head kidney cell line (SHK-1). PMID:14502397

  10. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  11. Comparative analysis of ADS gene promoter in seven Artemisia species

    Indian Academy of Sciences (India)

    Mojtaba Ranjbar; Mohammad Reza Naghavi; Hoshang Alizadeh

    2014-12-01

    Artemisinin is the most effective antimalarial drug that is derived from Artemisia annua. Amorpha-4,11-diene synthase (ADS) controls the first committed step in artemisinin biosynthesis. The ADS gene expression is regulated by transcription factors which bind to the cis-acting elements on the ADS promoter and are probably responsible for the ADS gene expression difference in the Artemisia species. To identify the elements that are significantly involved in ADS gene expression, the ADS gene promoter of the seven Artemisia species was isolated and comparative analysis was performed on the ADS promoter sequences of these species. Results revealed that some of the cis-elements were unique or in terms of number were more in the high artemisinin producer species, A. annua, than the other species. We have reported that the light-responsive elements, W-box, CAAT-box, 5′-UTR py-rich stretch, TATA-box sequence and tandem repeat sequences have been identified as important factors in the increased expression of ADS gene.

  12. Isolation and characterization of the murine Nanog gene promoter

    Institute of Scientific and Technical Information of China (English)

    Da Yong WU; Zhen YAO

    2005-01-01

    Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and embryonic germ cells but disappeared in differentiated ES cells. In this study, we have isolated, sequenced, and performed the first characterization of the Nanog promoter. The transcription start sites were mapped by primer extension analysis. Two promoter regions were found upstream the transcription start sites and the expression of major Nanog promoter/reporter gene construct is abolished in differentiated F9 EC cells as compared to the undifferentiated counterpart. We also showed that a putative octamer motif (ATGCAAAA) is necessary for the major promoter activity. Gel shift and supershift assays showed that Oct-1, Oct-4 and Oct-6 protein selectively bind to the octamer motif.

  13. THE EFFECT OF EXERCISE PRECONDITION INDUCED MIR-21 AND BAX GENE EXPRESSIONS ON RATS' CARDIAC MYOCYTE AFTER EXHAUSTIVE EXERCISES%运动预适应对力竭运动后心肌mir-21和bax基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    黄雅雯

    2011-01-01

    [目的]探讨运动预适应(excerise preconditioning,EP)对大鼠一次性力竭运动后心肌mir-21和bax基因表达的影响.[方法]健康雄性SD大鼠36只,随机分为对照组(C组)、一次性力竭运动组(E组)、运动预适应+一次性力竭运动组(EP组).力竭运动后即刻和24 h取各组心肌采用real-time PCR检测各心肌内mir-21和bax基因表达情况.[结果]EP可以显著抑制一次性力竭运动后心肌内mir-21基因的下调表达(P<0.05)和bax基因的上调表达(P<0.05).[结论]EP可以通过抑制mir-21基因下调从而抑制bax基因的上调来发挥保护作用.%[ Objective] To explore the effect of exercise preconditioning induced mir-21 and bax gene expressions on rats' cardiac myocyte after exhaustive exercises. [Methods] 36 healthy male SD rats were randomly divided into control group (C group) , exhaustive exercises (E group) , exercise preconditioning+ exhaustive exercises (EP group) .Used real-time PCR to detect expressions of mir-21 and bax after exhaustive exercises and 24h. [Results] The results of real-time PCR suggested that in EP group, down-regulation of mir-21 and up-regulation of bax cardiac myocyte were obviously inhibited by exhaustive exercises. [Conclusion] EP could inhibit the down-regulation of bax gene through inhibiting up-regulation of mir-21 gene.

  14. The effect of radiation on bcl-2 and bax in hyperplastic prostatic tissues

    International Nuclear Information System (INIS)

    Aim: To investigate the expressions of bcl-2 and bax in benign prostatic hyperplasia (BPH) and the effect of β-rays on bcl-2 and bax. Methods: The expressions of bcl-2 and bax are studied by means of immunohistochemical method in 9 normal prostate (NP) and 15 BPH and 35 patients treated with 90Sr/90Y Prostatic Hyperplasia Applicator. Results: The expressions of bcl-2 in epithelia of NP and BPH are higher than that in stroma P<0.01=. The expressions of bcl-2 in epithelia and stroma of BPH are higher than that in NP P<0.01=. The expressions of bax in epithelia of NP are higher than that in BPH P<0.05=. However ,the expressions of bcl-2 in epithelia and stroma of BPH are higher than bax P<0.01 =. Compared with the control group, the expressions of bcl-2 in epithelia and stroma of BPH treated with 90Sr/90Y Prostatic Hyperplasia Applicator decreased and the expressions of bax increased P<0.01=. Conclusion: bcl-2 gene and bax gene play an important role in the regulation of prostatic apoptosis and the treatment of β-rays can accelerate the apoptosis of prostatic tissues. (authors)

  15. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen

    2005-01-01

    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  16. Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.

    Science.gov (United States)

    Zhang, J; Huang, K; O'Neill, K L; Pang, X; Luo, X

    2016-01-01

    How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid(-/)(-)Bim(-/)(-)Puma(-/)(-) (TKO), TKO/Bax(-/)(-)/Bak(-/)(-) (PentaKO), and PentaKO/Mcl-1(-/-) (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53(-/-) (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized. PMID:27310874

  17. Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain.

    Directory of Open Access Journals (Sweden)

    Patrick O McGowan

    Full Text Available BACKGROUND: Alterations in gene expression in the suicide brain have been reported and for several genes DNA methylation as an epigenetic regulator is thought to play a role. rRNA genes, that encode ribosomal RNA, are the backbone of the protein synthesis machinery and levels of rRNA gene promoter methylation determine rRNA transcription. METHODOLOGY/PRINCIPAL FINDINGS: We test here by sodium bisulfite mapping of the rRNA promoter and quantitative real-time PCR of rRNA expression the hypothesis that epigenetic differences in critical loci in the brain are involved in the pathophysiology of suicide. Suicide subjects in this study were selected for a history of early childhood neglect/abuse, which is associated with decreased hippocampal volume and cognitive impairments. rRNA was significantly hypermethylated throughout the promoter and 5' regulatory region in the brain of suicide subjects, consistent with reduced rRNA expression in the hippocampus. This difference in rRNA methylation was not evident in the cerebellum and occurred in the absence of genome-wide changes in methylation, as assessed by nearest neighbor. CONCLUSIONS/SIGNIFICANCE: This is the first study to show aberrant regulation of the protein synthesis machinery in the suicide brain. The data implicate the epigenetic modulation of rRNA in the pathophysiology of suicide.

  18. Gene activation regresses atherosclerosis, promotes health, and enhances longevity

    Directory of Open Access Journals (Sweden)

    Luoma Pauli V

    2010-07-01

    Full Text Available Abstract Background Lifestyle factors and pharmacological compounds activate genetic mechanisms that influence the development of atherosclerotic and other diseases. This article reviews studies on natural and pharmacological gene activation that promotes health and enhances longevity. Results Living habits including healthy diet and regular physical activity, and pharmacotherapy, upregulate genes encoding enzymes and apolipoprotein and ATP-binding cassette transporters, acting in metabolic processes that promote health and increase survival. Cytochrome P450-enzymes, physiological factors in maintaining cholesterol homeostasis, generate oxysterols for the elimination of surplus cholesterol. Hepatic CTP:phosphocholine cytidylyltransferase-α is an important regulator of plasma HDL-C level. Gene-activators produce plasma lipoprotein profile, high HDL-C, HDL2-C and HDL-C/cholesterol ratio, which is typical of low risk of atherosclerotic disease, and also of exceptional longevity together with reduced prevalence of cardiovascular, metabolic and other diseases. High HDL contributes to protection against inflammation, oxidation and thrombosis, and associates with good cognitive function in very old people. Avoiding unhealthy stress and managing it properly promotes health and increases life expectancy. Conclusions Healthy living habits and gene-activating xenobiotics upregulate mechanisms that produce lipoprotein pattern typical of very old people and enhance longevity. Lipoprotein metabolism and large HDL2 associate with the process of living a very long life. Major future goals for health promotion are the improving of commitment to both wise lifestyle choices and drug therapy, and further the developing of new and more effective and well tolerated drugs and treatments.

  19. GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

    Science.gov (United States)

    Honda, Tsuyoshi; Coppola, Simona; Ghibelli, Lina; Cho, Song H; Kagawa, Shunsuke; Spurgers, Kevin B; Brisbay, Shawn M; Roth, Jack A; Meyn, Raymond E; Fang, Bingliang; McDonnell, Timothy J

    2004-04-01

    The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. PMID:15002033

  20. Analysis of promoter activity in transgenic plants by normalizing expression with a reference gene: anomalies due to the influence of the test promoter on the reference promoter

    Indian Academy of Sciences (India)

    Simran Bhullar; Suma Chakravarthy; Deepak Pental; Pradeep Kumar Burma

    2009-12-01

    Variations in transgene expression due to position effect and copy number are normalized when analysing and comparing the strengths of different promoters. In such experiments, the promoter to be tested is placed upstream to a reporter gene and a second expression cassette is introduced in a linked fashion in the same transfer DNA (T-DNA). Normalization in the activity of the test promoter is carried out by calculating the ratio of activities of the test and reference promoters. When an appropriate number of independent transgenic events are analysed, normalization facilitates assessment of the relative strengths of the test promoters being compared. In this study, using different modified versions of the Cauliflower Mosaic Virus (CaMV) 35S promoter expressing the reporter gene -glucuronidase (gus) (test cassette) linked to a chloramphenicol acetyl transferase (cat) gene under the wild-type 35S promoter (reference cassette) in transgenic tobacco lines, we observed that cat gene expression varied depending upon the strength of the modified 35S promoter expressing the gus gene. The 35S promoter in the reference cassette was found to have been upregulated in cases where the modified 35S promoter was weaker than the wild-type 35S promoter. Many studies have been carried out in different organisms to study the phenomenon of transcriptional interference, which refers to the reduced expression of the downstream promoter by a closely linked upstream promoter. However, we observed a positive interaction wherein the weakened activity of a promoter led to upregulation of a contiguous promoter. These observations suggest that, in situations where the promoters of the test and reference gene share the same transcription factors, the activity of the test promoter can influence the activity of the reference promoter in a way that the test promoter’s strength is underestimated when normalized by the reference promoter.

  1. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    International Nuclear Information System (INIS)

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca2+ was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca2+]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway, which may

  2. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  3. Genome-wide analysis of regions similar to promoters of histone genes

    KAUST Repository

    Chowdhary, Rajesh

    2010-05-28

    Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

  4. Influence of Apoptin on Up-regulation of the Expression of Bad and Bax

    Institute of Scientific and Technical Information of China (English)

    GUO Tai; YANG Qian

    2005-01-01

    The chicken anemia virus protein, apoptin, which manifests selectivity and specificity to tumor cells, induces a p53-independent and Bcl-2-insensitive type of apoptosis in various human tumor cells. In this study, the apoptin gene was cloned from the total DNA of chicken anemia virus, and the recombinant vector was constructed. We used oligonucleotide microarray to study the changes of four genes, including Bcl-2, Bcl-xL, Bad and Bax. The post-transfection with the recombinant was also studied. The pro-apoptotic genes(Bad and Bax) and anti-apoptosis genes(Bcl-2 and Bcl-xL) were up-regulated in contrast to the controls. According to the published data, either Bcl-2 or Bcl-xL can form non-functional heterodimers by Bad and Bax binding together, resulting in blocking partly the release of cytochrome c from mitochondria. However, apoptosis could be inhibited by neither the endogenous Bcl-xL nor Bcl-2 over-expression. The experiments show that the apoptin-induced apoptotic pathway is related to the up-regulation of Bad and Bax. Bad was up-regulated by apoptin; then this up-regulated product of Bad was in favor of displacing Bax from binding to Bcl-xL or Bcl-2. Consequently, Bax exerted a pro-apoptotic dysfunction to mitochondria, thereby inducing the release of cytochrome c. Finally, apoptin induced the apoptosis of HHCC cells. These results indicate that the oligonucleotide microarray can reveal the genes related to the apoptosis induced by apoptin in HHCC cells.

  5. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  6. Development of chimeric gene promoters responsive to hypoxia and ionizing radiation

    International Nuclear Information System (INIS)

    The authors describe two systems that make use of gene-directed enzyme prodrug therapy, regulated by radiation or hypoxic-responsive promoters. The use of treatment-, condition- or tumor-specific promoters to control gene-directed enzyme prodrug therapy is one such method for targeting gene expression to the tumor. The development of such strategies that achieve tumor targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy

  7. Keratin promoter based gene manipulation in the murine conducting airway

    Directory of Open Access Journals (Sweden)

    Stephen P. Malkoski, Timothy G. Cleaver, Shi-Long Lu, Jessyka G. Lighthall, Xiao-Jing Wang

    2010-01-01

    Full Text Available Systems capable of targeting genetic manipulations to keratin-positive airway basal cells are more poorly developed than systems targeting other airway epithelial cell populations and this has likely hindered development of animal models of diseases such as lung squamous cell carcinoma. Although keratin promoter driven-Cre recombinase constructs are potentially useful for targeting these cells, these constructs have substantially higher activity in the skin and oral epithelium than in the airways. We developed a method for delivering RU486, the conditional activator of Cre recombinase progesterone receptor (CrePR fusion proteins to the lung and then examined the activity of three keratin-driven CrePR constructs in the conducting airways. We also developed a technique for survival bronchioalveolar lavage on non-ventilated animals to examine the effects of the acetone/oil vehicle required to deliver RU486 to the lung. K5CrePR1 and K14CrePR1 constructs differ only in the keratin promoter used to target CrePR1 expression while K5Cre*PR contains a truncated progesterone receptor designed to reduce RU486-independent Cre activity. While all three constructs demonstrate RU486-inducible Cre activity in the conducting airways, both construct activity and tightness of regulation vary considerably. K5Cre*PR is the most tightly regulated Cre driver making it ideal for targeting somatic mutations to the airway epithelia while K5CrePR1 and K14CrePR1 may be better suited to studying diseases of the conducting airways where gene targeting of keratin expressing cells and their derivatives is desired.

  8. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  9. IL6 gene promoter polymorphisms and type 2 diabetes

    DEFF Research Database (Denmark)

    Huth, Cornelia; Heid, Iris M; Vollmert, Caren; Gieger, Christian; Grallert, Harald; Wolford, Johanna K; Langer, Birgit; Thorand, Barbara; Klopp, Norman; Hamid, Yasmin H; Pedersen, Oluf; Hansen, Torben; Lyssenko, Valeriya; Groop, Leif; Meisinger, Christa; Döring, Angela; Löwel, Hannelore; Lieb, Wolfgang; Hengstenberg, Christian; Rathmann, Wolfgang; Martin, Stephan; Stephens, Jeffrey W; Ireland, Helen; Mather, Hugh; Miller, George J; Stringham, Heather M; Boehnke, Michael; Tuomilehto, Jaakko; Boeing, Heiner; Möhlig, Matthias; Spranger, Joachim; Pfeiffer, Andreas; Wernstedt, Ingrid; Niklason, Anders; López-Bermejo, Abel; Fernández-Real, José-Manuel; Hanson, Robert L; Gallart, Luis; Vendrell, Joan; Tsiavou, Anastasia; Hatziagelaki, Erifili; Humphries, Steve E; Wichmann, H-Erich; Herder, Christian; Illig, Thomas

    2006-01-01

    countries, making this the largest association analysis for type 2 diabetes reported to date. The GC and CC genotypes of IL6 -174G>C were associated with a decreased risk of type 2 diabetes (odds ratio 0.91, P = 0.037), corresponding to a risk modification of nearly 9%. No evidence for association was found......Several lines of evidence indicate a causal role of the cytokine interleukin (IL)-6 in the development of type 2 diabetes in humans. Two common polymorphisms in the promoter of the IL-6 encoding gene IL6, -174G>C (rs1800795) and -573G>C (rs1800796), have been investigated for association with type...... 2 diabetes in numerous studies but with results that have been largely equivocal. To clarify the relationship between the two IL6 variants and type 2 diabetes, we analyzed individual data on >20,000 participants from 21 published and unpublished studies. Collected data represent eight different...

  10. Apoptosis repressor with a CARD domain (ARC restrains Bax-mediated pathogenesis in dystrophic skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Jennifer Davis

    Full Text Available Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc, in dystrophic mouse models. Nol3 (Arc protein genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP, as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3 (-/- Sgcd (-/- double targeted mice. Mechanistically, Nol3 (-/- Sgcd (-/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.

  11. A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

    DEFF Research Database (Denmark)

    Kampranis, S C; Damianova, R; Atallah, M;

    2000-01-01

    for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein...... was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the...... mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI...

  12. Ernest Belfort Bax: Marxist, Idealist, Positivist

    OpenAIRE

    Bevir, Mark

    1993-01-01

    Bax was the leading philosopher of the socialist revival in Britain during the 1880s. He saw Marxism as an economic and historical science that lacked a philosophical and ethical basis. Consequently, he tried to justify the Marxian dialectic by using a philosophy indebted to German idealism to show that the dialectic was a fact about reality itself, and he also tried to provide an ethical defence of Marxism in terms of a positivist ethic enshrining the goals of the French Revolution. Such ...

  13. HOX Gene Promoter Prediction and Inter-genomic Comparison: An Evo-Devo Study

    Directory of Open Access Journals (Sweden)

    Marla A. Endriga

    2010-10-01

    Full Text Available Homeobox genes direct the anterior-posterior axis of the body plan in eukaryotic organisms. Promoter regions upstream of the Hox genes jumpstart the transcription process. CpG islands found within the promoter regions can cause silencing of these promoters. The locations of the promoter regions and the CpG islands of Homeo sapiens sapiens (human, Pan troglodytes (chimpanzee, Mus musculus (mouse, and Rattus norvegicus (brown rat are compared and related to the possible influence on the specification of the mammalian body plan. The sequence of each gene in Hox clusters A-D of the mammals considered were retrieved from Ensembl and locations of promoter regions and CpG islands predicted using Exon Finder. The predicted promoter sequences were confirmed via BLAST and verified against the Eukaryotic Promoter Database. The significance of the locations was determined using the Kruskal-Wallis test. Among the four clusters, only promoter locations in cluster B showed significant difference. HOX B genes have been linked with the control of genes that direct the development of axial morphology, particularly of the vertebral column bones. The magnitude of variation among the body plans of closely-related species can thus be partially attributed to the promoter kind, location and number, and gene inactivation via CpG methylation.

  14. Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

    Directory of Open Access Journals (Sweden)

    Farré Domènec

    2007-12-01

    Full Text Available Abstract Background The arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes. Results We observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters. Conclusion The study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

  15. The Promoter of the Cereal VERNALIZATION1 Gene Is Sufficient for Transcriptional Induction by Prolonged Cold

    OpenAIRE

    Alonso-Peral, María M.; Oliver, Sandra N.; Casao Acerete, Cristina; Greenup, Aaron A.; Trevaskis, Ben

    2011-01-01

    The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expressio...

  16. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    OpenAIRE

    V Shilpa; Rahul Bhagat; C S Premalata; V R Pallavi; Ramesh, G.; Lakshmi Krishnamoorthy

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hyperme...

  17. 黄芪多糖对力竭运动大鼠心肌细胞凋亡及BcL-2、Bax蛋白表达的影响%Effects of acute exercise to exhaustion on BcL-2,Bax gene of rat, sheart after supplyment of astragalus polysaccharide

    Institute of Scientific and Technical Information of China (English)

    马兰军; 田振军

    2012-01-01

    为了探讨黄芪多糖(APS)对力竭运动大鼠机体的保护作用,通过建立大强度力竭运动大鼠模型,选取成年健康雄性SD大鼠24只,随机分为安静对照组,运动组和运动加药组,运动组和运动加药组按照训练模型进行为期6周的耐力训练,最后1次训练进行1次力竭运动,随后取心肌组织并进行样本处理.检测大鼠心肌MDA含量,用缺口末端标记法(TUNEL法)及免疫组织化学法检大鼠心肌细胞凋亡指数(AI)及BcL-2、Bax蛋白表达的变化.结果表明:运动组MDA含量、AI和BcL-2、Bax蛋白表达水平均明显高于安静对照组(P<0.05);与运动组比较,运动加药组MDA含量,AI与Bax蛋白表达水平均下降,而BcL-2蛋白表达水平显著增高(P<0.05).黄芪多糖可有效抑制力竭运动大鼠心肌细胞凋亡,此作用可能与其减轻氧化应激、上调BcL-2和下调Bax蛋白表达水平有关.%To study the supplementary astragalus polysaccharide (APS) to exhaustion movement rats myocardial cell apoptosis, observe the influence of myocardial cell apop-tosis BcL-2, the regulation of gene Bax changes, explore astragalus polysaccharide (APS) to exhaustion movement rats airframe of protection. By establishing high intensity exhaustion movement model of rats, the selection of adult health male SD rats 24 only randomly divided into quiet in the exercise group and control group, sports dosing group, the exercise group and exercise dosing groups according to training model six-week endurance training, last training once exhaustion movement, exhaustion after motion myocardial organize and sample processing. Detection rats myocardial MDA content, using gap end labeling (TUNEL law) and the method of immunohis to chemistry methods were used by rats myocardial cell apoptosis I,ndex (AI) and BcL-2, Bax protein expression changes. In the exercise group MDA content, AI and BcL-2, Bax protein expression levels were significantly higher in quiet in control group (P

  18. Molecular cloning and characterization of the promoter region of the porcine apolipoprotein E gene.

    Science.gov (United States)

    Xia, Jihan; Hu, Bingjun; Mu, Yulian; Xin, Leilei; Yang, Shulin; Li, Kui

    2014-05-01

    Apolipoprotein E (APOE), a component of lipoproteins plays an important role in the transport and metabolism of cholesterol, and is associated with hyperlipoproteinemia and Alzheimer's disease. In order to further understand the characterization of APOE gene, the promoter of APOE gene of Landrace pigs was analyzed in the present study. The genomic structure and amino acid sequence in pigs were analyzed and found to share high similarity in those of human but low similarity in promoter region. Real-time PCR revealed the APOE gene expression pattern of pigs in diverse tissues. The highest expression level was observed in liver, relatively low expression in other tissues, especially in stomach and muscle. Furthermore, the promoter expressing in Hepa 1-6 was significantly better at driving luciferase expression compared with C2C12 cell. After analysis of porcine APOE gene promoter regions, potential transcription factor binding sites were predicted and two GC signals, a TATA box were indicated. Results of promoter activity analysis indicated that one of potential regulatory elements was located in the region -669 to -259, which was essential for a high expression of the APOE gene. Promoter mutation and deletion analysis further suggested that the C/EBPA binding site within the APOE promoter was responsible for the regulation of APOE transcription. Electrophoretic mobility shift assays also showed the binding site of the transcription factor C/EBPA. This study advances our knowledge of the promoter of the porcine APOE gene. PMID:24464129

  19. Investigation of Interleukin-17 gene polymorphism on DAP-Kinase gene promoter methylation in Patients with Breast Cancer

    Directory of Open Access Journals (Sweden)

    S Naeimi

    2016-02-01

    Full Text Available Background & Aim: Breast cancer is the most common malignancy in women . Studies have shown that increased in methylation of CpG islands (CpG island hyper methylation, CIHM, is one of the important mechanisms in gene down regulation. DAP-Kinase protein plays an important role in the process of Apoptosis. Interleukin-17 is an proinflammatory cytokine and inflammation,is one of the factors  that affect on gene methylation . the purpose of this study was to evaluate the polymorphism of the IL-17 gene promoter methylation Dap-kinase and its relationship to breast cancer. Methods: In this case - control study, A total of 40 Women with Breast cancer and 40 healthy women in Iran were examined.DNA was extracted by saluting out method and Single nucleotide Polymorphisms of the IL-17 gene were analyzed by the PCR-RFLP method and To study gene promoter methylation Dap-kinase, MSPCR method was used.data were compared in both groups by using Pearson’s chi-square and Hardy-weinberg equilibrium test. RESULTS: Results confirm the fact that, there is a relationship between DAP-kinase gene promoter methylation and breast cancer disease So that the promoter of this gene in patients than in healthy individuals was much more methylated( p0.05 Conclusion: Due to the fact, that promoter genes methylation is one of the mechanisms of epigenetic genes silencing, it seems that DAP-kinase gene promoter methylation increases is associated with the risk of breast cancer in women.

  20. Analysis of promoter regions of co-expressed genes identified by microarray analysis

    Directory of Open Access Journals (Sweden)

    Höglund Mattias

    2006-08-01

    Full Text Available Abstract Background The use of global gene expression profiling to identify sets of genes with similar expression patterns is rapidly becoming a widespread approach for understanding biological processes. A logical and systematic approach to study co-expressed genes is to analyze their promoter sequences to identify transcription factors that may be involved in establishing specific profiles and that may be experimentally investigated. Results We introduce promoter clustering i.e. grouping of promoters with respect to their high scoring motif content, and show that this approach greatly enhances the identification of common and significant transcription factor binding sites (TFBS in co-expressed genes. We apply this method to two different dataset, one consisting of micro array data from 108 leukemias (AMLs and a second from a time series experiment, and show that biologically relevant promoter patterns may be obtained using phylogenetic foot-printing methodology. In addition, we also found that 15% of the analyzed promoter regions contained transcription factors start sites for additional genes transcribed in the opposite direction. Conclusion Promoter clustering based on global promoter features greatly improve the identification of shared TFBS in co-expressed genes. We believe that the outlined approach may be a useful first step to identify transcription factors that contribute to specific features of gene expression profiles.

  1. Isolation and characterization of oil palm constitutive promoter derived from ubiquitin extension protein (uep1) gene.

    Science.gov (United States)

    Masura, Subhi Siti; Parveez, Ghulam Kadir Ahmad; Ismail, Ismanizan

    2010-09-30

    The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm. We have isolated and characterized the 5' region of the oil palm uep1 gene, which contains an 828 bp sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector, which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional analysis of the promoter through transient expression studies. It was found that the 5' region of uep1 functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested, including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf, mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was demonstrated to be capable of driving gusA gene expression in tobacco. PMID:20123048

  2. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.

    Science.gov (United States)

    Zheng, Xuelian; Deng, Wei; Luo, Keming; Duan, Hui; Chen, Yongqin; McAvoy, Richard; Song, Shuiqing; Pei, Yan; Li, Yi

    2007-08-01

    Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. PMID:17340093

  3. Effect of glucocorticoid on promoter of 11β-hydroxysteroid dehydrogenase I gene

    Institute of Scientific and Technical Information of China (English)

    何平; 孙刚

    2003-01-01

    Objective: To study the effect of glucocorticoid on the promoter of the pre-receptor glucocorticoid metabolizing enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) gene. Methods: The 1.2 kb length sequence upstream to the transcription start site of the 11β-HSD1 gene was amplified with polymerase chain reaction (PCR) and then was cloned into pBLCAT6 plasmid carrying chloramphenicol acetyltransferase (CAT) reporter gene. The plasmid pBLCAT6 carrying the promoter and reporter gene was used to transfect HeLa cells to study the regulation of 11β-HSD1 gene expression by glucocorticoids in terms of reporter gene expression. Results: PCR showed that there was a complete alignment of the amplified sequence with the sequence 1.2 kb upstream to the transcription start site of 11β-HSD1 gene. When cloned into pBLCAT6 plasmid carrying the reporter gene, this part of the promoter is functional in terms of regulation of reporter gene expression upon transfection into HeLa cells. The synthetic glucocorticoid-dexamethasone induced the reporter gene expression in the system described above, which was blocked by glucocorticoid receptor antagonist RU486. Conclusion: Glucocorticoids can modulate the expression of 11β-HSD1 through a mechanism involving activation of GR and interaction of the promoter of 11β-HSD1 gene.

  4. The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

    International Nuclear Information System (INIS)

    Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

  5. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    International Nuclear Information System (INIS)

    Research highlights: → Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. → Expression profiling of apoptosis-related genes in ApcMin/+ mice revealed the differential expression of pro-apoptotic Bok and Bax. → APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. → Blocking of β-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or β-catenin causes constitutively active β-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the ApcMin/+ mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of β-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which uncontrolled epithelial cell proliferation in the stem

  6. Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

    Science.gov (United States)

    Matsuyama, Shigemi; Palmer, James; Bates, Adam; Poventud-Fuentes, Izmarie; Wong, Kelvin; Ngo, Justine; Matsuyama, Mieko

    2016-06-01

    Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their

  7. Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain▿ †

    OpenAIRE

    Lu, Chenfeng; Jeffries, Thomas

    2007-01-01

    We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentati...

  8. Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene.

    Science.gov (United States)

    Joo, Yoo Jin; Kim, Jin-Ha; Baek, Joung Hee; Seong, Ki Moon; Lee, Jae Yung; Kim, Joon

    2009-01-01

    Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using beta-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene. PMID:19853675

  9. Association of Polymorphisms in the Hepatocyte Growth Factor Gene Promoter with Keratoconus

    OpenAIRE

    Burdon, Kathryn P; MacGregor, Stuart; Bykhovskaya, Yelena; Javadiyan, Sharhbanou; Li, Xiaohui; Laurie, Kate J.; Muszynska, Dorota; Lindsay, Richard; Lechner, Judith; Haritunians, Talin; Henders, Anjali K.; Dash, Durga; Siscovick, David; Anand, Seema; Aldave, Anthony

    2011-01-01

    This is a meta-analysis of two genome-wide association studies that found evidence of association of keratoconus with polymorphisms in the promoter of the HGF gene. One polymorphism is associated with higher levels of serum HGF.

  10. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François

    2009-10-01

    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  11. Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

    Directory of Open Access Journals (Sweden)

    Daniel Meier

    Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

  12. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU

    2004-01-01

    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  13. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    OpenAIRE

    Miyuki Uno; Sueli Mieko Oba-Shinjo; Anamaria Aranha Camargo; Ricardo Pereira Moura; Paulo Henrique Aguiar; Hector Navarro Cabrera; Marcos Begnami; Sérgio Rosemberg; Manoel Jacobsen Teixeira; Suely Kazue Nagahashi Marie

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty...

  14. Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

    OpenAIRE

    Varley, A.W.; Coulthard, M G; Meidell, R S; Gerard, R D; Munford, R S

    1995-01-01

    We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal lu...

  15. A promoter that drives gene expression preferentially in male transgenic rats

    OpenAIRE

    Li, Qiling; Ma, Yamin; Li, Wenzhi; Xu, Wei; Ma, Li; Fu, Guoxing; Tian, Xiaohua; Wang, Yueling; Li, Xu; Bythwood, Tameka; Richards, Jendai; Song, Qing

    2013-01-01

    Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein (CRP) transgene was under the cont...

  16. Positive Selection of Primate Genes that Promote HIV-1 Replication

    OpenAIRE

    Meyerson, Nicholas R.; Rowley, Paul A.; Swan, Christina H; Le, Dona T.; Wilkerson, Greg K; Sawyer, Sara L

    2014-01-01

    Evolutionary analyses have revealed that most host-encoded restriction factors against HIV-1 have experienced virus-driven selection during primate evolution. However, HIV also depends on the function of many human proteins, called host factors, for its replication. It is not clear whether virus-driven selection shapes the evolution of host factor genes to the extent that it is known to shape restriction factor genes. We show that 5 out of 40 HIV host factor genes (13%) analyzed do bear stron...

  17. Approach of combined cancer gene therapy and radiation: response of promoters to ionizing radiation

    International Nuclear Information System (INIS)

    Gene therapy is an emerging cancer treatment modality. We are interested in developing a radiation-inducible gene therapy system to sensitize the tumor vasculature to the effects of ionizing radiation (IR) treatment. An expression system based on irradiation-inducible promoters will drive the expression of anti-tumor genes in the tumor vasculature. Solid tumors are dependent on angio genesis, a process in which new blood vessels are formed from the pre-existing vasculature. Vascular endothelial cells are un transformed and genetically stable, thus avoiding the problem of resistance to the treatments. Vascular endothelial cells may therefore represent a suitable target for this therapeutic gene therapy strategy.The identification of IR-inducible promoters native to endothelial cells was performed by gene expression profiling using cDNA micro array technology. We describe the genes modified by clinically relevant doses of IR. The extension to high doses aimed at studying the effects of total radiation delivery to the tumor. The radio-inductiveness of the genes selected for promoter study was confirmed by RT-PCR. Analysis of the activity of promoters in response to IR was also assessed in a reporter plasmid. We found that authentic promoters cloned onto a plasmid are not suitable for cancer gene therapy due to their low induction after IR. In contrast, synthetic promoters containing repeated sequence-specific binding sites for IR-activated transcription factors such as NF-κB are potential candidates for gene therapy. The activity of five tandemly repeated TGGGGACTTTCCGC elements for NF-κB binding in a luciferase reporter was increased in a dose-dependent manner. Interestingly, the response to fractionated low doses was improved in comparison to the total single dose. Thus, we put present evidence that a synthetic promoter for NF-κB specific binding may have application in the radio-therapeutic treatment of cancer. (author)

  18. Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

    International Nuclear Information System (INIS)

    The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

  19. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B;

    1999-01-01

    were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A...

  20. Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter.

    OpenAIRE

    Xie, Y Q; Takimoto, K; Pitot, H. C.; Miskimins, W K; Lindahl, R

    1996-01-01

    The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and ge...

  1. Common Promoter Elements in Odorant and Vomeronasal Receptor Genes

    OpenAIRE

    Jussara S Michaloski; Galante, Pedro A. F.; Nagai, Maíra H.; Lucia Armelin-Correa; Ming-Shan Chien; Hiroaki Matsunami; Bettina Malnic

    2011-01-01

    In mammals, odorants and pheromones are detected by hundreds of odorant receptors (ORs) and vomeronasal receptors (V1Rs and V2Rs) expressed by sensory neurons that are respectively located in the main olfactory epithelium and in the vomeronasal organ. Even though these two olfactory systems are functionally and anatomically separate, their sensory neurons show a common mechanism of receptor gene regulation: each neuron expresses a single receptor gene from a single allele. The mechanisms unde...

  2. Different promoter affinities account for specificity in MYC-dependent gene regulation

    Science.gov (United States)

    Lorenzin, Francesca; Benary, Uwe; Baluapuri, Apoorva; Walz, Susanne; Jung, Lisa Anna; von Eyss, Björn; Kisker, Caroline; Wolf, Jana; Eilers, Martin; Wolf, Elmar

    2016-01-01

    Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. DOI: http://dx.doi.org/10.7554/eLife.15161.001 PMID:27460974

  3. The promoter of the cereal VERNALIZATION1 gene is sufficient for transcriptional induction by prolonged cold.

    Directory of Open Access Journals (Sweden)

    Maria M Alonso-Peral

    Full Text Available The VERNALIZATION1 (VRN1 gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare. A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops.

  4. Rice Mitochondrial Genes Are Transcribed by Multiple Promoters That Are Highly Diverged

    Institute of Scientific and Technical Information of China (English)

    Qun-Yu Zhang; Yao-Guang Liu

    2006-01-01

    Plant mitochondrial genes are often transcribed into complex sets of mRNA. To characterize the transcription initiation and promoter structure, the transcript termini of four mitochondrial genes, atp1, atp6, cob,rps7, in rice (Oryza sativa L.), were determined by using a modified circularized RNA reverse transcriptionpolymerase chain reaction method. The results revealed that three genes (atp1, atp6, rps7) were transcribed from multiple initiation sites, indicating the presence of multiple promoters. Two transcription termination sites were detected in three genes (atp6, cob, rps7), respectively. Analysis on the promoter architecture showed that the YRTA (Y=T or C, R=A or G) motifs that are widely present in the mitochondrial promoters of other monocot and dicot plant species were detected only in two of the 12 analyzed promoters.Our data suggest that the promoter sequences in the rice mitochondrial genome are highly diverged in comparison to those in other plants, and the YRTA motif is not an essential element for the promoter activity.

  5. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2008-10-01

    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  6. Tissue specific promoters improve the localization of radiation-inducible gene expression

    International Nuclear Information System (INIS)

    Purpose: Site-specific activation of gene expression can be achieved by the use of a promoter that is induced by physical agents such as x-rays. The purpose of the present study was to determine whether site-specific activation of gene therapy can also be achieved within the vascular endothelium by use of radiation-inducible promoters. We studied induction of promoter-reporter gene constructs using previously identified radiation-promoters from c-jun, c-fos, Egr-1, ICAM-1, ELAM-1 after transfection into in the vascular endothelium. Methods: The following radiation-inducible genetic constructs were created: The ELAM-1 promoter fragment was cloned into pOGH to obtain the pE-sel(-587 +35)GH reporter construct. The ICAM-1 promoter fragment (-1162/+1) was cloned upstream of the CAT coding region of the pCAT-plasmid (Promega) after removal of the SV40 promoter by Bgl2/Stu1 digestion to create the pBS-CAT plasmid. The 132 to +170 bp segment of the 5' untranslated region of the c-jun promoter was cloned to the CAT reporter gene to create the -132/+170 cjun-CAT. The Egr-1 promoter fragment (-425/+75) was cloned upstream of the CAT coding region to create the pE425-CAT plasmid. Tandem repeats of the AP-1 binding site were cloned upstream of the CAT coding region (3 xTRE-CAT). Tandem repeats of the Egr binding site (EBS) were cloned upstream of the CAT coding region (EBS-CAT). Human vascular endothelial cells from both large vessel and small vessel origin (HUVEC and HMEC), as well as human tumor cell lines were transfected with plasmids -132/+170 cjun-CAT, pE425-CAT, 3 xTRE-CAT, EBS-CAT, pE-sel-GH and pBS-CAT by use of liposomes. Humor tumor cell lines included SQ20B (squamous), RIT3 (sarcoma), and HL525 (leukemia). Each plasmid was cotransfected with a plasmid containing a CMV promoter linked to the LacZ gene (1 μg). Transfected cells were treated with mock irradiation or x-rays. Cell extracts were assayed for reporter gene expression. Results: Radiation-induced gene

  7. Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors

    OpenAIRE

    Catia Pilon; Andrea Rebellato; Riccardo Urbanet; Vincenza Guzzardo; Rocco Cappellesso; Hironobu Sasano; Ambrogio Fassina; Francesco Fallo

    2015-01-01

    We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortica...

  8. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs.

    Science.gov (United States)

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-07-18

    In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV

  9. Gene promoter hypermethylation in leukoplakia of the oral mucosa

    OpenAIRE

    Liu, Mingli

    2010-01-01

    Mingli Liu1, Lei Feng2, Ximing Tang3, Shanchun Guo41Department of Physics, Tufts University School of Medicine, Boston, Massachussetts; 2Department of Thoracic/Head and Neck Medical Oncology, 3Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Sylvester Cancer Center, University of Miami School of Medicine, Florida, USAAbstract: To examine whether aberrant DNA methylation in the promoter region might occur earlier in tumorigenesis, particularly in premalignant ...

  10. Modulation of gene expression by promoter mutagenesis in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Elišáková, Veronika; Prouza, Marek; Phensaijai, M.; Nešvera, Jan; Pátek, Miroslav

    Prague: Institute of Microbiology ASCR, Prague, Czech Republic, 2006, s. 132-132. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Grant ostatní: EU(XE) ESF project No. CZ.04.3.07/4.2.01.1/0039 Institutional research plan: CEZ:AV0Z50200510 Keywords : c. glutamicum * promoters Subject RIV: EE - Microbiology, Virology

  11. Gene promoter methylation patterns throughout the process of cervical carcinogenesis

    NARCIS (Netherlands)

    Yang, Nan; Nijhuis, Esther R.; Volders, Haukeline H.; Eijsink, Jasper J. H.; Lendvai, Agnes; Zhang, Bo; Hollema, Harry; Schuuring, Ed; Wisman, G. Bea A.; van der Zee, Ate G. J.

    2010-01-01

    Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). Methods: QMSP was performed in normal cervix, low-grade ( L) SIL, high-grade (H) SIL, adenocarcinomas and squamous cell cervical

  12. Transcriptional targeting in cancer gene therapy using the p21/WAF1 promoter

    International Nuclear Information System (INIS)

    Lack of target cell specificity is the main limitation with gene therapy. Tissue-specific receptors (Kashara et al. 1994) and enhancers (Manome et al. 1994) have been used to localise therapy to the desired area. However low levels of gene expression are often associated with these methods. There has been a great deal of interest in the use of exogenous inducible promoters that operate at the transcriptional level, such as the Egr-1 promoter (Manome et al. 1998). This investigation focuses on the promoter for the x-ray inducible gene, p21/WAF1 (wild type p53 activated fragment). The WAF1 promoter has a p53 binding site which has been shown to confer p53 dependent inducibility upon the gene (El-Deiry et al. 1993). However a p53 independent activation pathway also exists (Wu et al. 2000). The current study examines the radiation and hypoxia inducibility of the WAF1 promoter in several human tumour cell lines of which the p53 status is known. Using the pEGFP reporter gene, we have established that the promoter is induced by radiation at clinically relevant doses (Worthington et al, 2000) and also by hypoxia. Results indicate that the WAF1 promoter is not leaky in normal human cells but is leaky in human tumour cells regardless of p53 status, indicating a level of tumour specificity not yet fully explored. This observation, coupled with its hypoxia and radiation inducibility at clinically relevant radiation doses, suggest that the WAF1 promoter could have significant advantages in overcoming the target specificity associated with gene therapy

  13. A Meta-analysis of Association between MGMT GenePromoter Methylation and Non-small Cell Lung Cancer

    OpenAIRE

    Fang, Nianzhen; Gu, Jundong; Wei, Huijun; Jiacong YOU; Zhou, Qinghua

    2014-01-01

    Backgroud and objective DNA promoter methylation of the tumor suppressor genes was one of the key mechanism for gene silence. The aim of this study is to investigate the difference of MGMT gene promoter methylation rate in tumor tissue and autologous controls (serum, normal lung tissue and bronchial lavage fluid) in patients with non-small cell lung cancer (NSCLC). Methods The databases of Medline, EMBSE, CNKI and Wanfang were searched for selection of published articles of MGMT gene promoter...

  14. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    Directory of Open Access Journals (Sweden)

    Miyuki Uno

    2011-01-01

    Full Text Available OBJECTIVES: 1 To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT promoter to its gene and protein expression levels in glioblastoma and 2 to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001. However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297. The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing, and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

  15. A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Li Mingji

    2012-02-01

    Full Text Available Abstract Background For metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-tac-promoters (MCPtacs in tandem. Results Through a uniquely designed in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtacs promoter clusters in polyhydroxybutyrate (PHB production proved that it was efficient. Integration of the phaCAB genes with the 5CPtacs promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation. Conclusions The transcription strength of the MCPtacs promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtacs promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.

  16. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation.

    Science.gov (United States)

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    BACKGROUND To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. MATERIAL AND METHODS Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. CONCLUSIONS Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  17. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    International Nuclear Information System (INIS)

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  18. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  19. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.

    2013-02-19

    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

  20. Culex tarsalis vitellogenin gene promoters investigated in silico and in vivo using transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available INTRODUCTION: Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes. RESULTS: Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b. Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2. CONCLUSIONS: These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.

  1. Cloning and characterizing of the murine IRF-3 gene promoter region.

    Science.gov (United States)

    Xu, Hua-Guo; Liu, Lifei; Gao, Shan; Jin, Rui; Ren, Wei; Zhou, Guo-Ping

    2016-08-01

    The interferon regulatory factor 3 (IRF-3) plays essential roles in inflammation and immune response. Here, we cloned the nucleotide sequence of the 5'-flanking region of the murine IRF-3 gene (mIRF-3) and characterized the molecular mechanisms controlling the mIRF-3 transcriptional activity in NIH3T3 cells. Analyses of a series of 5' deletion constructs demonstrated that a 301 bp region (-255/+46) of the mIRF-3 gene is sufficient for full promoter activity. This region contains IK1, Egr2, Cmyb, E2F1 and YY1 putative transcription factor binding sites. Mutation of Egr2 or YY1 site led to 52-68 % decrease of the mIRF-3 promoter activity, and double Egr2 and YY1 mutation reduced the promoter activity to 20 % of the wild-type promoter activity. Furthermore, knockingdown of endogenous Egr2 or YY1 by a siRNA strategy markedly inhibited the mIRF-3 promoter activity. Chromatin immunoprecipitation assays showed that Egr2 and YY1 interact with the mIRF-3 promoter in vivo. These results suggested that the basal promoter activity of the mIRF-3 gene is regulated by transcription factors Egr2 and YY1 in NIH3T3 cells. PMID:26740329

  2. Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

    Science.gov (United States)

    Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang

    2016-06-01

    Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1. PMID:27067405

  3. Characterization of promoter sequence of toll-like receptor genes in Vechur cattle

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    R. Lakshmi

    2016-06-01

    Full Text Available Aim: To analyze the promoter sequence of toll-like receptor (TLR genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. Materials and Methods: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. Results: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. Conclusion: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases.

  4. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Science.gov (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  5. The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter

    Institute of Scientific and Technical Information of China (English)

    WangHao; BaiYongyan

    1990-01-01

    The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expression vector which can be used in Ti plasmid system of Agrobacterium iumefaciens.The original plasmid,which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facilitate the selection of recombinant with Ti plasmid.Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter.This chimaeric gene was introduced into integrative Ti plasmid vector pGV 3850,and then transformed into Nicotiana tobaccum the chimaeric gene into tobacco cells.In both cases,the expression of ocs gene was demonstrated.The amount of octopine was much more than the nopaline synthesized by nopaline synthase (nos) gene transferred at the same time with Ti plasmid vector.This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the promoter of nos in tobacco cells.

  6. Adenovirus E4-34kDa requires active proteasomes to promote late gene expression

    International Nuclear Information System (INIS)

    A complex of the Adenovirus (Ad) early region 1b 55-kDa protein (E1b-55kDa) and the early region 4 ORF6 34-kDa protein (E4-34kDa) promotes viral late RNA accumulation in the cytoplasm while inhibiting the transport of most newly synthesized cellular mRNA. The E4 ORF3 11-kDa protein (E4-11kDa) functionally compensates for at least some of the activities of this complex. We find that the same large central region of E4-34kDa that is required for proteasome-mediated degradation of p53 (J. Virol. 75, (2001) 699-709) is also required to promote viral late gene expression in a complementation assay. E4-34kDa does not promote late gene expression in complementation assays performed in the presence of proteasome inhibitors. A proteasome inhibitor also dramatically reduced late gene expression by a virus that lacks the E4-11kDa gene and therefore relies on E4-34kDa for late gene expression. Our results suggest that E4-34kDa activity in promoting late gene expression depends on the proteasome

  7. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

    Science.gov (United States)

    Edman, R M; Linger, R J; Belikoff, E J; Li, F; Sze, S-H; Tarone, A M; Scott, M J

    2015-02-01

    The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests. PMID:25225046

  8. Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

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    Ansaya Thonpho

    Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

  9. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

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    V Shilpa

    2014-01-01

    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  10. Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

    Institute of Scientific and Technical Information of China (English)

    QIN Hui-juan; PAN Hong; FAN Xian-wei; WU Qiao; LI You-zhi

    2014-01-01

    Maize (Zea mays L.) is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog (GenBank accession no. EC864166) of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements (ECREs). Pro66 could drive expression of theβ-glucuronidase (GUS)-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

  11. NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Shen Shourong

    2010-04-01

    Full Text Available Abstract Background Nasopharyngeal carcinoma associated gene 6 (NGX6 is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC. In this study, we explored the role of DNA methylation in regulation of NGX6 transcription. Methods In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation. Results The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P Conclusions Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.

  12. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    Science.gov (United States)

    Shilpa, V.; Bhagat, Rahul; Premalata, C.S.; Pallavi, V.R.; Ramesh, G.; Krishnamoorthy, Lakshmi

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression. PMID:25579142

  13. NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

    International Nuclear Information System (INIS)

    Nasopharyngeal carcinoma associated gene 6 (NGX6) is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC). In this study, we explored the role of DNA methylation in regulation of NGX6 transcription. In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation. The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P < 0.05). An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the NGX6 promoter was associated with colorectal cancer patients age (P < 0.05). Moreover, a trend was shown toward metastasis status and primary site in colorectal carcinomas with NGX6 promoter methylation (p = 0.056 and P = 0.067, respectively). In addition, 5-Aza-dC could induce NGX6 mRNA expression and NGX6 promoter demethylation in HT-29 cells. Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might

  14. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

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    Kevin Pruitt

    2006-03-01

    Full Text Available The class III histone deactylase (HDAC, SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively, had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

  15. Inducible Cardiomyocyte-Specific Gene Disruption Directed by the Rat Tnnt2 Promoter in the Mouse

    OpenAIRE

    Wu, Bingruo; Wang, Yidong; Cheng, Hsiu-Ling; Hang, Calvin T.; Pu, William T.; Chang, Ching-Pin; Zhou, Bin

    2010-01-01

    We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by co-injection of two transgenes, a “reverse” tetracycline-controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline-responsive promoter (TetO). Here, Tnnt2-rtTA activated TetO-Cre expression takes place in cardiomyocytes following doxycycline treatme...

  16. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-γ) gene promoter and its effect on IFN-γ expression in chronic hepatitis B. Method The authors recruited 30 patients with HBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequencing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expres...

  17. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

    Science.gov (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

    2016-05-20

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  18. Association of polymorphisms of interleukin-18 gene promoter region with polycystic ovary syndrome in chinese population

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    Li Mei-zhi

    2010-10-01

    Full Text Available Abstract Background Recent research shows that polycystic ovary syndrome (PCOS may have an association with low-grade chronic inflammation, and that PCOS may induce an increase in serum interleukin-18 (IL-18 levels. Methods To investigate the polymorphisms of the IL-18 gene promoters with PCOS, two single nucleotide polymorphisms (SNPs in the promoter of the IL-18 gene (at positions -607C/A and -137G/C in 118 Chinese women with PCOS and 79 controls were evaluated using polymerase chain reaction (PCR. Results No significant differences were found in the genotype distribution, allele frequency and haplotype frequency between the PCOS and control groups. Further analysis demonstrated a relationship between IL-18 gene promoter polymorphisms and PCOS insulin resistance (IR. Regarding the -137 allele frequency, G and C allele frequencies were 93.5% and 6.5%, respectively, in the PCOS with IR patients; G and C allele frequencies were 85.4% and 14.6%, respectively, in PCOS patients without IR (chi2 = 3.601, P = 0.048. Conclusions The presence of a polymorphism in the IL-18 gene was found to have no correlation with the occurrence of PCOS. Carriage of the C allele at position -137 in the promoter of the IL-18 gene may play a protective role from the development of PCOS IR.

  19. Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene

    Institute of Scientific and Technical Information of China (English)

    CHENG; Shijun; (程世军); WANG; Zongyang(王宗阳); HONG; Mengmin(洪孟民)

    2002-01-01

    A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.

  20. Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

    OpenAIRE

    Coppe, Alessandro; Ferrari, Francesco; Bisognin, Andrea; Danieli, Gian Antonio; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, Stefania

    2008-01-01

    Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method...

  1. NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

    Directory of Open Access Journals (Sweden)

    Chao Lin

    Full Text Available Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

  2. PUMA and Bax-induced Autophagy Contributes to Apoptosis

    OpenAIRE

    Yee, Karen S.; Wilkinson, Simon; James, John; Ryan, Kevin M.; Vousden, Karen H.

    2009-01-01

    The p53-inducible BH3-only protein PUMA is a key mediator of p53-dependent apoptosis, and PUMA has been shown to function by activating Bax and mitochondrial outer membrane permeabilization. In this study we describe an ability of PUMA to induce autophagy that leads to the selective removal of mitochondria. This function of PUMA depends on Bax/Bak and can be reproduced by overexpression of Bax. The induction of autophagy coincides with cytochrome c release, and taken together the results sugg...

  3. PUMA- and Bax-induced autophagy contributes to apoptosis

    OpenAIRE

    Yee, K S; Wilkinson, S; James, J; Ryan, K M; Vousden, K H

    2009-01-01

    The p53-inducible BH3-only protein PUMA is a key mediator of p53-dependent apoptosis, and PUMA has been shown to function by activating Bax and mitochondrial outer membrane permeabilization. In this study, we describe an ability of PUMA to induce autophagy that leads to the selective removal of mitochondria. This function of PUMA depends on Bax/Bak and can be reproduced by overexpression of Bax. The induction of autophagy coincides with cytochrome c release, and taken together the results sug...

  4. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX1

    Science.gov (United States)

    Ruan, Hangjun; Wang, Jingli; Hu, Lily; Lin, Ching-Shwun; Lamborn, Kathleen R; Deen, Dennis F

    1999-01-01

    Abstract The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE), which can be activated through hypoxia-inducible factor-1 (HIF-1). We transfected plasmids containing multiple copies of HRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HRE copy number, and the degree of hypoxia. PMID:10933058

  5. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    1999-11-01

    Full Text Available The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE, which can be activated through hypoxia-inducible factor-1 (HIF-1. We transfected plasmids containing multiple copies of HIRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HIRE copy number, and the degree of hypoxia.

  6. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

    OpenAIRE

    Zomerdijk, J C; Ouellette, M; ten Asbroek, A L; Kieft, R.; Bommer, A M; Clayton, C E; Borst, P

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region m...

  7. Nuclear proteins from lactating mammary glands bind to the promoter of a milk protein gene.

    OpenAIRE

    LUBON, H.; Hennighausen, L

    1987-01-01

    The gene for the whey acidic protein (WAP) is expressed specifically in the lactating mammary glands of rodents. We present evidence that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the WAP gene promoter/upstream region. As monitored by mobility shifts, nuclear proteins from lactating mammary glands and from the mammary cell line MCF-7 form four high affinity complexes with a fragment spanning the region between nucleotides -175 and -88. Nuclear p...

  8. Mediterranean river buffalo oxytocin-neurophysin I (OXT) gene: structure, promoter analysis and allele detection

    OpenAIRE

    Ramunno, L.; D. Di Berardino; L. Zicarelli; R. Di Palo; NICODEMO, D.; MANCUSI, A.; A. Pauciullo; G. Cosenza

    2010-01-01

    Oxytocin (OXT) is a very abundant nonapeptide neurohypophysial hormone implicated in several aspects of reproduction, including social, sexual and maternal behaviour, induction of labour and milk ejection. The nucleotide sequence of the whole OXTneurophysin I encoding gene (OXT) in Mediterranean river buffalo was determined, plus 993 nucleotides at the 5’ flanking region. Buffalo oxytocin gene sequence analysis showed two transitions in the promoter region (C→T in position – ...

  9. Effects of curcumin on hippocampal Bax and Bcl-2 expression and cognitive function of a rat model of Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Yunliang Wang; Honglei Yin; Jiyu Lou; Bing Han; Xinyue Qin; Fanchao Meng; Shuang Geng; Yajun Liu

    2011-01-01

    We tested the effects of curcumin treatment on a rat model of Alzheimer's disease induced by beta-amlyoid (Aβ1-40) expression. We investigated alterations in the expression of the apoptosis-related genes Bax and Bcl-2 in the hippocampus, as well as changes in the spatial memory and cognitive function of the rats. Reverse transcription-polymerase chain reaction and immunohistochemistry results showed that Bax expression was remarkably decreased and Bcl-2 expression was increased in the rat Alzheimer's disease model after curcumin treatment. Morris water maze results showed that the average time of escape latency was shortened in the curcumin treated model rats. Our study shows that curcumin can significantly improve spatial learning and memory functions in rats with Aβ1-40-induced Alzheimer's disease by modulating Bax and Bcl-2 expression.

  10. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

    Science.gov (United States)

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-12-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

  11. L1 Antisense Promoter Drives Tissue-Specific Transcription of Human Genes

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Transcription of transposable elements interspersed in the genome is controlled by complex interactions between their regulatory elements and host factors. However, the same regulatory elements may be occasionally used for the transcription of host genes. One such example is the human L1 retrotransposon, which contains an antisense promoter (ASP driving transcription into adjacent genes yielding chimeric transcripts. We have characterized 49 chimeric mRNAs corresponding to sense and antisense strands of human genes. Here we show that L1 ASP is capable of functioning as an alternative promoter, giving rise to a chimeric transcript whose coding region is identical to the ORF of mRNA of the following genes: KIAA1797, CLCN5, and SLCO1A2. Furthermore, in these cases the activity of L1 ASP is tissue-specific and may expand the expression pattern of the respective gene. The activity of L1 ASP is tissue-specific also in cases where L1 ASP produces antisense RNAs complementary to COL11A1 and BOLL mRNAs. Simultaneous assessment of the activity of L1 ASPs in multiple loci revealed the presence of L1 ASP-derived transcripts in all human tissues examined. We also demonstrate that L1 ASP can act as a promoter in vivo and predict that it has a heterogeneous transcription initiation site. Our data suggest that L1 ASP-driven transcription may increase the transcriptional flexibility of several human genes.

  12. Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The relationship between hypermethylation of CpG islands in the promoter regions of O6methylguanine DNA methyltransferase (MGMT)genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, t issues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 (34.8 %) laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades (x2= 3. 130, P=0. 077) or in samples from patients with different TNM status (x2=3. 957, P=0. 138). No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

  13. Novel strong tissue specific promoter for gene expression in human germ cells

    Directory of Open Access Journals (Sweden)

    Kuzmin Denis

    2010-08-01

    Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

  14. Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

    Directory of Open Access Journals (Sweden)

    Muh. Alias L. Rajamuddin

    2016-02-01

    Full Text Available Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP, caulifl ower mosaic virus (pCaMV-GFP,medaka β-actin (pmBA-GFP and Japanese fl ounder keratin (pJfKer-GFP promoters were introduced by electroporationmethod. Electroporation was performed using a gene pulser (BIORAD with voltage of 300 V, pulse length of 0.5 ms,pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expressionlevel using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus(34.10%±1.49 expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activitywith CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25. mBA promoter drove GFP expressionat medium level and similar number of fi lament callus (8.85%±2.31 expressing GFP with CaMV, while JfKer promoterhad lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26 and GFP expression level. PCRanalysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressingfi lament callus in the same size (about 0.6 kb with positive control of plasmid. Thus, CMV and CaMV promoters wasan appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combiningthis achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.alvarezii can be feasible.

  15. The pea END1 promoter drives anther-specific gene expression in different plant species.

    Science.gov (United States)

    Gómez, María D; Beltrán, José-Pío; Cañas, Luis A

    2004-10-01

    END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (beta-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the -330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity. PMID:15221384

  16. Promotion

    OpenAIRE

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed.

  17. The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene

    Institute of Scientific and Technical Information of China (English)

    JIAN; YING; GUO; JIAN; XU; DA; QIN; MAO; LI; LI; FU; JIAN; REN; GU; JING; DE; ZHU

    2002-01-01

    The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a regiondisplaying a high score of loss of heterozygosity within chromosome 17p13.3 in human hepatocellular car-cinoma in China[1]. To unveil the underlying mechanisms for the transcription regulation of this gene andunderstand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by bothbioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 genewas evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its tran-scription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragmentcontaining the transcription start, extending from -112 to -55, represents the minimal promoter; 4, Theconsensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of theminimal promoter; 5, The bioinformatic analysis suggests that the C17orf25 gene may encode a protein inthe family of the glyoxalase. Our data has provided some deep insight into both function and regulation ofthe C17orf25 gene in the context of the normal liver and hepatocellular carcinoma.

  18. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    Science.gov (United States)

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. PMID:25800674

  19. Association of an Osteopontin gene promoter polymorphism with susceptibility to diabetic nephropathy in Asian Indians

    DEFF Research Database (Denmark)

    Cheema, Balneek Singh; Iyengar, Sreenivasa; Ahluwalia, Tarun Veer Singh;

    2012-01-01

    genetic polymorphisms in OPN with diabetic nephropathy is lacking. Thus, the present study was designed with the aim to examine the association of an OPN gene promoter polymorphism with diabetic nephropathy in Asian Indians. OPN C-443T (rs11730582) polymorphism was determined in 1115 type 2 diabetic...

  20. Specific myeloprotection via multidrug resistance 1 gene controlled by aminopeptidase N myeloid promoter

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In the treatment of tumor patients introduction of multidrug resistance genes into hematopoietic cells has been reported as an approach for reducing myelotoxicity created by antitumor drugs. However, the nonspecific expression of the genes can also increase the chemoresistance of the tumor cells invaded into bone marrow, which influences seriously the effectiveness of chemotherapy. In this study, a new strategy is described for specific myeloprotection. The recombinant retroviral vector containing multidrug resistance 1 (MDR1) gene regulated by aminopeptidase N (APN) myeloid promoter was constructed and then introduced into myeloblastic cells KG1a and tumor cell line BEL7402. The specific transcript of MDR1 was detected in KG1a cells transduced with MDR1 gene and rhodamine 123 was effectively extruded by Pgp, the protein of MDR1 gene. The resistance elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in MDR1 gene-transduced KG1a cells to chemotherapeutic drugs such as cochicine, VP-16, vincristine, doxorubi- cin and paclitaxel, respectively. In contrast, the chemoresistance had no significant changes in BEL7402 cells transduced with MDR1 gene. Expression of MDR1 directed by APN myeloid promoter resulted in myelospecific protection during the killing of tumor cells treated with antitumor drugs. The study would provide a new mean for circumventing myelosuppression of tumor patients undergoing chemotherapy.

  1. Identification of a Gene Sharing a Promoter and Peroxisome Proliferator-Response Elements With Acyl-CoA Oxidase Gene

    Directory of Open Access Journals (Sweden)

    Mst. Hasina Akter

    2006-01-01

    Full Text Available Many mammalian genes are clustered on the genomes, and hence the genes in the same cluster can be regulated through a common regulatory element. We indeed showed previously that the perilipin/PEX11α gene pair is transactivated tissue-selectively by PPARγ and PPARα, respectively, through a common binding site. In the present study, we identified a gene, named GSPA, neighboring a canonical PPAR target, acyl-CoA oxidase (AOX gene. GSPA expression was induced by a peroxisome proliferator, Wy14,643, in the liver of wild-type mice, but not PPARα-null mice. GSPA and AOX share the promoter and two peroxisome proliferator-response elements. GSPA mRNA was also found in the heart and skeletal muscle, as well as 3T3-L1 cells. GSPA encodes a protein of 161 amino acids that is enriched in 3T3-L1 cells. Even other gene pairs might be regulated through common sequence elements, and conversely it would be interesting how each gene is aptly regulated in clusters.

  2. Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, D.S. [Beth Israel Hospital, Boston, MA (United States)]|[Children`s Hospital, Boston, MA (United States); Vidal-Puig, A.; Moller, D.E. [Beth Israel Hospital, Boston, MA (United States)] [and others

    1996-02-01

    Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation of polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed. 40 refs., 1 figs., 1 tab.

  3. Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

    Science.gov (United States)

    Coppe, Alessandro; Ferrari, Francesco; Bisognin, Andrea; Danieli, Gian Antonio; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, Stefania

    2009-01-01

    Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation. PMID:19059999

  4. Diffusion limited aggregation and the fractal evolution of gene promoter networks

    Directory of Open Access Journals (Sweden)

    Preston R. Aldrich

    2011-09-01

    Full Text Available Gene promoter networks (GPNs are systems-level representations of the base pair-sharing relationships (graph edges among promoters (graph nodes. It has been shown in the bacterium E. coli that these networks can contain a fractal nucleus of strong associations suggesting a self-organizing complexity. Here I report results of twenty seven in silico simulations for a diffusion limited aggregation model which accounts for much of the fractal structure previously observed in GPNs. Parameters varied in the model included (a the frequency of gene duplication events, and the extent of (b attraction and (c repulsion presented by the DNAprotein binding chemistry. Both duplication and attraction had significant effects on fractal topology of the GPN nucleus, whereas repulsion due to DNA-protein binding chemistry did not, at least for the levels explored in these simulations. Since repulsion is thought to be a key feature of fractal networks, it is likely that the repulsion in GPNs arises from the sparseness of the promoter space. The generation of a finite random set of promoters leads to sparse occupancy of promoter space which itself presents a considerable repulsion away from the consensus motif, working against the DNA-binding protein's efforts to organize the system of promoters over evolutionary time. This interplay between attractive and repulsive forces in a GPN is sufficient to generate a fractal topology.

  5. Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

    Directory of Open Access Journals (Sweden)

    Gianfranco Cosenza

    2010-01-01

    Full Text Available The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2 plus 1,476 nt at the 5' flanking region and 51 nt at the 3' flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5 to 498 bp (exon 7 and 8 introns from 92 bp (intron 5 to 22 59 bp (intron 1. Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene were also identified at 3 different locations of the sequenced region: 5' untranscribed region, intron 1, and intron 4.

  6. Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects

    Institute of Scientific and Technical Information of China (English)

    Ji-Ping Shan; Xiao-Li Wang; Yuan-Gang Qiao; Hong-Xin Wan Yan; Wen-Hui Huang; Shu-Chao Pang; Bo Yan

    2014-01-01

    Background: Congenital heart disease (CHD) is the most common human birth defect. Genetic causes for CHD remain largely unknown. GATA transcription factor 5 (GATA 5) is an essential regulator for the heart development. Mutations in the GATA5 gene have been reported in patients with a variety of CHD. Since misregulation of gene expression have been associated with human diseases, we speculated that changed levels of cardiac transcription factors, GATA5, may mediate the development of CHD. Methods: In this study, GATA5 gene promoter was genetically and functionally analyzed in large cohorts of patients with ventricular septal defect (VSD) (n=343) and ethnic-matched healthy controls (n=348). Results: Two novel and heterozygous DNA sequence variants (DSVs), g.61051165A>G and g.61051463delC, were identified in three VSD patients, but not in the controls. In cultured cardiomyocytes, GATA5 gene promoter activities were significantly decreased by DSV g.61051165A>G and increased by DSV g.61051463delC. Moreover, fathers of the VSD patients carrying the same DSVs had reduced diastolic function of left ventricles. Three SNPs, g.61051279C>T (rs77067995), g.61051327A>C (rs145936691) and g.61051373G>A (rs80197101), and one novel heterozygous DSV, g.61051227C>T, were found in both VSD patients and controls with similar frequencies. Conclusion: Our data suggested that the DSVs in the GATA5 gene promoter may increase the susceptibility to the development of VSD as a risk factor.

  7. Novel and Functional DNA Sequence Variants within the GATA6 Gene Promoter in Ventricular Septal Defects

    Directory of Open Access Journals (Sweden)

    Chunyu Li

    2014-07-01

    Full Text Available Congenital heart disease (CHD is the most common birth defect in humans. Genetic causes and underlying molecular mechanisms for isolated CHD remain largely unknown. Studies have demonstrated that GATA transcription factor 6 (GATA6 plays an essential role in the heart development. Mutations in GATA6 gene have been associated with diverse types of CHD. As GATA6 functions in a dosage-dependent manner, we speculated that changed GATA6 levels, resulting from DNA sequence variants (DSVs within the gene regulatory regions, may mediate the CHD development. In the present study, GATA6 gene promoter was genetically and functionally analyzed in large groups of patients with ventricular septal defect (VSD (n = 359 and ethnic-matched healthy controls (n = 365. In total, 11 DSVs, including four SNPs, were identified in VSD patients and controls. Two novel and heterozygous DSVs, g.22169190A>T and g.22169311C>G, were identified in two VSD patients, but in none of controls. In cultured cardiomyocytes, the activities of the GATA6 gene promoter were significantly reduced by the DSVs g.22169190A>T and g.22169311C>G. Therefore, our findings suggested that the DSVs within the GATA6 gene promoter identified in VSD patients may change GATA6 levels, contributing to the VSD development as a risk factor.

  8. Activity Analysis and Preliminary Inducer Screening of the Chicken DAZL Gene Promoter

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    Lei Zhang

    2015-03-01

    Full Text Available This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was −383 to −39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA, a retinoic acid receptor alpha agonist (tamibarotene/Am80, or estradiol (E2 could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly; whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro.

  9. Activity analysis and preliminary inducer screening of the chicken DAZL gene promoter.

    Science.gov (United States)

    Zhang, Lei; Zhu, Rui; Zuo, Qisheng; Li, Dong; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-01-01

    This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was -383 to -39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA), a retinoic acid receptor alpha agonist (tamibarotene/Am80), or estradiol (E2) could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly; whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC)-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro. PMID:25807265

  10. Synthetic gene involving azobenzene-tethered T7 promoter for the photocontrol of gene expression by visible light.

    Science.gov (United States)

    Kamiya, Yukiko; Takagi, Toshiki; Ooi, Hideaki; Ito, Hiroshi; Liang, Xingguo; Asanuma, Hiroyuki

    2015-04-17

    In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4'-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4'-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340-360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage. PMID:25144622

  11. High level transgenic expression of soybean (Glycine max GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

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    Jones Michelle L

    2010-11-01

    Full Text Available Abstract Background Although numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max. Results In this research, a bioinformatics approach was first performed to identify members of the Gmubi (G.max ubiquitin and the GmERF (G. max Ethylene Response Factor gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression. Conclusion In this study, we present expression intensity data on 20 novel soybean promoters

  12. Over-represented localized sequence motifs in ribosomal protein gene promoters of basal metazoans.

    Science.gov (United States)

    Perina, Drago; Korolija, Marina; Roller, Maša; Harcet, Matija; Jeličić, Branka; Mikoč, Andreja; Cetković, Helena

    2011-07-01

    Equimolecular presence of ribosomal proteins (RPs) in the cell is needed for ribosome assembly and is achieved by synchronized expression of ribosomal protein genes (RPGs) with promoters of similar strengths. Over-represented motifs of RPG promoter regions are identified as targets for specific transcription factors. Unlike RPs, those motifs are not conserved between mammals, drosophila, and yeast. We analyzed RPGs proximal promoter regions of three basal metazoans with sequenced genomes: sponge, cnidarian, and placozoan and found common features, such as 5'-terminal oligopyrimidine tracts and TATA-boxes. Furthermore, we identified over-represented motifs, some of which displayed the highest similarity to motifs abundant in human RPG promoters and not present in Drosophila or yeast. Our results indicate that humans over-represented motifs, as well as corresponding domains of transcription factors, were established very early in metazoan evolution. The fast evolving nature of RPGs regulatory network leads to formation of other, lineage specific, over-represented motifs. PMID:21457775

  13. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori

    International Nuclear Information System (INIS)

    The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter

  14. A database of annotated promoters of genes associated with common respiratory and related diseases

    KAUST Repository

    Chowdhary, Rajesh

    2012-07-01

    Many genes have been implicated in the pathogenesis of common respiratory and related diseases (RRDs), yet the underlying mechanisms are largely unknown. Differential gene expression patterns in diseased and healthy individuals suggest that RRDs affect or are affected by modified transcription regulation programs. It is thus crucial to characterize implicated genes in terms of transcriptional regulation. For this purpose, we conducted a promoter analysis of genes associated with 11 common RRDs including allergic rhinitis, asthma, bronchiectasis, bronchiolitis, bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, eczema, psoriasis, and urticaria, many of which are thought to be genetically related. The objective of the present study was to obtain deeper insight into the transcriptional regulation of these disease-associated genes by annotating their promoter regions with transcription factors (TFs) and TF binding sites (TFBSs). We discovered many TFs that are significantly enriched in the target disease groups including associations that have been documented in the literature. We also identified a number of putative TFs/TFBSs that appear to be novel. The results of our analysis are provided in an online database that is freely accessible to researchers at http://www.respiratorygenomics.com. Promoter-associated TFBS information and related genomic features, such as histone modification sites, microsatellites, CpG islands, and SNPs, are graphically summarized in the database. Users can compare and contrast underlying mechanisms of specific RRDs relative to candidate genes, TFs, gene ontology terms, micro-RNAs, and biological pathways for the conduct of metaanalyses. This database represents a novel, useful resource for RRD researchers. Copyright © 2012 by the American Thoracic Society.

  15. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2015-12-01

    To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression. PMID:26706070

  16. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

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    Catherine eRAVEL

    2014-11-01

    Full Text Available The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator. In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

  17. Identification of a binding protein to the X gene promoter region of hepatitis B virus.

    Science.gov (United States)

    Nakamura, I; Koike, K

    1992-12-01

    The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes. PMID:1448911

  18. Transactivation of the proximal promoter of human oxytocin gene by TR4 orphan receptor

    International Nuclear Information System (INIS)

    The human testicular receptor 4 (TR4) shares structural homology with members of the nuclear receptor superfamily. Some other members of this superfamily were able to regulate the transcriptional activity of the human oxytocin (OXT) promoter by binding to the first DR0 regulatory site. However, little investigation was conducted systematically in the study of the second dDR4 site of OXT proximal promoter, and the relationship between the first and the second sites of OXT promoter. Here, we demonstrated for the first time that TR4 could increase the proximal promoter activity of the human OXT gene via DR0, dDR4, and OXT (both DR0 and dDR4) elements, respectively. TR4 might induce OXT gene expression through the OXT element in a dose-dependent manner. However, there is no synergistic effect between DR0 and dDR4 elements during TR4 transactivation. Taken together, these results suggested that TR4 should be one of important regulators of OXT gene expression

  19. Functional Characterization of a Putative Nitrate Transporter Gene Promoter from Rice

    Institute of Scientific and Technical Information of China (English)

    Ting-Zhang HU; Kai-Ming CAO; Mian XIA; Xi-Ping WANG

    2006-01-01

    Drought is one of the most significant abiotic stresses that influence plant growth and development. Expression analysis revealed that OsNRT1.3, a putative nitrate transporter gene in rice, was induced by drought. To confirm if the OsNRT1.3 promoter can respond to drought stress, a 2019 bp upstream sequence of OsNRT1.3 was cloned. Three OsNRT1.3 promoter fragments were generated by 5'-deletion, and fused to the β-glucuronidase (GUS) gene. The chimeric genes were introduced into rice plants. NRT2019::GUS, NRT1196: :GUS and NRT719::GUS showed similar expression patterns in seeds,roots, leaves and flowers in all transgenic rice, and GUS activity conferred by different OsNRT1.3 promoter fragments was significantly upregulated by drought stress, indicating that OsNRT1.3 promoter responds to drought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.

  20. c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells

    International Nuclear Information System (INIS)

    The BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc. c-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay. Depletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein. Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity. The distal BRCA1 promoter region is associated with c

  1. Characterization of the Human Insulin-induced Gene 2 (INSIG2) Promoter

    Science.gov (United States)

    Fernández-Alvarez, Ana; Soledad Alvarez, María; Cucarella, Carme; Casado, Marta

    2010-01-01

    Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5′-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver. PMID:20145255

  2. Development of radiation-inducible promoters for use in nitric oxide synthase gene therapy of cancer

    International Nuclear Information System (INIS)

    Full text: The free radical nitric oxide (NO) at nM concentrations performs multiple signaling roles that are essential for survival. These processes are regulated via the enzymes nNOS and eNOS, but another isoform, inducible nitric oxide synthase (iNOS) is capable of generating much higher concentrations (mM) over longer periods, resulting in the generation of very toxic species such as peroxynitrite. At high concentrations NO has many of the characteristics of an ideal anticancer molecule: it is cytotoxic (pro-apoptotic via peroxynitrite), it is a potent chemical radiosensitizer, it is anti-angiogenic and anti-metastatic. Thus, we see iNOS gene therapy as a strategy for targeting the generation of high concentrations of NO to tumours for therapeutic benefit. iNOS gene therapy should be used in combination with radiotherapy; so it is logical that the use of a radiation-inducible promoter should be part of the targeting strategy. We have tested several candidate promoters in vitro and in vivo. The WAF1 promoter has many of the properties desirable for therapeutic use including: rapid 3-4 fold induction at X-ray doses of 2 and 4Gy and no significant leakiness. WAF1 also has the advantage of being inducible by hypoxia and by the final product, NO. We have also tested the synthetic CArG promoter and demonstrated that, in addition to a high level of radiation inducibility, it is also inducible by NO. We have also been able to demonstrate potent radiosensitization (SER 2.0-2.5) in tumour cells in vitro and in vivo using iNOS gene transfer with constitutive or radiation-inducible promoters. We have also tested the use of iNOS gene therapy in combination with cisplatin and shown significant enhancement

  3. Promoter regulatory domain identification of cassava starch synthase IIb gene in transgenic tobacco.

    Science.gov (United States)

    Guan, Zhihui; Chen, Xin; Xie, Hairong; Wang, Wenquan

    2016-05-01

    Soluble starch synthase is a key enzyme in the starch biosynthesis pathway, and its enzyme activity significantly influences starch components in cassava storage root. However, studies on the regulation mechanism of soluble starch synthase gene are rare. In this study, we cloned the 5' flanking sequence of the MeSSIIb gene and predicted the distribution of cis-elements. The region from -453 to -1 was considered the primary core promoter by the quantitative detection of GUS activity in transgenic tobacco plants containing 5' truncated promoters fused with the GUS gene. Analysis results clarified that the region from -531 to -454 significantly repressed promoter activity. The region from -453 to -388 was a repressive domain of ethylene, and some unknown drought responsive cis-elements were located in the region from -387 to -1. These findings will provide useful information on the functional assay and transcriptional regulation mechanisms of the MeSSIIb gene. PMID:26919397

  4. Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

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    Parveen Kumar

    Full Text Available The myeloid translocation gene 16 (MTG16 co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1 heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1. Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.

  5. A multistep bioinformatic approach detects putative regulatory elements in gene promoters

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    Pizzi Cinzia

    2005-05-01

    Full Text Available Abstract Background Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs. Results Novel software (COOP was developed for extraction of sequence motifs, based on clustering of exact or approximate patterns according to the frequency of their overlapping occurrences. Genomic sequences of 1 Kb upstream of 91 genes differentially expressed and/or encoding proteins with relevant function in adult human retina were analyzed. Methodology and results were tested by analysing 1,000 groups of putatively unrelated sequences, randomly selected among 17,156 human gene promoters. When applied to a sample of human promoters, the method identified 279 putative motifs frequently occurring in retina promoters sequences. Most of them are localized in the proximal portion of promoters, less variable in central region than in lateral regions and similar to known regulatory sequences. COOP software and reference manual are freely available upon request to the Authors. Conclusion The approach described in this paper seems effective for identifying a tractable number of sequence motifs with putative regulatory role.

  6. A multistep bioinformatic approach detects putative regulatory elements in gene promoters

    Science.gov (United States)

    Bortoluzzi, Stefania; Coppe, Alessandro; Bisognin, Andrea; Pizzi, Cinzia; Danieli, Gian Antonio

    2005-01-01

    Background Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs. Results Novel software (COOP) was developed for extraction of sequence motifs, based on clustering of exact or approximate patterns according to the frequency of their overlapping occurrences. Genomic sequences of 1 Kb upstream of 91 genes differentially expressed and/or encoding proteins with relevant function in adult human retina were analyzed. Methodology and results were tested by analysing 1,000 groups of putatively unrelated sequences, randomly selected among 17,156 human gene promoters. When applied to a sample of human promoters, the method identified 279 putative motifs frequently occurring in retina promoters sequences. Most of them are localized in the proximal portion of promoters, less variable in central region than in lateral regions and similar to known regulatory sequences. COOP software and reference manual are freely available upon request to the Authors. Conclusion The approach described in this paper seems effective for identifying a tractable number of sequence motifs with putative regulatory role. PMID:15904489

  7. Implication of bidirectional promoters containing duplicated GGAA motifs of mitochondrial function-associated genes

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    Fumiaki Uchiumi

    2013-12-01

    Full Text Available Mitochondria are well known as the primary required organelle in all eukaryotic cells. They have their own mtDNA containing genes that encode tRNAs, rRNAs and a set of functional proteins required for energy (ATP production. However, almost all (99% of mitochondrial proteins are encoded by host nuclear genes. Therefore, expression of mitochondrial protein-encoding genes should be regulated similarly to genes that are present in the host nuclear chromosomes. Interestingly, from genomic database assisted surveillance, it was revealed that a lot of mitochondrial function associated protein-encoding genes are oppositely linked in a head-head manner. If the two head-head conjugated genes are regulated by the same transcription factor(s, their expression would be dependent on the direction of transcription machinery that contains RNA polymerase II to execute mRNA synthesis. In this article, we will focus on several examples of the mitochondrial and the partner gene sets and discuss putative functions of transcription factor binding elements in the bidirectional promoters of mitochondrial function-associated genes in chromosomes.

  8. Simian virus 40 promoters direct expression of the tetracycline gene in plasmid pACYC184.

    OpenAIRE

    Jenkins, F J; Howett, M K; Rapp, F

    1983-01-01

    Insertion of HindIII DNA fragments into the HindIII site of plasmid pACYC184 destroys the promoter of the plasmid tetracycline resistance gene and causes Escherichia coli cells harboring recombinant plasmids to be tetracycline sensitive and chloramphenicol resistant. The HindIII-C DNA fragment of simian virus 40 contains the two virus promoters and the virus origin of replication. We report the isolation of recombinant plasmids that contained the simian virus 40 HindIII-C DNA fragment at the ...

  9. Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter

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    Rodríguez Mario A

    2007-09-01

    Full Text Available Abstract Background The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. Results To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1 of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE. Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. Conclusion The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.

  10. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

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    Skiriute Daina

    2012-06-01

    Full Text Available Abstract Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3% of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p CASP8 with older (p MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p CASP8 was more frequent in patients who survived shorter than 36 months (p MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.

  11. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

    International Nuclear Information System (INIS)

    Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. The overwhelming majority (97.3%) of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p < 0.05), while CASP8 with older (p < 0.01). MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p < 0.05), while methylation of CASP8 was more frequent in patients who survived shorter than 36 months (p < 0.05). Cox regression analysis showed patient age, treatment, MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p < 0.05). MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome

  12. Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype

    OpenAIRE

    Van der Auwera, I; Laere, S.J.; Van den Bosch, S M; Van den Eynden, G. G.; Trinh, B X; van Dam, P A; Colpaert, C G; van Engeland, M; Van Marck, E A; Vermeulen, P B; Dirix, L Y

    2008-01-01

    Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal b...

  13. Plumbagin reduces chronic lymphocytic leukemia cell survival by downregulation of Bcl-2 but upregulation of the Bax protein level.

    Science.gov (United States)

    Fu, Chunling; Gong, Yanqing; Shi, Xuanxuan; Sun, Zengtian; Niu, Mingshan; Sang, Wei; Xu, Linyan; Zhu, Feng; Wang, Ying; Xu, Kailin

    2016-09-01

    Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and mainly originates from an accumulation of abnormal B cells caused by the dysregulation of cell proliferation and apoptosis rates. The aberration of apoptosis-related genes in CLL cells results in defective apoptosis of CLL cells in response to traditional therapeutic medicine. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a natural compound from Plumbago zeylinica, has been shown to exhibit pro-apoptotic activities in tumor cells. In the present study, we report that plumbagin effectively inhibited CLL cell viability with a lower dose compared to fludarabine, and inhibited cell proliferation in a dose-dependent manner. In addition, plumbagin promoted accumulation of MEC-1 cells in the S phase, and blocked cell cycle transition of HG3 cells from G0/G1 to S phase. Molecularly, plumbagin markedly induced CLL cell apoptosis through reduction of Bcl-2, but through an increase in the Bax protein level. These results suggest that plumbagin may be considered as a potential anticancer agent for CLL therapy. PMID:27461100

  14. Characterization of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

    Indian Academy of Sciences (India)

    Nandini Vasudevan; Urvashi Bahadur; Paturu Kondaiah

    2001-03-01

    The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yolk and white protein. Eggs from hens which have a splice mutation in RCP gene fail to hatch, indicating an absolute requirement of RCP for the transport of riboflavin to the oocyte. In order to understand the mechanism of regulation of this gene by estrogen, the chicken RCP gene including 1 kb of the 5′ flanking region has been isolated. Characterization of the gene structure shows that it contains six exons and five introns, including an intron in the 5′ untranslated region. Sequence analysis of the 5′ flanking region does not show the presence of any classical, palindromic estrogen response element (ERE). However, there are six half site ERE consensus elements. Four deletion constructs of the 5′ flanking region with varying number of ERE half sites were made in pGL3 basic vector upstream of the luciferase-coding region. Transient transfection of these RCP promoter deletion constructs into a chicken hepatoma cell line (LMH2A) showed 6-12-fold transcriptional induction by a stable estrogen analogue, moxesterol. This suggests that the RCP gene is induced by estrogen even in the absence of a classical ERE and the half sites of ERE in this promoter may be important for estrogen induction.

  15. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Chandler, Ronald L. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Fritz, David T. [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Mortlock, Douglas P. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Rogers, Melissa B., E-mail: rogersmb@umdnj.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States)

    2010-02-05

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  16. Study on the Polymorphisms of Porcine Myostatin Gene in Promoter Region by PCR-RFLPS

    Institute of Scientific and Technical Information of China (English)

    YANG Xiu-qin; LIU Di

    2005-01-01

    In order to further study functions of the porcine myostatin gene, we analyzed the polymorphisms of porcine myostatin gene in promoter region among different breeds including Yorkshire, Landrace, Duroc, Junmu, Min pig and Sanjiang white pig by PCR-RFLPs. The allele T dominated in the imported lean-type pig breeds such as Yorkshire, Landrace and Duroc. No allele A was detected in Junmu and Sanjiang white pig, and the frequencies of three genotypes were about equal in Min pig. The result using X2 analysis showed that the distribution of three genotypes was related to pig breeds.

  17. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    International Nuclear Information System (INIS)

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  18. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters

    OpenAIRE

    Song, Tianqiao; MA, ZHENCHUAN; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-01-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana en...

  19. Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene*

    OpenAIRE

    Wang, Xiaofu; Gulhati, Pat; Li, Jing; Dobner, Paul R.; Weiss, Heidi; Townsend, Courtney M.; Evers, B. Mark

    2010-01-01

    Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels...

  20. Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans

    OpenAIRE

    Conine, Colin C.; Moresco, James J.; Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Yates, John R.; Mello, Craig C

    2013-01-01

    During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expre...

  1. Maximal Expression of the Evolutionarily Conserved Slit2 Gene Promoter Requires Sp1.

    Science.gov (United States)

    Saunders, Jacquelyn; Wisidagama, D Roonalika; Morford, Travis; Malone, Cindy S

    2016-08-01

    Slit2 is a neural axon guidance and chemorepellent protein that stimulates motility in a variety of cell types. The role of Slit2 in neural development and neoplastic growth and migration has been well established, while the genetic mechanisms underlying regulation of the Slit2 gene have not. We identified the core and proximal promoter of Slit2 by mapping multiple transcriptional start sites, analyzing transcriptional activity, and confirming sequence homology for the Slit2 proximal promoter among a number of species. Deletion series and transient transfection identified the Slit2 proximal promoter as within 399 base pairs upstream of the start of transcription. A crucial region for full expression of the Slit2 proximal promoter lies between 399 base pairs and 296 base pairs upstream of the start of transcription. Computer modeling identified three transcription factor-binding consensus sites within this region, of which only site-directed mutagenesis of one of the two identified Sp1 consensus sites inhibited transcriptional activity of the Slit2 proximal promoter (-399 to +253). Bioinformatics analysis of the Slit2 proximal promoter -399 base pair to -296 base pair region shows high sequence conservation over twenty-two species, and that this region follows an expected pattern of sequence divergence through evolution. PMID:26456684

  2. Characterization and regulation of the bovine stearoyl-CoA desaturase gene promoter

    International Nuclear Information System (INIS)

    The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36 bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid

  3. Dissection of a locus on mouse chromosome 5 reveals arthritis promoting and inhibitory genes

    DEFF Research Database (Denmark)

    Lindvall, Therese; Karlsson, Jenny; Holmdahl, Rikard;

    2009-01-01

    ABSTRACT: INTRODUCTION: In a cross between the susceptible B10.RIII (H-2r) and resistant RIIIS/J (H-2r) mouse strains, a locus on mouse chromosome 5 (Eae39) was previously shown to control experimental autoimmune encephalomyelitis (EAE). Recently, quantitative trait loci (QTL), linked to disease in...... Eae39 congenic- and sub-interval congenic mice, carrying RIIIS/J genes on the B10.RIII genetic background, revealed three loci within Eae39 that control disease and anti-collagen antibody titers. Two of the loci promoted disease and the third locus was protecting from collagen induced arthritis...... development. By further breeding of mice with small congenic fragments, we identified a 3.2 Megabasepair (Mbp) interval that regulates disease. CONCLUSIONS: Disease promoting- and protecting genes within the Eae39 locus on mouse chromosome 5, control susceptibility to collagen induced arthritis. A disease...

  4. Protein-DNA interactions in the cAMP responsive promoter region of the murine ornithine decarboxylase gene.

    OpenAIRE

    Palvimo, J J; Eisenberg, L M; Jänne, O A

    1991-01-01

    To evaluate the function of the murine ornithine decarboxylase (ODC) gene promoter, expression of chimeric ODC-chloramphenicol acetyltransferase (CAT) plasmids (pODCcat) containing 1,658 nt of the ODC promoter sequence and its various 5'-deletions was analyzed. In transient expression assays with NIH/3T3 mouse cells, pODCcat constructs exhibited fairly strong promoter activity yielding CAT values up to 40% of those obtained with the viral promoter RSV. Interestingly, 5'-deletions of the pODCc...

  5. Analysis of the sericin1 promoter and assisted detection of exogenous gene expression efficiency in the silkworm Bombyx mori L.

    OpenAIRE

    Ye, Lupeng; Qian, Qiujie; Zhang, Yuyu; You, Zhengying; Che, Jiaqian; Song, Jia; Zhong, Boxiong

    2015-01-01

    In genetics, the promoter is one of the most important regulatory elements controlling the spatiotemporal expression of a target gene. However, most studies have focused on core or proximal promoter regions, and information on regions that are more distant from the 5′-flanking region of the proximal promoter is often lacking. Here, approximately 4-kb of the sericin1 (Ser1) promoter was predicted to contain many potential transcriptional factor binding sites (TFBSs). Transgenic experiments hav...

  6. Association between Osteopontin Promoter Gene Polymorphisms and Haplotypes with Risk of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Balneek Singh Cheema

    2015-06-01

    Full Text Available Background: Osteopontin (OPN C-443T promoter polymorphism has been shown as a genetic risk factor for diabetic nephropathy (DN in type 2 diabetic patients (T2D. Methods: In the present study we investigated the association of three functional promoter gene polymorphisms C-443T, delG-156G, and G-66T and their haplotypes with the risk of DN and estimated Glomerular Filtration Rate (eGFR in Asian Indians T2D patients using Real time PCR based Taqman assay. A total of 1165 T2D patients, belonging to two independently ascertained Indian Asian cohorts, were genotyped for three OPN promoter polymorphisms C-443T (rs11730582, delG-156G (rs17524488 and G-66T (rs28357094. Results: -156G allele and GG genotypes (delG-156G and haplotypes G-C-G and T-C-G (G-66T, C-443T, delG-156G were associated with decreased risk of DN and higher eGFR. Haplotype G-T-delG and T-T-delG (G-66T, C-443T, delG-156G were identified as risk haplotypes, as shown by lower eGFR. Conclusion: This is the first study to report an association of OPN promoter gene polymorphisms; G-66T and delG-156G and their haplotypes with DN in T2D. Our results suggest an association between OPN promoter gene polymorphisms and their haplotypes with DN.

  7. Sox3 binds to 11β-hydroxysteroid dehydrogenase gene promoter suggesting transcriptional interaction in catfish.

    Science.gov (United States)

    Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

    2016-04-01

    In fishes, the expression of steroidogenic enzyme genes and their related transcription factors (TFs) are critical for the regulation of steroidogenesis and gonadal development. 11-KT is the potent androgen and hence, 11β-hsd, enzyme involved in 11-KT production is important. Regulation of 11β-hsd gene was never studied in any fishes. At first 11β-hsd was cloned and recombinant protein was tested for enzyme activity prior to expression and promoter motif analysis. Expression changes revealed stage- and sex-dependent increase in the ontogenic studies. Further, 11β-hsd expression was higher during spawning phase of reproductive cycle and was found to be gonadotropin inducible both in vivo and in vitro. ∼2kb of 5' upstream region of 11β-hsd, was cloned from catfish genomic DNA library and in silico promoter analysis revealed putative TF binding sites such as Sox3, Wt1, Pax2, Dmrt1 and Ad4BP/SF-1. Luciferase reporter assay using the sequential deletion constructs in human embryonic kidney and Chinese hamster ovary cells revealed considerable promoter activity of the constructs containing Sox3, but not with other motifs largely. Site-directed mutagenesis, Sox3 over expression, electrophoretic mobility shift and chromatin immunoprecipitation assays further substantiated the binding of Sox3 to its corresponding cis-acting element in the upstream promoter motif of 11β-hsd. This is the first report to show that Sox3 binds to the 11β-hsd gene promoter and transactivates to regulate male reproduction in a teleost. PMID:26772480

  8. ACC interleukin-10 gene promoter haplotype as a breast cancer risk factor predictor among Jordanian females

    OpenAIRE

    Atoum, Manar

    2016-01-01

    Manar Fayiz Atoum Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, Zarqa, Jordan Introduction: Interleukin-10 (IL-10) is a multifactorial cytokine with a complex biological role in breast cancer. The aims of this study were to investigate any association between IL-10 gene promoter polymorphisms, 1082A>/G, -819T>C, and -592A>C, or haplotypes and breast cancer risk among Jordanian women and to evaluate any associatio...

  9. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    Directory of Open Access Journals (Sweden)

    Oliveira Jorge

    2007-07-01

    Full Text Available Abstract Background Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. Methods A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC, 13 papillary (pRCC, 10 chromophobe (chRCC, and 10 oncocytomas and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Results Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007, PTGS2 (p = 0.002, and RASSF1A (p = 0.0001. CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively, whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004. RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035. In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031 and nuclear grade (p = 0.022, respectively. Conclusion The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

  10. Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

    OpenAIRE

    Haltiner, M M; Smale, S T; Tjian, R

    1986-01-01

    A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this...

  11. A Common Variation in the Promoter Region of Interleukin-6 Gene Shows Association with Exercise Performance

    OpenAIRE

    Antti Huuskonen; Minna Tanskanen; Jani Lappalainen; Niku Oksala; Heikki Kyröläinen; Mustafa Atalay

    2009-01-01

    Skeletal muscle-derived interleukin-6 (IL-6) is a pleiotropic cytokine which regulates body metabolism during strenuous physical exercise. OBJECTIVE: The effect of a potentially functional single nucleotide polymorphism (SNP) -174G/C of the IL6 gene (rs1800795) promoter was examined on maximal oxygen uptake (VO2max), body mass index (BMI) and plasma IL-6 levels in response to physical training. Fifty four male military conscripts were studied for 8 weeks during their basic training. At weeks ...

  12. PROMOTERS WITH CANCER CELL-SPECIFIC ACTIVITY FOR MELANOMA GENE THERAPY

    OpenAIRE

    Pleshkan, V.; Alekseenko, I.; Zinovyeva, M.; Vinogradova, T.; Sverdlov, E.

    2011-01-01

    Melanoma is one of the most aggressive tumors. It develops from pigment-forming cells (melanocytes) and results in a high number of lethal outcomes. The use of genetic constructs with the ability to specifically kill melanoma cells, but not normal cells, might increase the lifespan of patients, as well as improve their quality of life. One of the methods to achieve a selective impact for therapeutic genes on cancer cells is to utilize a transcriptional control mechanism using promoters that a...

  13. Finding Combination of Features from Promoter Regions for Ovarian Cancer-related Gene Group Classification

    KAUST Repository

    Olayan, Rawan S.

    2012-12-01

    In classification problems, it is always important to use the suitable combination of features that will be employed by classifiers. Generating the right combination of features usually results in good classifiers. In the situation when the problem is not well understood, data items are usually described by many features in the hope that some of these may be the relevant or most relevant ones. In this study, we focus on one such problem related to genes implicated in ovarian cancer (OC). We try to recognize two important OC-related gene groups: oncogenes, which support the development and progression of OC, and oncosuppressors, which oppose such tendencies. For this, we use the properties of promoters of these genes. We identified potential “regulatory features” that characterize OC-related oncogenes and oncosuppressors promoters. In our study, we used 211 oncogenes and 39 oncosuppressors. For these, we identified 538 characteristic sequence motifs from their promoters. Promoters are annotated by these motifs and derived feature vectors used to develop classification models. We made a comparison of a number of classification models in their ability to distinguish oncogenes from oncosuppressors. Based on 10-fold cross-validation, the resultant model was able to separate the two classes with sensitivity of 96% and specificity of 100% with the complete set of features. Moreover, we developed another recognition model where we attempted to distinguish oncogenes and oncosuppressors as one group from other OC-related genes. That model achieved accuracy of 82%. We believe that the results of this study will help in discovering other OC-related oncogenes and oncosuppressors not identified as yet.

  14. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    International Nuclear Information System (INIS)

    Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses

  15. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    张友才

    2006-01-01

    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  16. Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

    Directory of Open Access Journals (Sweden)

    Catherine E Winbanks

    Full Text Available Recombinant adeno-associated viral vectors (rAAV vectors are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal, human placental alkaline phosphatase (hPLAP, and green fluorescent protein (GFP as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

  17. Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences.

    Science.gov (United States)

    Yang, Shu Yuan; Fugmann, Sebastian D; Schatz, David G

    2006-12-25

    It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light chain (IgL) transcriptional control elements in GCV/SHM in the chicken B cell line DT40. Promoter substitution experiments led to identification of a strong RNA polymerase II promoter incapable of supporting efficient GCV/SHM. This surprising finding indicates that high levels of transcription are not sufficient for robust GCV/SHM in Ig loci. Deletion of the IgL enhancer in a context in which high-level transcription was not compromised showed that the enhancer is not necessary for GCV/SHM. Our results indicate that cis-acting elements are important for Ig gene diversification, and we propose that targeting specificity is achieved through the combined action of several Ig locus elements that include the promoter. PMID:17178919

  18. Promoter and enhancer elements in the rearranged alpha chain gene of the human T cell receptor.

    Science.gov (United States)

    Luria, S; Gross, G; Horowitz, M; Givol, D

    1987-11-01

    We cloned and compared the sequence of a rearranged human T cell receptor (TCR) V alpha J alpha gene and its germline counterparts. The only difference in the coding region sequence was confined to the joining region where three nucleotides, TTG, unaccountable by either V alpha or J alpha sequence, were present. By nuclease S1 mapping we identified the mRNA start of the alpha chain 70 nucleotides upstream from the initiator ATG. A 600 bp fragment containing the sequences upstream to the ATG drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene. This promoter activity is T cell specific since it can be demonstrated in human T cells but not in B cells or HeLa cells. A 1.1 kb BamHI- HindIII fragment located 5' to the first exon of the C alpha gene was found to enhance transcription from either the heterologous SV40 promoter or the homologous TCR alpha chain promoter. This enhancement activity was independent of the location of the fragment with respect to CAT and was specific to lymphoid cells (either T or B cells) but cannot be demonstrated in HeLa cells. PMID:3501368

  19. Promoter Hypermethylation of the EMP3 Gene in a Series of 229 Human Gliomas

    Directory of Open Access Journals (Sweden)

    Marta Mellai

    2013-01-01

    Full Text Available The epithelial membrane protein 3 (EMP3 is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate the EMP3 promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations of EMP3 hypermethylation with total 1p/19q codeletion, MGMT promoter hypermethylation, IDH1/IDH2 and TP53 mutations, and EGFR amplification were studied, as well as its prognostic significance. The EMP3 promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with both IDH1/IDH2 mutations and total 1p/19q codeletion and inversely with EGFR gene amplification. No association was found with MGMT hypermethylation and TP53 mutations. In the whole series, the EMP3 hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of the EMP3 promoter hypermethylation was found in patients with oligodendroglial tumors.

  20. Analysis of Hypothetical Promoter Domains of DKFZp564A1164, NPHS1 and HSPOX1 Genes

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, S S

    2003-11-29

    For this study, a high throughput method for identifying and testing regulatory elements was examined. In addition, the validity of promoters predicted by FirstEF was tested. It was found that by combining computer based promoter and first exon predictions from FirstEF (Davuluri et al., 2001) with PCR-based cloning to generate luciferase reporter constructs, and by testing reporter activity in cultured mammalian cells plated in a 96 well format one could identify promoter activity in a relatively high throughput manner. The data generated in this study suggest that FirstEF predictions are sometimes incorrect. Therefore, having a strategy for defining which FirstEF predicted promoters to test first may accelerate the process. Initially testing promoters that are at a confirmed transcription start site for a gene, at a possible alternate transcription start site or in a region of conserved sequence would be the best candidates, while promoters predicted in gene desert regions may not be as easy to confirm. The luciferase assay lent itself very well to the high throughput search, however the subcloning did not always go smoothly. The numerous steps that this traditional subcloning method requires were time consuming and increased the opportunities for errors. A faster method that skips many of the traditional subcloning steps, such as the Creator{trademark} system by Clontech is currently being investigated by our lab. The development and testing of substantially larger enhancer/silencer regulatory elements may not be possible at this time using these high throughput methods. These regulatory elements are generally GC rich making them more difficult to PCR and subclone. Additionally, confirming upstream untranslated first exons was not possible within this time scale using the SMART RACE protocol. It will be necessary to further explore the limitations within these procedures in order to confirm these and future regulatory elements. Alterations and modifications to

  1. Genomic organization and promoter cloning of the human X11α gene APBA1.

    LENUS (Irish Health Repository)

    Chai, Ka-Ho

    2012-05-01

    X11α is a brain specific multi-modular protein that interacts with the Alzheimer\\'s disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer\\'s disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer\\'s disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer\\'s disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

  2. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels

    Science.gov (United States)

    Steinacher, Arno; Bates, Declan G.; Akman, Ozgur E.; Soyer, Orkun S.

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  3. Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Sergey V., E-mail: serge@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Mogilenko, Denis A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Shavva, Vladimir S. [Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Dizhe, Ella B.; Ignatovich, Irina A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Perevozchikov, Andrej P., E-mail: app@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation)

    2010-07-23

    Research highlights: {yields} TNF{alpha} stimulates the distal alternative promoter of human apoA-I gene. {yields} TNF{alpha} acts by weakening of promoter competition within apoA-I gene (promoter switching). {yields} MEK1/2 and nuclear receptors PPAR{alpha} and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1{beta} and TNF{alpha}. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNF{alpha}-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNF{alpha} on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNF{alpha} leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNF{alpha}. The MEK1/2-ERK1/2 cascade and nuclear receptors PPAR{alpha} and LXRs are important for TNF{alpha}-mediated apoA-I promoter switching.

  4. Dexamethasone down-regulates, the activity of promoter from human al (Ⅱ) procollagen gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of dexamethasone on the promoter activity of human al(1) procollagen gene.Methods Fibroblasts from human skin were primary cultured and subcultured. (1) The effects of dexamethasone on the human skin fibroblasts were determined by BrdU incorporation into DNA of fibroblasts. (2) Three plasmids containing various engths of 5' flanksequence of human al(1) procollagen gene and CAT as reporter gene were constructed, and were transfected into the human skin fi-broblasts by FuGENE Transfecfion Reagent. The effects of dexamethasone on 3 plasmids were determined by CAT - ELlSA. Results (1)After 24h of treatment on the fibroblasts with 110-9 ~110-4mol/L examethasone in DMEM containing 2% or 10% FCS, BrdU in-corporation into DNA showed no difference ( P > 0.05) . (2) the 3 plasmids were transfected into fibroblasts and then treated with 110-5mol/L and 110-6mol/L dexamethasones for 24h, relative CAT values were different belwent dexamethasone and control,higher dexamethasone(110 -5mol/L} and lower examethasone(110 -6mol/L) ( P <0. 05) . ConcluSion Dexamethasone has noeffects on the proliferation of human skin fibroblasts, and it has negative effect on the promoter activity of human al(1) procollagengene, which is dose- dependent.

  5. Structure of the gene for human β2-adrenergic receptor: expression and promoter characterization

    International Nuclear Information System (INIS)

    The genomic gene coding for the human β2-adrenergic receptor (β2AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with β2AR properties. Southern blot analyses with β2AR-specific probes show that a single β2AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the β2AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins

  6. Symmetry in the Language of Gene Expression: A Survey of Gene Promoter Networks in Multiple Bacterial Species and Non-σ Regulons

    Directory of Open Access Journals (Sweden)

    Stefan M. Turcic

    2011-11-01

    Full Text Available The language of gene expression displays topological symmetry. An important step during gene expression is the binding of transcriptional proteins to DNA promoters adjacent to a gene. Some proteins bind to many promoters in a genome, defining a regulon of genes wherein each promoter might vary in DNA sequence relative to the average consensus. Here we examine the linguistic organization of gene promoter networks, wherein each node in the network represents a promoter and links between nodes represent the extent of base pair-sharing. Prior work revealed a fractal nucleus in several σ-factor regulons from Escherichia coli. We extend these findings to show fractal nuclei in gene promoter networks from three bacterial species, E. coli, Bacillus subtilis, and Pseudomonas aeruginosa. We surveyed several non-σ transcription factors from these species and found that many contain a nucleus that is both visually and numerically fractal. Promoter footprint size scaled as a negative power-law with both information entropy and fractal dimension, while the latter two parameters scaled positively and linearly. The fractal dimension of the diffuse networks (dB = ~1.7 was close to that expected of a diffusion limited aggregation process, confirming prior predictions as to a possible mechanism for development of this structure.

  7. Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

    Directory of Open Access Journals (Sweden)

    Betsy M. Martinez- Vaz

    2010-04-01

    Full Text Available A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation.

  8. Association of Interleukin-10 Gene Promoter Polymorphisms in Saudi Patients with Vitiligo

    Directory of Open Access Journals (Sweden)

    Abdullah Abanmi

    2008-01-01

    Full Text Available The promoter region of human Interleukin −10 gene is highly polymorphic and has been associated with numerous autoimmune diseases. Recent studies have linked vitiligo with defective autoimmune system. This study is aimed to explore a possible association between IL-10 gene polymorphism and vitiligo in Saudi population. This case control study consisted of 184 Saudi subjects including 83 vitiligo patients (40 males, 43 females mean age 27.85 ± 12.43 years and 101 matched controls. Genomic DNA was extracted from the blood samples of healthy controls and Vitiligo patients visiting out patient clinic of Department of Dermatology, Riyadh Armed Forces Hospital, using QIA ampR DNA mini kit (Qiagen CA, USA. Interleukin-10 gene was amplified by polymerase chain reaction (PCR using Arms primers to detect any polymorphism involved at positions −592, −819 and −1082.

  9. An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

    Science.gov (United States)

    Larsen, Camilla; Franch-Marro, Xavier; Hartenstein, Volker; Alexandre, Cyrille; Vincent, Jean-Paul

    2006-01-01

    Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit. PMID:17093046

  10. Promoter characterization and genomic organization of the human X11β gene APBA2.

    LENUS (Irish Health Repository)

    Hao, Yan

    2012-02-15

    Overexpression of neuronal adaptor protein X11β has been shown to decrease the production of amyloid-β, a toxic peptide deposited in Alzheimer\\'s disease brains. Therefore, manipulation of the X11β level may represent a potential therapeutic strategy for Alzheimer\\'s disease. As X11β expression can be regulated at the transcription level, we determined the genomic organization and the promoter of the human X11β gene, amyloid β A4 precursor protein-binding family A member 2 (APBA2). By RNA ligase-mediated rapid amplification of cDNA ends, a single APBA2 transcription start site and the complete sequence of exon 1 were identified. The APBA2 promoter was located upstream of exon 1 and was more active in neurons. The core promoter contains several CpG dinucleotides, and was strongly suppressed by DNA methylation. In addition, mutagenesis analysis revealed a putative Pax5-binding site within the promoter. Together, APBA2 contains a potent neuronal promoter whose activity may be regulated by DNA methylation and Pax5.

  11. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    Science.gov (United States)

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output. PMID:25085421

  12. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

    Directory of Open Access Journals (Sweden)

    Xian-E Peng

    Full Text Available Liver fatty acid-binding protein (L-FABP, also known as fatty acid-binding protein 1 (FABP1, is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182 from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032.Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05. The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003, while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014. Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

  13. Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

    Science.gov (United States)

    Peng, Xian-E; Wu, Yun-Li; Zhu, Yi-Bing; Huang, Rong-Dong; Lu, Qing-Qing; Lin, Xu

    2015-01-01

    Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans. PMID:26439934

  14. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    International Nuclear Information System (INIS)

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study

  15. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    International Nuclear Information System (INIS)

    Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue

  16. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  17. Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    CHEN XiaoGuang; ZHANG YaJing; ZHENG XueLi; WANG ChunMei

    2007-01-01

    The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles stephensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I digestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively conserved in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.

  18. P07.04PROMOTER METHYLATION OF THE LATS1 AND LATS2 GENES IN SCHWANNOMAS

    Science.gov (United States)

    Ohta, T.; Oh, J.; Mittelbronn, M.; Paulus, W.; Ohgaki, H.

    2014-01-01

    Schwannoma is a benign nerve sheath tumor that is typically encapsulated and composed of well-differentiated Schwann cellswhich comprises 5-10% of all intracranial tumors in adults. Approximately 90% of schwannomas are solitary and sporadic, whereas ∼4% are considered to arise in the setting of neurofibromatosis type 2 (NF2) syndrome by NF2 germline mutations. The molecular basis of sporadic schwannomas is not fully understood, other than frequent NF2 mutations (∼60%). LATS1 and the related LATS2 are downstream molecules of NF2 and negative regulators of the YAP oncogene in the Salvador/Warts/Hippo (SWH) signaling pathway. Expression of these genes is reduced due to promoter methylation in a variety of neoplasms including gliomas. In the present study, methylation-specific PCR revealed promoter methylation of the LATS1 and LATS2 in 15 of 91 (16%) and 32 of 91 (35%) schwannomas, respectively. These alterations were significantly more frequent in spinal than in peripheral schwannomas (23% vs 3% for LATS1, P = 0.0171; 42% vs 21% for LATS2, P = 0.0386). LATS1 methylation was also detected in 3 of 4 schwannomatosis cases. Furthermore, neurofibroma / schwannoma hybrid tumors showed promoter methylation in LATS1 (3/14; 21%) and LATS2 (8/14; 57%). LATS1 and LATS2 promoter methylation were largely mutually exclusive, and there was a significant negative correlation (P = 0.003); only 10 cases had methylation in both genes. These results suggest that LATS1 and LATS2 promoter methylation may be additional molecular mechanisms resulting in an abnormal SWH pathway in schwannomas and related tumors.

  19. Cloning and functional analysis of human acyl coenzyme A: Cholesterol acyltransferase1 gene P1 promoter.

    Science.gov (United States)

    Ge, Jing; Cheng, Bei; Qi, Benling; Peng, Wen; Wen, Hui; Bai, Lijuan; Liu, Yun; Zhai, Wei

    2016-07-01

    Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) catalyzes the conversion of free cholesterol (FC) to cholesterol ester. The human ACAT1 gene P1 promoter has been cloned. However, the activity and specificity of the ACAT1 gene P1 promoter in diverse cell types remains unclear. The P1 promoter fragment was digested with KpnI/XhoI from a P1 promoter cloning vector, and was subcloned into the multiple cloning site of the Firefly luciferase vector pGL3‑Enhancer to obtain the construct P1E‑1. According to the analysis of biological information, the P1E‑1 plasmid was used to generate deletions of the ACAT1 gene P1 promoter with varying 5' ends and an identical 3' end at +65 by polymerase chain reaction (PCR). All the 5'‑deletion constructs of the P1 promoter were identified by PCR, restriction enzyme digestion mapping and DNA sequencing. The transcriptional activity of each construct was detected after transient transfection into THP‑1, HepG2, HEK293 and Hela cells using DEAE‑dextran and Lipofectamine 2000 liposome transfection reagent. Results showed that the transcriptional activity of the ACAT1 gene P1 promoter and deletions of P1 promoter in THP‑1 and HepG2 cells was higher than that in HEK293 and HeLa cells. Moreover, the transcriptional activity of P1E‑9 was higher compared with those of other deletions in THP‑1, HepG2, HEK293 and HeLa cells. These findings indicate that the transcriptional activity of the P1 promoter and the effects of deletions vary with different cell lines. Thus, the P1 promoter may drive ACAT1 gene expression with cell‑type specificity. In addition, the core sequence of ACAT1 gene P1 promoter was suggested to be between -125 and +65 bp. PMID:27220725

  20. Upregulation of Bax and Bcl-2 following prenatal cocaine exposure induces apoptosis in fetal rat brain

    Directory of Open Access Journals (Sweden)

    DaLiao Xiao, Lubo Zhang

    2008-01-01

    Full Text Available Cocaine abuse during pregnancy has been associated with numerous adverse perinatal outcomes. Aims: The present study was to determine whether prenatal cocaine exposure induced apoptosis and the possible role of Bcl-2 family genes in the programming cell death in fetal rat brain. Main methods: Pregnant rats were treated with cocaine subcutaneously (30 & 60 mg/kg/day from day 15 to 21 of gestation. Then the fetal and maternal brains were isolated. Key findings: Cocaine produced a dose-dependent decrease in fetal brain weight and brain/body weight ratio (P<0.05. Apoptotic nuclei in fetal brain were increased from 2.6 ± 0.1 (control to 8.1± 0.6 (low dose and 10.4 ± 0.2% (high dose (P<0.05. In accordance, cocaine dose dependently increased activities of caspase-3, caspase-8, and caspase-9 (% of control in the fetal brain by 177%, 155%, 174%, respectively, at 30 mg/kg/day, and by 191%, 176%, 274%, respectively, at 60 mg/kg/day. In contrast, cocaine showed no effect on caspase activities in the maternal brain. Cocaine produced a dose-dependent increase in both Bcl-2 and Bax protein expression in the fetal brain, and increased the ratio of Bax/Bcl-2 at dose of 30 mg/kg/day (P<0.05. Significance: Our study has demonstrated that prenatal cocaine exposure induces apoptosis in the fetal brain, and suggested that up-regulating Bax/Bcl-2 gene expression may be involved in cocaine-induced apoptosis. The increased apoptosis of neuronal cells in the fetal brain is likely to play a key role in cocaine-induced neuronal defects during fetal development.

  1. 15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers

    Science.gov (United States)

    Leng, Shuguang; Liu, Yushi; Weissfeld, Joel L.; Thomas, Cynthia L.; Han, Younghun; Picchi, Maria A.; Edlund, Christopher K.; Willink, Randall P.; Gaither Davis, Autumn L.; Do, Kieu C.; Nukui, Tomoko; Zhang, Xiequn; Burki, Elizabeth A.; Van Den Berg, David; Romkes, Marjorie; Gauderman, W. James; Crowell, Richard E.; Tesfaigzi, Yohannes; Stidley, Christine A.; Amos, Christopher I.; Siegfried, Jill M.; Gilliland, Frank D.

    2015-01-01

    Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. Methods: A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. Results: Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10–8). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10–9). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. Conclusions: A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing. PMID:25713168

  2. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

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    Rowe J

    2009-08-01

    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  3. Coactivator CBP/p300 increased the activity of C/EBP-mediated human interleukin-5 gene promoter

    Institute of Scientific and Technical Information of China (English)

    LIU Chunyan; LU Jun; LI Lin; TAN Jiang; SHAO Yangguang; HUANG Baiqu

    2004-01-01

    Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an essential role in the development of allergic diseases, such as allergic asthma. Histone acetyltransferase CBP/p300 remodels chromatin by acetylating histones, resulting in open structure of chromatin and active transcription. Adenovirus protein E1A inhibits the activity of CBP/p300. In this study, we analyzed the effects of E1A on IL-5 gene promoter/luciferase reporter activity. The results showed that E1A protein inhibited the activity of PMA/ionomycin-stimulated IL-5 gene promoter/luciferase reporter construct. In contrast, overexpression of the CBP/p300-binding defective E1A A2-36 protein did not inhibit IL-5 gene promoter activity. These data demonstrated for the first time that transcriptional coactivator CBP/p300was involved in the activation of IL-5 gene promoter. E1A protein can modulate CBP/p300 function to activate the transcription of IL-5 gene promoter/luciferase reporter plasmid. Furthermore, in collaboration with transcription factor C/EBP, CBP/p300 activated IL-5 gene promoter/luciferase reporter expression. This study provides further insight into the mechanisms of transcriptional regulation of IL-5 gene.

  4. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

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    Tung Shu-Yun

    2011-04-01

    Full Text Available Abstract Background Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. Results Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR. A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI genes (OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. Conclusions By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

  5. Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus

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    Yin Weilun

    2010-01-01

    Full Text Available Abstract Background CBL1 is a calcium sensor that regulates drought, cold and salt signals in Arabidopsis. Overexpression of CBL1 gene in Arabidopsis and in Ammopiptanthus mongolicus showed different tolerant activities. We are interested in understanding the molecular mechanism of the upstream region of the CBL1 gene of A. mongolicus (AmCBL1. We investigated and characterized the promoter of the AmCBL1 gene, for promoters play a very important role in regulating gene expression in eukaryotes. Results A 1683-bp 5' flanking region was isolated from A. mongolicus. The sequence was identified as AmCBL1 promoter. Analysis of the promoter sequence indicated a 690-bp intron and some basic cis-acting elements were related to various environmental stresses and plant hormones. To identify the functional region of the AmCBL1 promoter, five plant expression vectors fused with the GUS (β-glucuronidase gene, driven by series deleted fragments of AmCBL1 promoter at different lengths from -1659, -1414, -1048, -296 to -167 bp relative to the transcriptional start site were constructed and transformed into Nicotiana tabacum L. cv. 89. Functional properties of each promoter segment were examined by GUS staining and fluorescence quantitative analyses using at least three single-copy PCR-positive plants of transgenic tobacco, treated with various environmental stresses and plant hormones for different times. We demonstrated that the AmCBL1 promoter was a vascular-specific and multiple-stress-inducible promoter. Our results further imply that the promoter fragment B1S3 possessed sufficient essential cis-acting elements, accounting for vascular-specific and stress-induced expression patterns. It may also indicate that for response to some stresses certain cis-elements are required in tissues outside the region of the B1S3 construct. Conclusions To help resolve uncertainties about the upstream regulatory mechanism of the CBL1 gene in desert plants, we suggest that

  6. ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

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    de Souza Emanuel M

    2009-03-01

    Full Text Available Abstract Background ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. Methods First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP. Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. Results The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM; tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC was 76.2% compared with 25.5% in invasive ductal carcinoma

  7. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy

    Science.gov (United States)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine

    1997-01-01

    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  8. Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

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    Meiya Li

    2015-10-01

    Full Text Available Phytoene synthase (PSY catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg2+ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

  9. Association of the Resistin Gene Promoter Region Polymorphism with Kawasaki Disease in Chinese Children

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    Ruixi Liu

    2012-01-01

    Full Text Available Objectives. The −420C>G polymorphism located in the resistin gene (RETN promoter has recently been suggested to play a potential role in proinflammatory conditions and cardiovascular disease. This study investigated the association of the RETN promoter polymorphism with Kawasaki disease (KD and its clinical parameters in Chinese children. Methods. We compared patients with complete KD to incomplete KD children. Genotyping of the RETN promoter polymorphism was performed using MassARRAY system, and serum resistin levels were estimated using the sandwich enzyme immunoassay method. Results. There was no significant difference in RETN (−420C>G genotypes between KD and control groups. However, the frequency of the G allele was higher in iKD patients than in cKD children due to a significantly increased frequency of the GG genotypes. Serum levels of resistin were significantly higher in KD patients than in controls regardless of the presence of coronary artery lesions (CALs. Conclusion. The present findings suggest that while resistin may play a role in the pathogenesis of KD, there is no apparent association between CAL and the RETN (−420C>G gene polymorphism in KD children. However, the diagnosis of iKD is challenging but can be supported by the presence of the G allele and the GG genotypes.

  10. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir

    2012-07-01

    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  11. Structural and functional analysis of the mouse mdr1b gene promoter.

    Science.gov (United States)

    Cohen, D; Piekarz, R L; Hsu, S I; DePinho, R A; Carrasco, N; Horwitz, S B

    1991-02-01

    The overproduction of P-glycoprotein, an integral membrane protein thought to function as a drug efflux pump, is the hallmark of the multidrug resistance phenotype. In murine multidrug resistant J774.2 cell lines, distinct mdr genes, mdr1a and mdr1b, encode unique P-glycoprotein isoforms. To examine the transcriptional regulation of the mdr1b gene, its promoter was isolated and characterized. The transcription initiation site was mapped by primer extension, and the 5'-flanking region was sequenced. Several potential regulatory elements were identified in this region. A transient expression vector was constructed by fusion of 540 base pairs of 5'-flanking sequence and part of the first untranslated exon to the chloramphenicol acetyltransferase (CAT) gene. When transfected into monkey kidney COS-1, rat pituitary GH3 or T47D human breast cells, the mdr1b 5'-flanking sequences were capable of driving CAT expression. Transient transfection studies using deletion subclones of the mdr1b-CAT construct were done to locate potential cis-acting sequences. The studies indicate the presence of cis-acting elements in the 5'-flanking region of the mdr1b gene. The implications of these findings for expression and regulation of the mdr1b gene are discussed. PMID:1671222

  12. Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

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    C.K. Moreira

    2004-02-01

    Full Text Available Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

  13. Variants in the dopamine-4-receptor gene promoter are not associated with sensation seeking in skiers.

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    Cynthia J Thomson

    Full Text Available Sensation seeking is a personality trait that has been associated with disinhibited behaviours including substance use and gambling, but also with high-risk sport practices including skydiving, paragliding, and downhill skiing. Twin studies have shown that sensation seeking is moderately heritable, and candidate genes encoding components involved in dopaminergic transmission have been investigated as contributing to this type of behaviour. To determine whether variants in the regulatory regions of the dopamine-4-receptor gene (DRD4 influenced sport-specific sensation seeking, we analyzed five polymorphisms (-1106T/C, -906T/C, -809G/A, -291C/T, 120-bp duplication in the promoter region of the gene in a cohort of skiers and snowboarders (n = 599 that represented a broad range of sensation seeking behaviours. We grouped subjects by genotype at each of the five loci and compared impulsive sensation seeking and domain-specific (skiing sensation seeking between groups. There were no significant associations between genotype(s and general or domain-specific sensation seeking in the skiers and snowboarders, suggesting that while DRD4 has previously been implicated in sensation seeking, the promoter variants investigated in this study do not contribute to sensation seeking in this athlete population.

  14. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

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    Dan Liu

    2016-01-01

    Full Text Available HtrA serine peptidase 2 (HtrA2, also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1 sites, two Sp1 transcription factor (SP1sites, one activator protein (AP site, seven p53 sites, and four YY1 transcription factor(YY1 sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

  15. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter.

    Science.gov (United States)

    Liu, Dan; Liu, Xin; Wu, Ye; Wang, Wen; Ma, Xinliang; Liu, Huirong

    2016-01-01

    HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at -1205~-838 bp and -146~+93 bp, with the -838~-649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at -709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases. PMID:26784188

  16. Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma.

    Science.gov (United States)

    Brigliadori, Giovanni; Foca, Flavia; Dall'Agata, Monia; Rengucci, Claudia; Melegari, Elisabetta; Cerasoli, Serenella; Amadori, Dino; Calistri, Daniele; Faedi, Marina

    2016-06-01

    Despite advances in the treatment of glioblastoma (GBM), median survival is 12-15 months. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status is acknowledged as a predictive marker for temozolomide (TMZ) treatment. When MGMT promoter values fall into a "methylated" range, a better response to chemotherapy is expected. However, a cutoff that discriminates between "methylated" and "unmethylated" status has yet to be defined. We aimed to identify the best cutoff value and to find out whether variability in methylation profiles influences the predictive capacity of MGMT promoter methylation. Data from 105 GBM patients treated between 2008 and 2013 were analyzed. MGMT promoter methylation status was determined by analyzing 10 CpG islands by pyrosequencing. Patients were treated with radiotherapy followed by TMZ. MGMT promoter methylation status was classified into unmethylated 0-9 %, methylated 10-29 % and methylated 30-100 %. Statistical analysis showed that an assumed methylation cutoff of 9 % led to an overestimation of responders. All patients in the 10-29 % methylation group relapsed before the 18-month evaluation. Patients with a methylation status ≥30 % showed a median overall survival of 25.2 months compared to 15.2 months in all other patients, confirming this value as the best methylation cutoff. Despite wide variability among individual profiles, single CpG island analysis did not reveal any correlation between single CpG island methylation values and relapse or death. Specific CpG island methylation status did not influence the predictive value of MGMT. The predictive role of MGMT promoter methylation was maintained only with a cutoff value ≥30 %. PMID:27029617

  17. Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

    OpenAIRE

    Li, B.; Weber, J. A.; Chen, Y; Greenleaf, A L; Gilmour, D S

    1996-01-01

    Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reacti...

  18. Identification of novel motif patterns to decipher the promoter architecture of co-expressed genes in Arabidopsis thaliana

    OpenAIRE

    López, Yosvany; Patil, Ashwini; Nakai, Kenta

    2013-01-01

    Background The understanding of the mechanisms of transcriptional regulation remains a challenge for molecular biologists in the post-genome era. It is hypothesized that the regulatory regions of genes expressed in the same tissue or cell type share a similar structure. Though several studies have analyzed the promoters of genes expressed in specific metazoan tissues or cells, little research has been done in plants. Hence finding specific patterns of motifs to explain the promoter architectu...

  19. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

    OpenAIRE

    Shimada, T.; Inokuchi, K; Nienhuis, A W

    1987-01-01

    The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites ...

  20. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

    Science.gov (United States)

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

    2015-12-01

    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

  1. CypA, a gene downstream of HIF-1α, promotes the development of PDAC.

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    Huan Zhang

    Full Text Available Hypoxia-inducible factor-1α (HIF-1α is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC. In response to tumor hypoxia, cyclophilin A (CypA is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.

  2. Polymorphisms of the ELANE Gene Promoter Region in End-Stage Chronic Kidney Disease Patients

    Science.gov (United States)

    Fernandes, Rafael; Freitas, Bruno; Miranda, Vasco; Costa, Elísio; Santos-Silva, Alice; Bronze-da-Rocha, Elsa

    2016-01-01

    End-stage renal disease (ESRD) patients have a high mortality rate that exceeds that of non-ESRD population. The hemodialysis procedure induces neutrophil activation and elastase release, which might have a role in the inflammatory process and in the development of oxidative stress. The ELANE gene encodes the neutrophil elastase. We analyzed the effect of ELANE promoter region polymorphisms and its relation with the circulating levels of elastase, as well as several clinical, biochemical and inflammatory markers in 123 ESRD patients. We found two duplications in heterozygosity in the promoter region and a new polymorphism, the c.-801G>A. ESRD patients heterozygous for the c.-903T>G polymorphism had no changes in the circulating levels of elastase or other evaluated variables, and those homozygous for the c.-741G>A polymorphism showed significant effects on neutrophils count, as well as in neutrophils/lymphocytes ratio, which might be associated with an increased inflammatory process. PMID:27136588

  3. A regulatory potential of the Xist gene promoter in vole M. rossiaemeridionalis.

    Directory of Open Access Journals (Sweden)

    Konstantin E Orishchenko

    Full Text Available X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5' flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.

  4. MGMT-B gene promoter hypermethylation in patients with inflammatory bowel disease - a novel finding.

    Science.gov (United States)

    Mokarram, Pooneh; Kavousipour, Soudabeh; Sarabi, Mostafa Moradi; Mehrabani, Golnosh; Fahmidehkar, Mohammad Ali; Shamsdin, Seyedeh Azra; Alipour, Abbas; Naini, Mahvash Alizade

    2015-01-01

    Inflammatory bowel disease (IBD) is a disease strongly associated with colorectal cancer (CRC) as a well-known precancerous condition. Alterations in DNA methylation and mutation in K-ras are believed to play an early etiopathogenic role in CRC and may also an initiating event through deregulation of molecular signaling. Epigenetic silencing of APC and SFRP2 in the WNT signaling pathway may also be involved in IBD-CRC. The role of aberrant DNA methylation in precancerous state of colorectal cancer (CRC) is under intensive investigation worldwide. The aim of this study was to investigate the status of promoter methylation of MGMT-B, APC1A and SFRP2 genes, in inflamed and normal colon tissues of patients with IBD compared with control normal tissues. A total of 52 IBD tissues as well as corresponding normal tissues and 30 samples from healthy participants were obtained. We determined promoter methylation status of MGMT-B, SFRP2 and APC1A genes by chemical treatment with sodium bisulfite and subsequent MSP. The most frequently methylated locus was MGMT-B (71%; 34 of 48), followed by SFRP2 (66.6 %; 32 of 48), and APC1A (43.7%; 21 of 48). Our study demonstrated for the first time that hypermethylation of the MGMT-B and the SFRP2 gene promoter regions might be involved in IBD development. Methylation of MGMT-B and SFRP2 in IBD patients may provide a method for early detection of IBD-associated neoplasia. PMID:25773792

  5. Molecular and functional characterization of the promoter of ETS2, the human c-ets-2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Mavrothalassitis, G.J.; Watson, D.K.; Papas, T.S. (National Cancer Institute, Frederick, MD (USA))

    1990-02-01

    The 5{prime} end of the human c-ets-2 gene, ETS2, was cloned and characterized. The major transcription initiation start sites were identified, and the pertinent sequences surrounding the ETS2 promoter were determined. The promoter region of ETS2 does not possess typical TATA and CAAT elements. However, this promoter contains several repeat regions, as well as two consensus AP2 binding sites and three putative Sp1 sites. There is also a palindromic region similar to the serum response element of the c-fos gene, located 1,400 base pairs (bp) upstream from the first major transcription initiation site. A G+C-rich sequence (GC element) with dyad symmetry can be seen in the ETS2 promoter, immediately following an unusually long polypurine-polypyrimidine tract. A series of deletion fragments from the putative promoter region were ligated in front of the bacterial chloramphenicol acetyltransferase gene and tested for activity following transfection into HeLa cells. The 5{prime} boundary of the region needed for maximum promoter activity was found to be 159 bp upstream of the major initiation site. The promoter of ETS2 (within the polypyrimidine tract) serves to illustrate an alternative structure that may be present in genes with TATA-less promoters.

  6. Molecular and functional characterization of the promoter of ETS2, the human c-ets-2 gene

    International Nuclear Information System (INIS)

    The 5' end of the human c-ets-2 gene, ETS2, was cloned and characterized. The major transcription initiation start sites were identified, and the pertinent sequences surrounding the ETS2 promoter were determined. The promoter region of ETS2 does not possess typical TATA and CAAT elements. However, this promoter contains several repeat regions, as well as two consensus AP2 binding sites and three putative Sp1 sites. There is also a palindromic region similar to the serum response element of the c-fos gene, located 1,400 base pairs (bp) upstream from the first major transcription initiation site. A G+C-rich sequence (GC element) with dyad symmetry can be seen in the ETS2 promoter, immediately following an unusually long polypurine-polypyrimidine tract. A series of deletion fragments from the putative promoter region were ligated in front of the bacterial chloramphenicol acetyltransferase gene and tested for activity following transfection into HeLa cells. The 5' boundary of the region needed for maximum promoter activity was found to be 159 bp upstream of the major initiation site. The promoter of ETS2 (within the polypyrimidine tract) serves to illustrate an alternative structure that may be present in genes with TATA-less promoters

  7. Quantitative global and gene-specific promoter methylation in relation to biological properties of neuroblastomas

    Directory of Open Access Journals (Sweden)

    Kiss Nimrod B

    2012-09-01

    Full Text Available Abstract Background In this study we aimed to quantify tumor suppressor gene (TSG promoter methylation densities levels in primary neuroblastoma tumors and cell lines. A subset of these TSGs is associated with a CpG island methylator phenotype (CIMP in other tumor types. Methods The study panel consisted of 38 primary tumors, 7 established cell lines and 4 healthy references. Promoter methylation was determined by bisulphate Pyrosequencing for 14 TSGs; and LINE-1 repeat element methylation was used as an indicator of global methylation levels. Results Overall mean TSG Z-scores were significantly increased in cases with adverse outcome, but were unrelated to global LINE-1 methylation. CIMP with hypermethylation of three or more gene promoters was observed in 6/38 tumors and 7/7 cell lines. Hypermethylation of one or more TSG (comprising TSGs BLU, CASP8, DCR2, CDH1, RASSF1A and RASSF2 was evident in 30/38 tumors. By contrast only very low levels of promoter methylation were recorded for APC, DAPK1, NORE1A, P14, P16, TP73, PTEN and RARB. Similar involvements of methylation instability were revealed between cell line models and neuroblastoma tumors. Separate analysis of two proposed CASP8 regulatory regions revealed frequent and significant involvement of CpG sites between exon 4 and 5, but modest involvement of the exon 1 region. Conclusions/significance The results highlight the involvement of TSG methylation instability in neuroblastoma tumors and cell lines using quantitative methods, support the use of DNA methylation analyses as a prognostic tool for this tumor type, and underscore the relevance of developing demethylating therapies for its treatment.

  8. E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

    OpenAIRE

    Stubenrauch, F.; Leigh, I M; Pfister, H

    1996-01-01

    The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mu...

  9. Overlapping TATA-dependent and TATA-independent early promoter activities in the baculovirus gp64 envelope fusion protein gene.

    OpenAIRE

    Kogan, P H; Chen, X.; Blissard, G W

    1995-01-01

    In previous studies to characterize basal and activated transcription from the early promoter of the gp64 envelope fusion protein (efp) gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus, the TATA box was identified as a functional element, essential for basal transcription from a minimal promoter construct. In the current study, we used discrete deletions and multiple point mutations that removed the functional TATA box from larger promoter constructs of the gp64 efp gen...

  10. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-Li JIANG; Jian-Ye ZHANG; Charles YOUNG; Xiao-Yan HU; Yong-Mei WANG; Zhi-Fang LIU; Mei-Lan HAO

    2004-01-01

    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development andprostate cancer. To study its regulation of transcription, 1.06 kb 5 ′ flanking region of Nkx3.1 gene and its5 ′deletion mutants (861,617, 417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, apromoter-less luciferase reporter vector, to examine their promoter activities driving the reporter genetranscription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-controland pGL3-basic were used as positive and negative control respectively. The promoter activities were deter-mined by dual-luciferase reporter assay 48 h after pGL3 constructs were cotransfected with pRL-TK intoprostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M1/M2) of pGL3-1.06 kbcotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotrans-fection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The resultsalso showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861 bp,pGL3-617 bp, pGL3-417 bp, pGL3-238 bp, the last one still had 80% promoter activity compared with pGL3-1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. Theconclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoteractivity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain someimportant positive or negative regulatory elements. It will be important to identify the elements within theNkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  11. Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

    Institute of Scientific and Technical Information of China (English)

    Jaeseok Han; Eung-Hwi Kim; Woohyuk Choi; Hee-Sook Jun

    2012-01-01

    AIM:To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.METHODS:We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes.We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter (rAd-SP-rINSfur) into diabetic Leprdb/db mice.A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control (rAd-CMV-βgal).Blood glucose levels and body weights were monitored for 50 d.Glucose and insulin tolerance tests were performed.Immunohistochemical staining was performed to investigate islet morphology and insulin content.RESULTS:Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d,whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic.Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve (AUC):21 508.80 ± 2248.18 vs 62 640.00 ± 5014.28,P < 0.01] and at 6 wk (AUC:29 956.60 ± 1757.33 vs 60 016.60 ± 3794.47,P < 0.01).In addition,insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice (AUC:9150.17±1007.78 vs 11 994.20 ± 474.40,P < 0.05).The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.CONCLUSION:Based on these results,we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

  12. The bidirectional promoter of the divergently transcribed mouse Surf-1 and Surf-2 genes.

    OpenAIRE

    Lennard, A C; Fried, M

    1991-01-01

    The ubiquitously expressed mouse Surf-1 and Surf-2 genes are divergently transcribed, and their heterogeneous start sites are separated by up to a maximum of only 73 bp. By using in vitro DNase I, dimethyl sulfate methylation, and gel retardation assays, we have identified five putative promoter control elements between and around the Surf-1 and Surf-2 start sites. The effects of each site on the regulation of Surf-1 and Surf-2 transcription have been studied in vivo, and four sites were foun...

  13. Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization.

    Science.gov (United States)

    Bevilacqua, M A; Giordano, M; D'Agostino, P; Santoro, C; Cimino, F; Costanzo, F

    1992-02-15

    We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5'-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed. PMID:1541403

  14. The analysis of the human hemopexin promoter defines a new class of liver-specific genes.

    OpenAIRE

    Poli, V.; Silengo, L; Altruda, F; Cortese, R

    1989-01-01

    Hemopexin (Hpx) is a plasma glycoprotein which is expressed only in the liver. It is synthesized at a lower rate in the fetal liver than in the adult, and its level increases during acute infections. As shown here, a fragment of the human hemopexin promoter spanning from positions -130 to +22 relative to the cap site is sufficient to direct cell-specific transcription of a reporter gene. Within this segment a short sequence, located between positions -120 and -104, is responsible for this eff...

  15. Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Knecht, Wolfgang; Warneboldt, J.;

    2004-01-01

    the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and...... mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase...

  16. Characterization of the bovine pregnancy-associated glycoprotein gene family – analysis of gene sequences, regulatory regions within the promoter and expression of selected genes

    Directory of Open Access Journals (Sweden)

    Walker Angela M

    2009-04-01

    Full Text Available Abstract Background The Pregnancy-associated glycoproteins (PAGs belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1 we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2 we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3 we determined relative transcript abundance of selected PAGs during pregnancy and, 4 we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo PAG-2. Results From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs, were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene. Conclusion PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed

  17. Mediterranean river buffalo oxytocin-neurophysin I (OXT gene: structure, promoter analysis and allele detection

    Directory of Open Access Journals (Sweden)

    L. Ramunno

    2010-02-01

    Full Text Available Oxytocin (OXT is a very abundant nonapeptide neurohypophysial hormone implicated in several aspects of reproduction, including social, sexual and maternal behaviour, induction of labour and milk ejection. The nucleotide sequence of the whole OXTneurophysin I encoding gene (OXT in Mediterranean river buffalo was determined, plus 993 nucleotides at the 5’ flanking region. Buffalo oxytocin gene sequence analysis showed two transitions in the promoter region (C→T in position – 966 and G→A in position – 790 and one transversion G→T at the 170th nucleotide of the second exon, responsible for the Arg97→Leu aa substitution which identifies an allele named OXT B. A PCR-RFLP based method for a rapid identification of carriers of these alleles has been developed.

  18. TRPC6 gene promoter polymorphisms in steroid resistant nephrotic syndrome children

    Directory of Open Access Journals (Sweden)

    Mahesh Kumar Kempanahalli Basappa

    2015-04-01

    Full Text Available Introduction: Nephrotic syndrome (NS is the most frequent cause of proteinuria in children and is emerging as a leading cause of uremia. Among idiopathic NS, 10% of children do not respond to steroids or to any other immunosuppressive therapy, and progress to end-stage renal disease (ESRD. Several studies have investigated the mutations in genes encoding podocyte proteins and their possible associations with several forms of hereditary NS. Objectives: The present study aimed to determine the distribution of the TRPC6 gene promoter polymorphisms in subjects with features of steroid resistant nephrotic syndrome (SRNS and controls. Patients and Methods: About 49 unrelated patients with SRNS and 45 age matched controls no renal or other disorders were included in the study. PCR-RFLP was used for genotyping rs3824934 (-254C>G and rs56134796 (-218C>T polymorphisms located in TRPC6 gene promoter region. Results: Both -254C>G and -218C>T are polymorphic in both SRNS patients and controls. No statistically significant differences in genotypes or allele frequencies between SRNS patients and controls were observed. Linkage disequilibrium was not strong and significant and haplotypes were not associated with SRNS. Interaction analysis by multifactor dimensionality reduction (MDR revealed a significant interaction between -254G>C and -218C>T in <10 years age group. Conclusion: The results demonstrate that the TRPC6 polymorphisms do not affect susceptibility of SRNS in Indian population. Further replications, preferably a systematic search for TRPC6 functional variants that affect gene expression are desirable for validation of our findings.

  19. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression.

    Science.gov (United States)

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A

    2012-03-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

  20. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

    Science.gov (United States)

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A.

    2012-01-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex—comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4—serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

  1. Abnormal promoter methylation of multiple genes in the malignant transformed PEP2D cells induced by alpha particles exposure

    Institute of Scientific and Technical Information of China (English)

    LiP; SuiJL

    2002-01-01

    The 5' promoter regions of some genes contain CpG-rich areas,known as CpG islands,Methylation of the cytosine in these dinuleotides has important regulatory effects on gene expression.The functional significance of promoter hypermethylation would play the same roles in carcinogenesis as gene mutations.The promoter methylations p14ARF,p16INK4a,MGMT,GSTP1,BUB3 and DAPK genes were analyzed with methylation specific PCR(MSP) in the transformed human bronchial epithelial cells(BEP2D) induced by α-particles.The results indicated that p14ARF gene was not methylated in BEP2D cells,but was methylated in the malignant transformed BERP35T-1 cells,and the level of its transcription was depressed remarkable in the latter.However p16INK4a gene,which shares two exons with p14ARF gene,was not methylated.MGMT gene was methylated in both BEP2D and BERP35T-1.DAPK gene was partially methylated in BEP2D cells and methylated completely in BERP35T1.GSTP1 was not methylated in BEP2D cells and was methylated partly in BERP35T-1.BUB3 gene was not methylated in BEP2D as well as BERP35T1 cells and was further proved by sequencing analysis.

  2. Isolation and characterization of a chalcone isomerase gene promoter from potato cultivars.

    Science.gov (United States)

    Chen, M; Zhu, W J; You, X; Liu, Y D; Kaleri, G M; Yang, Q

    2015-01-01

    Chalcone isomerase (CHI) is a key enzyme involved in anthocyanin metabolism. Previous research on CHI has mainly focused on cDNA cloning and gene expression. In the current study, the 1425-bp potato CHI promoter (PCP) was isolated from four potato cultivars (Heijingang, Zhongshu 7, Désirée, and Favorita) using PCR and DNA sequencing. The PCP contained many cis-regulatory elements (CREs) related to anthocyanin metabolism, tissue specificity, light response, stress, and hormone induction. Of the PCP CREs identified, 19 were common to those found in the higher plants examined, based on plant CRE databases. Multiple sequence alignment showed six single nucleotide variation sites in PCP among the potato cultivars examined, resulting in changes in the number of CREs connected with tissue specificity, anthocyanin metabolism, and light response. The 665-bp PCP fragments from Favorita and 1425-bp PCP fragments from Heijingang were used to construct plant expression vectors, which may be a useful tool for biological engineering. A transient expression assay demonstrated that the two PCP fragments from Heijingang could direct the expression of a green fluorescent protein gene in onion epidermis and a β-glucuronidase gene in all potato tuber tissues with different colors, suggesting that the single nucleotide variation in the PCP did not affect its activity, and that silencing of the CHI gene in Favorita may be attributed to other regulatory factors. PMID:26782538

  3. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris.

    Science.gov (United States)

    Hohenblum, Hubertus; Gasser, Brigitte; Maurer, Michael; Borth, Nicole; Mattanovich, Diethard

    2004-02-20

    The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source. PMID:14755554

  4. Structure of two solanum tuberosum steroidal glycoalkaloid glycosyltransferase genes and expression of their promoters in transgenic potatoes

    Science.gov (United States)

    The Sgt2 gene in potato encodes a solanidine glucosyltransferase and is present as two distinct alleles expressed in cultivated potatoes. Promoter regions upstream from both steroidal glycoalkaloid biosynthetic gene alleles, Sgt2.1 and Sgt2.2, were isolated from Solanum tuberosum cv. Russet Burbank ...

  5. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  6. Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

    Directory of Open Access Journals (Sweden)

    Loro L Kujjo

    Full Text Available BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii oocytes possess the machinery and capability for repairing such DNA damage; (iii Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in

  7. RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts.

    Science.gov (United States)

    Lopez, Pascal; Wagner, Kay-Dietrich; Hofman, Paul; Van Obberghen, Emmanuel

    2016-05-15

    RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions. PMID:26976645

  8. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes.

    Science.gov (United States)

    Müller, Gerd A; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Padi, Megha; Fischer, Martin; Litovchick, Larisa; DeCaprio, James A; Engeland, Kurt

    2012-02-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G(0)/G(1). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G(0). Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G(0)/G(1), but also for activation in S, G(2) and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle. PMID:22064854

  9. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

    Science.gov (United States)

    Müller, Gerd A.; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Padi, Megha; Fischer, Martin; Litovchick, Larisa; DeCaprio, James A.; Engeland, Kurt

    2012-01-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G0/G1. It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G0. Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G0/G1, but also for activation in S, G2 and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle. PMID:22064854

  10. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

    Science.gov (United States)

    Ribault, S; Neuville, P; Méchine-Neuville, A; Augé, F; Parlakian, A; Gabbiani, G; Paulin, D; Calenda, V

    2001-03-16

    Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders. PMID:11249869

  11. ΔNp73 enhances promoter activity of TGF-β induced genes.

    Directory of Open Access Journals (Sweden)

    Maarten Niemantsverdriet

    Full Text Available The p53 homolog p73 is frequently overexpressed in cancers. Especially the transactivation domain truncated isoform ΔNp73 has oncogenic properties and its upregulation is associated with poor patient survival. It has been shown that ΔNp73 has an inhibitory effect on the transactivation capacity of p53 and other p73 isoforms. Here, we confirm this finding but surprisingly find that ΔNp73 may also stimulate the expression of TGF-β signaling targets. Promoter-reporter analysis indicated that the presence of Smad Binding Elements (SBE in the promoter is sufficient for stimulation of gene expression by ΔNp73. TGF-β signaling was less efficient in ΔNp73 downregulated cells, whereas tetracycline induced ΔNp73 increased expression of endogenous TGF-β regulated genes PAI-1 and Col1a1. Pull-down assays with SBE DNA suggest that ΔNp73 enhances smad3/4 binding to SBEs, thereby stimulating TGF-β signaling. Chromatin immunoprecipitation assays confirmed a direct interaction between ΔNp73 and SBE. Given the role of TGF-β signaling in carcinogenesis, tumor invasion and metastasis via targets like PAI-1 and Col1a1, our data suggest a model on how this effect of ΔNp73 could be a contributing factor in cancer progression.

  12. Prolactin promoter gene as marker assisted selection (MAS for the control of broodiness of Kampung chicken

    Directory of Open Access Journals (Sweden)

    Tike Sartika

    2004-12-01

    Full Text Available Preliminary research about MAS (Marker Assisted Selection was conducted to detect broodiness trait of Kampung chicken. MAS currently is very important in situations, where the accuracy of selection is low, such as, traits with low heritability, e.g. broodiness trait and egg production. Prolactin promoter was selected as a marker gene for broodiness because it plays a critical part in the neuroendocrine cascade which is triggered at the onset of broodiness. DNA samples were collected from low and highbroodiness samples on basic population (G0 each 24 samples, and from selected population (G3 each 28 samples. As control population without broody behavior was used 16 samples White Leghorn (WL chicken. Prolactin promoter gene was amplified using polymerase chain reaction (PCR. PCR product was analyzed using electrophoresis agarose gel 2%. The results showed four types of bands represent in the Kampung chicken, three types called as wild type band and one type as the WL band. The chickens with low and high broodiness on G0 generation have 75 and 87.5% of wild type band while in the G3 generation was decreased to 25 and 75%. Conclusions of the research indicated that the selected breed of the Kampung chicken on G3 generation increased WL band like White Leghorn chicken as much as 31,25% from the G0 generation.

  13. In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

    LENUS (Irish Health Repository)

    Murphy, Therese M

    2011-01-01

    Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

  14. A study of tumor suppressor gene expression and promoter methylation for the identification of prognostic markers in glioblastoma

    OpenAIRE

    Vaitkienė Grigaitė, Paulina

    2013-01-01

    Glioblastoma (GBM) is the most common primary brain tumor in adults. This study attempted to identify the genes potentially regulated by promoter methylation in GBM. Therefore, we analyzed the expression of COX7A1, SPINT1, AREG, NPTX2, and KRT81 in glioblastomas and human brain tissue and investigated if there were any associations between the expression and methylation of these genes. Moreover, we aimed to determine the methylation frequency of 11 genes (AREG, CASP8, CD81, DcR...

  15. The CHR Promoter Element Controls Cell Cycle-Dependent Gene Transcription and Binds the DREAM and MMB Complexes

    OpenAIRE

    Müller, Gerd A.; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Fischer, Martin; Engeland, Kurt; Padi, Megha; Litovchick, Larisa; DeCaprio, James A.

    2011-01-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like \\(cyclin B, CDC2\\) and \\(CDC25C\\) are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in \\(G_0/G_1\\). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and...

  16. Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients

    Institute of Scientific and Technical Information of China (English)

    WANG Li-jia; BAI Yu; BAO Zhao-shi; CHEN Yan; YAN Zhuo-hong; ZHANG Wei; ZHANG Quan-geng

    2013-01-01

    Background Glioblastoma is the most common and lethal cancer of the central nervous system.Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies,but the effects of these methylation abnormalities on glioblastomas are still largely unclear.Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment.To better understand the relationship between CpG island methylation status and patient outcome,this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets.Methods We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients.Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM.Survival curves were calculated according to the Kaplan-Meier method,and differences between curves were assessed using the log-rank test.Then,we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors.Results Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P<0.05).Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P <0.05).This abnormality is also confirmed in glioblastoma cell lines (U87 and U251).Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P=0.013).Conclusions Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients.TES might represent a valuable prognostic marker

  17. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    Science.gov (United States)

    Wennier, Sonia T; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  18. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression.

    Science.gov (United States)

    DeCaprio, J A

    2014-07-31

    The study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. Although much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al., doi:10.1038/onc.2013.426, demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle. Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and can prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor-risk cervical cancers. PMID:24166507

  19. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression

    Science.gov (United States)

    DeCaprio, James A.

    2014-01-01

    Study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. While much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al. demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle (1). Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor risk cervical cancers. PMID:24166507

  20. Methylation state of the EDA gene promoter in Chinese X-linked hypohidrotic ectodermal dysplasia carriers.

    Directory of Open Access Journals (Sweden)

    Wei Yin

    Full Text Available INTRODUCTION: Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED which is caused by genetic ectodysplasin A (EDA deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom. METHODS: A large Chinese XLHED family was reported and the entire coding region and exon-intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers' tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system. RESULTS: A known frameshift mutation (c.573-574 insT was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI, 18 (78.26% carriers were hypermethylated at these 4 sites. CONCLUSION: Chinese XLHED carriers often have a hypermethylated EDA promoter.

  1. LINKAGE MAPPING OF CANDIDATE GENES FOR INDUCED RESISTANCE AND GROWTH PROMOTION BY Trichoderma koningiopsis (Th003 IN TOMATO Solanum lycopersicum

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    Cotes Prado Alba Marina

    2011-08-01

    Full Text Available Induced systemic resistance (ISR is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum is inoculated with several strains of Trichoderma ssp. This study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis Th003. Forty-nine candidate genes previously identified on tomato plants treated with Th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named “TOMATO EXPEN 2000”. The location of six unigenes was similar to the location of resistance gene analogs (RGAs, defense related ESTs and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.

  2. Linkage mapping of candidate genes for induce resistance and growth promotion by trichoderma koningiopsis (th003) in tomato solanum lycopersicum

    International Nuclear Information System (INIS)

    Induced systemic resistance (ISR) is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum) is inoculated with several strains of Trichoderma ssp. this study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis th003. Forty-nine candidate genes previously identified on tomato plants treated with th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named TOMATO EXPEN 2000. The location of six unigenes was similar to the location of resistance gene analogs (RGAS), defense related ests and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL) regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.

  3. Molecular analysis of the human SLC13A4 sulfate transporter gene promoter

    International Nuclear Information System (INIS)

    Highlights: ► Basal promoter activity of SLC13A4 −57 to −192 nt upstream of transcription initiation site. ► Human SLC13A4 5′-flanking region has conserved motifs with other placental species. ► Putative NFY, SP1 and KLF7 motifs in SLC13A4 5′-flanking region enhance transcription. -- Abstract: The human solute linked carrier (SLC) 13A4 gene is primarily expressed in the placenta where it is proposed to mediate the transport of nutrient sulfate from mother to fetus. The molecular mechanisms involved in the regulation of SLC13A4 expression remain unknown. To investigate the regulation of SLC13A4 gene expression, we analysed the transcriptional activity of the human SLC13A4 5′-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Basal transcriptional activity was identified in the region −57 to −192 nucleotides upstream of the SLC13A4 transcription initiation site. Mutational analysis of the minimal promoter region identified Nuclear factor Y (NFY), Specificity protein 1 (SP1) and Krüppel like factor 7 (KLF7) motifs which conferred positive transcriptional activity, as well as Zinc finger protein of the cerebellum 2 (ZIC2) and helix–loop–helix protein 1 (HEN1) motifs that repressed transcription. The conserved NFY, SP1, KLF7, ZIC2 and HEN1 motifs in the SLC13A4 promoter of placental species but not in non-placental species, suggests a potential role for these putative transcriptional factor binding motifs in the physiological control of SLC13A4 mRNA expression

  4. Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

    International Nuclear Information System (INIS)

    The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the at

  5. Enhanced levels of the apoptotic BAX/BCL-2 ratio in children with acute lymphoblastic leukemia and high-risk features

    Directory of Open Access Journals (Sweden)

    Maria Kaparou

    2013-01-01

    Full Text Available It has been suggested that leukemia is characterized by an impaired balance between the proliferation of blood cells and their capacity to undergo apoptosis. The aim of this study was to examine the expression of key molecules related to apoptosis (BCL-2, BAX, FAS, FAS-L in children with acute lymphoblastic leukemia (ALL. Measurement of BCL-2 and BAX mRNA was performed by quantitative real-time PCR, and membrane expression of FAS and FAS-L was assessed by flow cytometry in bone marrow mononuclear cells, both at diagnosis and at remission following induction chemotherapy. At diagnosis, increased levels of the apoptotic BAX/BCL-2 ratio were observed in children older than 10 years and with higher white blood cell counts. A DNA index < 1.16 was associated with increased BAX/BCL-2, both at diagnosis and at remission, and the del(9p chromosome abnormality with increased BAX/BCL-2 at remission. The expression of the apoptotic receptor FAS was significantly higher at remission compared to diagnosis, which might reflect enhanced sensitivity of the leukemic clone to apoptosis and response to treatment. Altogether, our results highlight the association of apoptosis-related genes with clinical and cytogenetic prognostic parameters in pediatric ALL. A better understanding of the mechanisms and regulation of apoptosis should enable the design of novel targeted therapies for these patients.

  6. The 14-3-3σ gene promoter is methylated in both human melanocytes and melanoma

    International Nuclear Information System (INIS)

    Recent evidence demonstrates that 14-3-3σ acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3σ has been largely overlooked in melanoma. The methylation status of 14-3-3σ CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3σ mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3σ gene expression. 14-3-3σ is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3σ gene expression and the active induction of 14-3-3σ mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3σ was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3σ gene. 14-3-3σ is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3σ is a rare event in melanoma, indicating 14-3-3σ might have a tentative role in the pathogenesis of melanoma

  7. In Vitro Cultured Rat Islets Express Genes That both Prevent and Promote Angiogenesis

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    Tillmar L

    2004-03-01

    Full Text Available CONTEXT: Endogenous pancreatic islets are supported by a dense sinusoidal capillary system which is disrupted following isolation and culture in vitro. A rapid and accurate revascularization is therefore crucial for the survival and functioning of the transplanted islet. Although a blood flow is established in islet grafts within 1-2 weeks, these islets show poor development of intra-islet capillaries. To improve the revascularization process and the arrangement of the new blood vessels, islet production of the factors governing these processes needs to be further characterized. OBJECTIVE: To study the expression of factors which regulate angiogenesis in cultured rat islets. DESIGN AND MAIN OUTCOME MEASURES: Rat islets were isolated and cultured for one week. After 6 hours of exposure to normoxic (21% O2 or hypoxic (1% O2 conditions, mRNA expression was evaluated by the GEArray Angiogenesis 1 and 2 systems. The expression of the vascular endothelial growth factor (VEGF, the tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1 (Tie1 and acidic fibroblast growth factor (aFGF, was further evaluated by semi-quantitative RT-PCR. RESULTS: We found the expression of 19 genes that code for factors either promoting or preventing angiogenesis. Only VEGF and Tie1 were upregulated in response to hypoxia. CONCLUSION: Hypoxia-induced islet vascularization may involve VEGF and Tie-induced signaling events. The results also show that cultured islets express genes which prevent angiogenesis concurrently with genes coding for factors stimulating angiogenesis. The balance between these factors is probably of vital importance for the revascularization process in transplanted islets. Thus, pharmacologic or genetic attenuation of islet-derived angiostatic factors may prove beneficial in promoting islet revascularization in future transplantation trials.

  8. The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit.

    Science.gov (United States)

    Romera-Branchat, Maida; Ripoll, Juan José; Yanofsky, Martin F; Pelaz, Soraya

    2013-01-01

    The Arabidopsis fruit forms a seedpod that develops from the fertilized gynoecium. It is mainly comprised of an ovary in which three distinct tissues can be differentiated: the valves, the valve margins and the replum. Separation of cells at the valve margin allows for the valves to detach from the replum and thus dispersal of the seeds. Valves and valve margins are located in lateral positions whereas the replum is positioned medially and retains meristematic properties resembling the shoot apical meristem (SAM). Members of the WUSCHEL-related homeobox family have been involved in stem cell maintenance in the SAM, and within this family, we found that WOX13 is expressed mainly in meristematic tissues including the replum. We also show that wox13 loss-of-function mutations reduce replum size and enhance the phenotypes of mutants affected in the replum identity gene RPL. Conversely, misexpression of WOX13 produces, independently from BP and RPL, an oversized replum and valve defects that closely resemble those of mutants in JAG/FIL activity genes. Our results suggest that WOX13 promotes replum development by likely preventing the activity of the JAG/FIL genes in medial tissues. This regulation seems to play a role in establishing the gradient of JAG/FIL activity along the medio-lateral axis of the fruit critical for proper patterning. Our data have allowed us to incorporate the role of WOX13 into the regulatory network that orchestrates fruit patterning. PMID:22946675

  9. Negative regulation of human parathyroid hormone gene promoter by vitamin D3 through nuclear factor Y

    International Nuclear Information System (INIS)

    The negative regulation of the human parathyroid hormone (PTH) gene by biologically active vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) was studied in rat pituitary GH4C1 cells, which express factors needed for the negative regulation. We report here that NF-Y binds to sequences downstream of the site previously reported to bind the vitamin D receptor (VDR). Additional binding sites for NF-Y reside in the near vicinity and were shown to be important for full activity of the PTH gene promoter. VDR and NF-Y were shown to exhibit mutually exclusive binding to the VDRE region. According to our results, sequestration of binding partners for NF-Y by VDR also affects transcription through a NF-Y consensus binding element in GH4C1 but not in ROS17/2.8 cells. These results indicate that 1,25(OH)2D3 may affect transcription of the human PTH gene both by competitive binding of VDR and NF-Y, and by modulating transcriptional activity of NF-Y

  10. Analysis of promoter activity of members of the PECTATE LYASE-LIKE (PLL gene family in cell separation in Arabidopsis

    Directory of Open Access Journals (Sweden)

    van Nocker Steven

    2010-07-01

    Full Text Available Abstract Background Pectate lyases depolymerize pectins by catalyzing the eliminative cleavage of α-1,4-linked galacturonic acid. Pectate lyase-like (PLL genes make up among the largest and most complex families in plants, but their cellular and organismal roles have not been well characterized, and the activity of these genes has been assessed only at the level of entire organs or plant parts, potentially obscuring important sub-organ or cell-type-specific activities. As a first step to understand the potential functional diversity of PLL genes in plants and specificity of individual genes, we utilized a reporter gene approach to document the spatial and temporal promoter activity for 23 of the 26 members of the Arabidopsis thaliana (Arabidopsis PLL gene family throughout development, focusing on processes involving cell separation. Results Numerous PLL promoters directed activity in localized domains programmed for cell separation, such as the abscission zones of the sepal, petal, stamen, and seed, as well as the fruit dehiscence zone. Several drove activity in cell types expected to facilitate separation, including the style and root endodermal and cortical layers during lateral root emergence. However, PLL promoters were active in domains not obviously programmed for separation, including the stipule, hydathode and root axis. Nearly all PLL promoters showed extensive overlap of activity in most of the regions analyzed. Conclusions Our results document potential for involvement of PLL genes in numerous aspects of growth and development both dependent and independent of cell separation. Although the complexity of the PLL gene family allows for enormous potential for gene specialization through spatial or temporal regulation, the high degree of overlap of activity among the PLL promoters suggests extensive redundancy. Alternatively, functional specialization might be determined at the post-transcriptional or protein level.

  11. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  12. Association between promoter polymorphisms of OPN gene and cancer risk: a meta-analysis

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    Liu JW

    2015-12-01

    Full Text Available Jingwei Liu,1–2 Caiyun He,1–2 Quan Yuan,1–2 Zhenning Wang,1–2 Chengzhong Xing,1–2 Yuan Yuan1–2 1Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University, 2Key Laboratory of Cancer Etiology and Prevention, China Medical University, Liaoning Provincial Education Department, Shenyang, People’s Republic of China Background: Results of the association between polymorphisms of osteopontin (OPN gene promoter region and risk of cancer were inconclusive. The aim of this meta-analysis was to elucidate whether OPN promoter polymorphisms were associated with cancer risk.Methods: Electronic databases including PubMed, Web of Science, and Chinese National Knowledge Infrastructure were systematically searched. Odd ratios (ORs and their 95% confidential interval (CI were used to assess the strength of association between OPN promoter polymorphisms and cancer risks.Results: Nine studies were finally included in this meta-analysis. For OPN rs17524488 polymorphism, carriers of GG or -/G genotype were significantly associated with increased cancer risk compared with wild-type -/- carriers, respectively (GG vs -/-: OR =1.40, 95% CI =1.03–1.91, P=0.033; -/G vs -/-: OR =1.22, 95% CI =1.07–1.40, P=0.002. Additionally, G allele was significantly associated with increased cancer risk compared with (- allele (OR =1.21, 95% CI =1.04–1.40, P=0.016. However, no significant association was observed of OPN rs11730582 polymorphism and cancer risk (CC vs TT: OR =0.98, 95% CI =0.49–1.97, P=0.964; CT vs TT: OR =0.88, 95% CI =0.54–1.43, P=0.610.Conclusion: Carriers of GG or -/G genotype of OPN promoter rs17524488 (-156-/G polymorphism might be associated with increased risk of cancer compared with wild-type -/- carriers, respectively. However, no significant association was observed between OPN promoter rs11730582 (-443C/T polymorphism and risk of cancer. Keywords: OPN

  13. The microarray expression analysis identifies BAX as a mediator of beta-carotene effects on apoptosis.

    Science.gov (United States)

    Bodzioch, Marek; Dembinska-Kiec, Aldona; Hartwich, Jadwiga; Lapicka-Bodzioch, Katarzyna; Banas, Agnieszka; Polus, Anna; Grzybowska, Joanna; Wybranska, Iwona; Dulinska, Joanna; Gil, Dorota; Laidler, Piotr; Placha, Wojciech; Zawada, Magdalena; Balana-Nowak, Agnieszka; Sacha, Tomasz; Kiec-Wilk, Beata; Skotnicki, Aleksander; Moehle, Christoph; Langmann, Thomas; Schmitz, Gerd

    2005-01-01

    Beta-carotene is a ubiquitous compound rich in foods. However, there are conflicting reports regarding its role in carcinogenesis. We performed a microarray expression analysis in normal [human umbilical vein endothelial cells (HUVECs)] and neoplastic (melanoma A375 and myelomonocytic leukemia U937) actively proliferating cells and found evidence that beta-carotene stimulated vital cellular functions in the former and suppressed them in the latter. These differential effects correlated with the expression of the proapoptotic BCL2-associated X protein (BAX), which was downregulated in HUVECs and upregulated in the two neoplastic cell lines. The quantitative expression analysis using real-time polymerase chain reaction largely confirmed the inhibition of B-cell CLL/lymphoma 2 (BCL2) pathway-mediated apoptosis in HUVECs and its activation in melanoma and leukemic cells. The assays for apoptosis, detecting DNA breaks and caspase activation, showed consistent proapoptotic and antiapoptotic effects in U937 and HUVEC lines, respectively. However, beta-carotene-induced expression changes of BAX and other BCL2 pathway genes did not lead to the predicted induction of apoptosis in the A375 cells. PMID:15860445

  14. Human PSENEN and U2AF1L4 genes are concertedly regulated by a genuine bidirectional promoter.

    Science.gov (United States)

    Didych, D A; Shamsutdinov, M F; Smirnov, N A; Akopov, S B; Monastyrskaya, G S; Uspenskaya, N Y; Nikolaev, L G; Sverdlov, E D

    2013-02-15

    Head-to-head genes with a short distance between their transcription start sites may constitute up to 10% of all genes in the genomes of various species. It was hypothesized that this intergenic space may represent bidirectional promoters which are able to initiate transcription of both genes, but the true bidirectionality was proved only for a few of them. We present experimental evidence that, according to several criteria, a 269 bp region located between the PSENEN and U2AF1L4 human genes is a genuine bidirectional promoter regulating a concerted divergent transcription of these genes. Concerted transcription of PSENEN and U2AF1L4 can be necessary for regulation of T-cell activity. PMID:23246698

  15. Isolation of Promoters and Fragments of Genes Controlling Endosperm Development Without Fertilization in Arabidopsis and Engineering of the Antisense Constructions

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    Grigory A. Gerashchenkov

    2015-06-01

    Full Text Available Apomixis is asexual seed reproduction without both meiosis and fertilization based on the complex developmental processes such as apomeiosis, parthenogenesis and specific endosperm development. This investigation is aimed at engineering of apomixis in Arabidopsis thaliana with sexual seed reproduction. The fragments of known genes of endosperm formation MEA, FIE, FIS2 and gene of apomeiosis DYAD (as control were isolated using Q5 high fidelity DNA polymerase. These gene fragments of interest at the antisense orientation were fused with isolated constitutive and meiosis specific promoters of Arabidopsis at NcoI sites. The fused promoter-gene fragment modules were cloned in pCambia1301 at SalI cites. The engineered constructions will be used for the floral dip transformation of Arabidopsis and down regulation of these genes at engineering of apomixis.

  16. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  17. ModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants

    Directory of Open Access Journals (Sweden)

    Whelan James

    2006-04-01

    Full Text Available Abstract Background Uncovering the key sequence elements in gene promoters that regulate the expression of plant genomes is a huge task that will require a series of complementary methods for prediction, substantial innovations in experimental validation and a much greater understanding of the role of combinatorial control in the regulation of plant gene expression. Results To add to this larger process and to provide alternatives to existing prediction methods, we have developed several tools in the statistical package R. ModuleFinder identifies sets of genes and treatments that we have found to form valuable sets for analysis of the mechanisms underlying gene co-expression. CoReg then links the hierarchical clustering of these co-expressed sets with frequency tables of promoter elements. These promoter elements can be drawn from known elements or all possible combinations of nucleotides in an element of various lengths. These sets of promoter elements represent putative cis-acting regulatory elements common to sets of co-expressed genes and can be prioritised for experimental testing. We have used these new tools to analyze the response of transcripts for nuclear genes encoding mitochondrial proteins in Arabidopsis to a range of chemical stresses. ModuleFinder provided a subset of co-expressed gene modules that are more logically related to biological functions than did subsets derived from traditional hierarchical clustering techniques. Importantly ModuleFinder linked responses in transcripts for electron transport chain components, carbon metabolism enzymes and solute transporter proteins. CoReg identified several promoter motifs that helped to explain the patterns of expression observed. Conclusion ModuleFinder identifies sets of genes and treatments that form useful sets for analysis of the mechanisms behind co-expression. CoReg links the clustering tree of expression-based relationships in these sets with frequency tables of promoter

  18. Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

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    Darja Henseler

    2011-10-01

    Full Text Available Normal 0 21 false false false MicrosoftInternetExplorer4 The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC, which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly. Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1. Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.

  19. Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea.

    Science.gov (United States)

    Zhu, Changfu; Yang, Qingjie; Ni, Xiuzhen; Bai, Chao; Sheng, Yanmin; Shi, Lianxuan; Capell, Teresa; Sandmann, Gerhard; Christou, Paul

    2014-04-01

    Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea. PMID:24256196

  20. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  1. ACC interleukin-10 gene promoter haplotype as a breast cancer risk factor predictor among Jordanian females

    Directory of Open Access Journals (Sweden)

    Atoum MF

    2016-06-01

    Full Text Available Manar Fayiz Atoum Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, Zarqa, Jordan Introduction: Interleukin-10 (IL-10 is a multifactorial cytokine with a complex biological role in breast cancer. The aims of this study were to investigate any association between IL-10 gene promoter polymorphisms, 1082A>/G, -819T>C, and -592A>C, or haplotypes and breast cancer risk among Jordanian women and to evaluate any association between the most common haplotype with clinicopathological features of breast cancer.Patients and methods: A total of 202 breast cancer patients and 210 age-matched healthy control subjects were genotyped for -1082A/G, -819T/C, and -592A/C single nucleotide polymorphisms in the promoter region of the IL-10 gene by polymerase chain reaction-restriction fragment length polymorphism. Study patients and control subjects were recruited from Prince Hamzah Hospital, Amman, Jordan (2012–2013. Ethical approval and signed consent forms were signed by all participants. DNA was extracted, and polymerase chain reaction fragments were amplified and restriction digested by MnII, MaeIII, and RsaI.Results: This study showed no statistically significant difference between -1082A/G, -819T/C, and -592A/C IL-10 genotypes or alleles among breast cancer patients or controls. Four different haplotypes ATA, ACC, GTA, and ACA within the IL-10 promoter gene were determined among both breast cancer and control groups. The most frequent haplotype was ACC among breast cancer patients and controls (41.6% and 40.7%, respectively. No statistical differences in these haplotypes among breast cancer patients or controls were determined. Analysis of the most common ACC haplotype showed statistical difference in positive estrogen receptor (P=0.022, positive progesterone receptor (P=0.004, cancer grade (P=0.0001, and cancer stage (P=0.009 among the ACC haplotype compared to non-ACC haplotype.Conclusion: To our

  2. Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

    Energy Technology Data Exchange (ETDEWEB)

    Palsamy, Periyasamy [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Ayaki, Masahiko [Shizuoka National Hospital, Saitama (Japan); Elanchezhian, Rajan [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Shinohara, Toshimichi, E-mail: tshinohara@unmc.edu [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer We found significant Keap1 promoter demethylation in diabetic cataractous lenses. Black-Right-Pointing-Pointer Demethylation of Keap1 gene upregulated the expression of Keap1 mRNA and protein. Black-Right-Pointing-Pointer Elevated levels of Keap1 are known to decrease the levels of Nrf2. Black-Right-Pointing-Pointer Thereby, the levels of antioxidant enzymes are suppressed by decreased Nrf2 level. -- Abstract: Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2 Prime deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which

  3. Spatial and temporal changes in Bax subcellular localization during NPe6-PDT-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Xing, Da; Chen, Wei R.; Wan, Qingling; Zhou, Feifan

    2008-02-01

    Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Bax, a member of the Bcl-2 family of proteins, is an essential regulator of apoptosis. Bax is normally found in the cytosol of healthy cells, and translocates to mitochondria in response to many apoptotic stimuli. In this study, using real-time single-cell analysis, we have investigated the kinetics of Bax distribution during NPe6-induced apoptosis in ASTC-a-1 cells. In order to monitor Bax subcellular distribution, cells were stained with GFP-Bax and Mito Tracker Red. The results show that Bax redistribution occurred at about 170 min after treated with NPe6-PDT, and then sequestered into clusters associated with the mitochondira within 30 min. Our data clearly showed the spatial and temporal changes in Bax distribution in living cells during NPe6-induced apoptosis.

  4. A Meta-analysis of Association between MGMT GenePromoter Methylation and Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Nianzhen FANG

    2014-08-01

    Full Text Available Backgroud and objective DNA promoter methylation of the tumor suppressor genes was one of the key mechanism for gene silence. The aim of this study is to investigate the difference of MGMT gene promoter methylation rate in tumor tissue and autologous controls (serum, normal lung tissue and bronchial lavage fluid in patients with non-small cell lung cancer (NSCLC. Methods The databases of Medline, EMBSE, CNKI and Wanfang were searched for selection of published articles of MGMT gene promoter methylation and non-small cell lung carcinoma risk. The pooled odds ratio (OR and percentage of MGMT for lung cancer tissue of NSCLC patients compared with normal lung tissue, plasma and the bronchial lavage fluid were pooled. Results 15 articles of association between MGMT gene promoter methylation and non small cell lung carcinoma risk were included in this meta-analysis. The combined results demonstrated the methylation rate of MGMT in NSCLC cancer tissue was 38% (95%CI: 23%-53%. For normal lung tissue, plasma and the bronchial lavage fluid were 16% (95%CI: 5%-27%, 23% (95%CI: 10%-34% and 39% (95%CI: 23%-55% respectively. The OR in cancer tissue was much higher than that in normal lung tissue and plasma odds ratio (OR 3.98 (95%CI: 2.71-5.84, P0.05. Conclusions Mehtylation rate in MGMT gene promoter of cancer tissue in NSCLC patients was much higher than that in normal lung tissue and plasma, which showed a close association between NSCLC cancer and MGMT gene promoter methylation.

  5. Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum.

    Science.gov (United States)

    Giacani, Lorenzo; Lukehart, Sheila; Centurion-Lara, Arturo

    2007-11-01

    In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct. PMID:17683506

  6. Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245.

    Science.gov (United States)

    Pothier, Joël F; Wisniewski-Dyé, Florence; Weiss-Gayet, Michèle; Moënne-Loccoz, Yvan; Prigent-Combaret, Claire

    2007-10-01

    Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1-3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant. PMID:17906157

  7. Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation.

    Directory of Open Access Journals (Sweden)

    Ilse M Beck

    Full Text Available Compound A possesses glucocorticoid receptor (GR-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1, upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.

  8. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

    OpenAIRE

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A.

    2012-01-01

    Gene expression is tightly regulated during cell cycle progression. DeCaprio and colleagues now provide a comprehensive model of the MuvB complex's dynamic control of cell cycle gene expression from quiescence through mitosis. They find that MuvB sequentially recruits B-Myb and then FoxM1 to gene promoters during G2/M. The study provides a definitive description of MuvB's central role as a part of three distinct transcription complexes in the coordinated expression of a large number of genes ...

  9. DNA methylation dynamics in the rat EGF gene promoter after partial hepatectomy

    Directory of Open Access Journals (Sweden)

    Deming Li

    2014-06-01

    Full Text Available Epidermal growth factor (EGF, a multifunctional growth factor, is a regulator in a wide variety of physiological processes. EGF plays an important role in the regulation of liver regeneration. This study was aimed at investigating the methylation level of EGF gene throughout liver regeneration. DNA of liver tissue from control rats and partial hepatectomy (PH rats at 10 time points was extracted and a 354 bp fragment including 10 CpG sites from the transcription start was amplified after DNA was modified by sodium bisulfate. The result of sequencing suggested that methylation ratio of four CpG sites was found to be significantly changed when PH group was compared to control group, in particular two of them were extremely striking. mRNA expression of EGF was down-regulated in total during liver regeneration. We think that the rat EGF promoter region is regulated by variation in DNA methylation during liver regeneration.

  10. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

    Science.gov (United States)

    Mohun, T; Garrett, N; Treisman, R

    1987-03-01

    Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution. PMID:3582369

  11. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons

    DEFF Research Database (Denmark)

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T;

    2004-01-01

    -expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists......We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures....... Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co...

  12. 8周中等强度低负荷量训练对老龄雌性大鼠骨骼肌Bax和Bcl-2蛋白及SIRT1/SIRT3信号轴基因表达的影响%Effects of 8-week medium intensity low load training on proteins Bax and Bcl-2 and the gene expression of signal axis SIRT1/SIRT3 of skeletal muscle of aged female rats

    Institute of Scientific and Technical Information of China (English)

    李方晖; 肖琳; 覃飞; 刘承宜

    2014-01-01

    In order to observe the effects of 8-week medium intensity low load training on the levels of proteins Bax and Bcl-2 and the gene messenger RNA (mRNA) expression of axis sirtuin 1 (SIRT1)/sirtuin 3 (SIRT3) of gastrocne-mius of aged rats, the authors divided 16 18-month old female SD rats randomly into a control group and an exercise group, each of which contained 8 rats, let the rats in the exercise group do an aerobic exercise on a treadmill for con-secutive 8 weeks, at a speed of 15 km/h (with 60%~75%VO2max), 15 minutes a day, 5 days a week, let the rats in the control group live freely, in 24 hours after rat exercising at the end of week 8, killed the rats, measured gastrocnemius index, measured the levels of proteins Bax and Bcl-2 of gastrocnemius by means of Western blot analysis, measured the mRNA levels of SIRT3, SIRT1, manganese superoxide dismutase (MnSOD), Caspase 3, peroxisome prolifera-tor-activated receptor-γcoactivator-1 (PGC-1α), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1) by means of RT-PCR, and revealed the following findings:as for the rats in the exercise group, their gastrocnemius mass and gastrocnemius index increased significantly (P<0.05 and P<0.01 respectively), their protein Bax level decreased significantly (P<0.05), their protein Bcl-2 level and Bcl-2/Bax ratio increased significantly (P<0.05);their mRNA levels of SIRT3, SIRT1, PGC-1α, NRF1, TFAM and MnSOD increased significantly (P<0.05), their mRNA level of Caspase-3 decreased significantly (P<0.05). The said findings indicated the followings:medium intensity low load training could delay the changing of muscle cell apoptosis signal of aged rats; the homeostatic mechanism mediated by axis SIRT1/SIRT3 played an important role in medium intensity low load training increasing the mitochondria refreshing rate and antioxidase level of skeletal muscle of aged rats.%观察8周中等强度低负荷量训练对老龄雌性大鼠腓肠肌Bax和Bcl-2

  13. Promoter analysis of intestinal genes induced during iron-deprivation reveals enrichment of conserved SP1-like binding sites

    Directory of Open Access Journals (Sweden)

    Hu Zihua

    2007-11-01

    Full Text Available Abstract Background Iron-deficiency leads to the induction of genes related to intestinal iron absorption and homeostasis. By analyzing a large GeneChip® dataset from the rat intestine, we identified a large cluster of 228 genes that was induced by iron-deprivation. Only 2 of these genes contained 3' iron-response elements, suggesting that other regulation including transcriptional may be involved. We therefore utilized computational methods to test the hypothesis that some of the genes within this large up-regulated cluster are co-ordinately regulated by common transcriptional mechanisms. We thus identified promoters from the up-regulated gene cluster from rat, mouse and human, and performed enrichment analyses with the Clover program and the TRANSFAC database. Results Surprisingly, we found a strong statistical enrichment for SP1 binding sites in our experimental promoters as compared to background sequences. As the TRANSFAC database cannot distinguish among SP/KLF family members, many of which bind similar GC-rich DNA sequences, we surmise that SP1 or an SP1-like factor could be involved in this response. In fact, we detected induction of SP6/KLF14 in the GeneChip® studies, and confirmed it by real-time PCR. Additional computational analyses suggested that an SP1-like factor may function synergistically with a FOX TF to regulate a subset of these genes. Furthermore, analysis of promoter sequences identified many genes with multiple, conserved SP1 and FOX binding sites, the relative location of which within orthologous promoters was highly conserved. Conclusion SP1 or a closely related factor may play a primary role in the genetic response to iron-deficiency in the mammalian intestine.

  14. The Placental Gene PEG10 Promotes Progression of Neuroendocrine Prostate Cancer

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    Shusuke Akamatsu

    2015-08-01

    Full Text Available More potent targeting of the androgen receptor (AR in advanced prostate cancer is driving an increased incidence of neuroendocrine prostate cancer (NEPC, an aggressive and treatment-resistant AR-negative variant. Its molecular pathogenesis remains poorly understood but appears to require TP53 and RB1 aberration. We modeled the development of NEPC from conventional prostatic adenocarcinoma using a patient-derived xenograft and found that the placental gene PEG10 is de-repressed during the adaptive response to AR interference and subsequently highly upregulated in clinical NEPC. We found that the AR and the E2F/RB pathway dynamically regulate distinct post-transcriptional and post-translational isoforms of PEG10 at distinct stages of NEPC development. In vitro, PEG10 promoted cell-cycle progression from G0/G1 in the context of TP53 loss and regulated Snail expression via TGF-β signaling to promote invasion. Taken together, these findings show the mechanistic relevance of RB1 and TP53 loss in NEPC and suggest PEG10 as a NEPC-specific target.

  15. Cloning and functional analysis of promoters of three GnRH genes in a cichlid

    International Nuclear Information System (INIS)

    Mechanisms regulating gonadotropin-releasing hormone (GnRH) types, a key molecule for reproductive physiology, remain unclear. In the present study, we cloned the promoters of GnRH1, GnRH2, and GnRH3 genes in the tilapia, Oreochromis niloticus; and found putative binding sites for glucocorticoid receptors, Sp1, C/EBP, GATA, and Oct-1, but not for androgen receptors in all three GnRH promoters using computer analysis. The presence of binding sites for progesterone receptors in GnRH1, estrogen receptors in GnRH1 and GnRH2, and thyroid hormone receptors in GnRH1 and GnRH3 suggests direct action of steroid hormones on GnRH types. Our observation of SOX and LINE-like sequences exclusively in GnRH1, COUP in GnRH2, and retinoid X receptors in GnRH3 suggests their role in sexual differentiation, midbrain segmentation, and visual cue integration, respectively. Thus, the characteristic binding sites for nuclear receptors and transcription factors support the notion that each GnRH type is regulated differently and has distinct physiological roles

  16. The stress-regulated transcription factor CHOP promotes hepatic inflammatory gene expression, fibrosis, and oncogenesis.

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    Diane DeZwaan-McCabe

    Full Text Available Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC. Although these conditions also lead to integrated stress response (ISR or unfolded protein response (UPR activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.

  17. Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

    Directory of Open Access Journals (Sweden)

    C.G. Barbosa

    2010-08-01

    Full Text Available Fetal hemoglobin (HbF, encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104 and an Afro-descendant one (N = 98, to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

  18. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    Directory of Open Access Journals (Sweden)

    Recillas-Targa Félix

    2011-06-01

    Full Text Available Abstract Background Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb gene promoter in different tumoral cell lines. Methods To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. Results We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. Conclusions This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.

  19. Characterization of a REST-Regulated Internal Promoter in the Schizophrenia Genome-Wide Associated Gene MIR137.

    Science.gov (United States)

    Warburton, Alix; Breen, Gerome; Rujescu, Dan; Bubb, Vivien J; Quinn, John P

    2015-05-01

    MIR137 has been identified as a candidate gene for schizophrenia from genome-wide association studies via association with an intronic single nucleotide polymorphism (SNP), rs1625579. The location of the SNP suggests one mechanism in which transcriptional or posttranscriptional regulation of miR-137 expression could underlie schizophrenia. We identified and validated a novel promoter of the MIR137 gene adjacent to miR-137 itself which can direct the expression of distinct mRNA isoforms encoding miR-137. Analysis of both endogenous gene expression and reporter gene assays determined that this internal promoter is regulated by repressor element-1 silencing transcription factor (REST), which has previously been associated with pathways linked to schizophrenia. Distinct isoforms of REST mediate differential expression at this locus, suggesting the relative levels of these isoforms are important for miR-137 expression profiles. The internal promoter contains a variable number tandem repeat (VNTR) domain adjacent to the pre-miR-137 sequence. The reporter gene activity directed by this promoter was modified by the genotype of the VNTR. Differential expression was also observed in response to cocaine, which is known to regulate the REST pathway in SH-SY5Y cells. Our data support the hypothesis that a "gene × environment" interaction could modify the level of miR-137 expression via this internal promoter and that the genotype of the VNTR could modulate transcriptional responses. We demonstrate that this promoter region is not in disequilibrium with rs1625579 and therefore would supply a distinct pathway to potentially alter miR-137 levels in response to environmental cues. PMID:25154622

  20. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    International Nuclear Information System (INIS)

    Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing

  1. CLONING PROMOTER OF HUMAN SATB1 GENE AND EFFECT OF ATRA AND CoCl2 ON ITS ACTIVITY

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the structure and activity of SATB1 promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5' untranslated sequence from human SATB1 gene, called pGL3-SP2946-luc, pGL3-SP1718-luc and pGL3-SP751-luc, and transfected into Jurkat T, K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of ATRA and CoCl2 treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-luc kept on the higher lever in Jurkat T, K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCl2 on SATB1 's mRNA expression and the relative luciferase expression from pGL3-SP751-luc in U937 cell was down-regulated obviously by ATRA and CoCl2 in the concentration- and time-dependent manners. Conclusion SATB1 promoter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 ~ - 9bp of 5' untranslated region of human SATB1 gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.

  2. Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

    OpenAIRE

    Bhat, R. A.; Metz, B.; Thimmappaya, B

    1983-01-01

    The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitutio...

  3. Brain-derived neurotrophic factor gene transfection promotes neuronal repair and neurite regeneration after diffuse axonal injury

    Institute of Scientific and Technical Information of China (English)

    Yin Yu; Chao Du; Xingli Zhao; Jiajia Shao; Qiang Shen; Tao Jiang; Wei Wu; Dong Zhu; Yu Tian; Yongchuan Guo

    2011-01-01

    This study sought to assess the potential of brain-derived neurotrophic factor (BDNF) to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.

  4. Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Junko [Kyoto Univ., Agricultural Chemistry Dept., Kyoto (Japan); Ozeki, Yoshihiro [Tokyo Univ. of Agriculture and Technology, Biotechnology Dept., Tokyo (Japan); Yoshida, Kazuichi [Kyoto Univ., Botany Dept., Kyoto (Japan)

    1997-10-01

    The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPALLI) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALI promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALI promoters and to CaMV35S promoter fusion with various parts 5` - upstream of the gDcPALI promoter. The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant responses. However, gDCPALI promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALI promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the downstream below -190 (UVB and C responsive). The overall UVB response of the gDcPALI full-length promoter is explained as the result of interaction of these three components. (Author).

  5. Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells

    International Nuclear Information System (INIS)

    The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPALLI) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALI promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALI promoters and to CaMV35S promoter fusion with various parts 5' - upstream of the gDcPALI promoter. The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant responses. However, gDCPALI promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALI promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the downstream below -190 (UVB and C responsive). The overall UVB response of the gDcPALI full-length promoter is explained as the result of interaction of these three components. (Author)

  6. Mutational Analysis of hOGG1 Gene Promoter in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xia LIU

    2011-03-01

    Full Text Available Background and objective 8-hydroxygumine DNA glycosylase 1 (OGG1 is a DNA repair enzyme, which can repair damaged DNA by excising 8-dihydro-8-oxoguanine (8-OH-G. Polymorphisms in human OGG1 gene (hOGG1 may alter glycosylase activity, thereby affects its repair to the damaged DNA, resulting in contribution to carcinogenesis. However, an association of genetic variants of hOGG1 promoter with non-small cell lung cancer (NSCLC remains unclear. The present study aims to explore whether there are mutations in the promoter region of hOGG1 and the association of the potential genetic variants with NSCLC. Methods Forty lung cancer patients were enrolled from January, 2003 to December, 2005 in the first affiliated hospital of Soochow University. PCR-SSCP followed by direct sequencing were performed to detect mutations within the promoter region of the hOGG1 gene in NSCLC and corresponding paracancerous lung tissues. Results No abnormal mutation was found in the promoter region of the hOGG1 gene in 40 patients with NSCLC. However, a SNP rs159153 in hOGG1 was significantly associated with higher TNM stage (P=0.008. Moreover, lower frequency of lymph node metastasis was observed in smoker patients with NSCLC (P=0.034. Conclusion The SNP rs159153 in the promoter region of the hOGG1 gene and smoking history may effectively forebode the aggressiveness and metastatic potential of NSCLC.

  7. β-Glucan synthase gene overexpression and β-glucans overproduction in Pleurotus ostreatus using promoter swapping.

    Directory of Open Access Journals (Sweden)

    Ran Chai

    Full Text Available Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR, and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

  8. The Relationship between FHIT Gene Promoter Methylation and Lung Cancer Risk: 
a Meta-analysis

    Directory of Open Access Journals (Sweden)

    Yichang SUN

    2014-03-01

    Full Text Available Background and objective Tumor-suppressor gene promoter DNA methylation in tumor cells is associated with its reduced expression. FHIT (fragile histindine triad was one of the important tumor suppressor genes which was found hypermethylated in the promoter region in most of tumors. The aim of this study is to evaluate the relationship between FIHT gene promother methylation and lung cancer risk by meta-analysis. Methods By searching Pubmed, CNKI and Wanfang, the open published articles related to FHIT gene promoter methylation and lung carcinoma risk were collected. The odds ratio (OR and range of FHIT gene of cancer tissue of lung cancer patients compared with normal lung tissue, plasma and the bronchial lavage fluid were pooled by statistical software Stata 11.0. Results Eleven studies were finally included in this meta-analysis. The median methylation rate were Pmedian=40.0% (0-68.3%, Pmedian=8.7% (0-35.0%, Pmedian=33.3% (17.1%-38.3% and Pmedian=35.9% (31.1%-50.8% in cancer tissue, NLT, BALF and plasm respectively. The pooled results showed the methylation rate in tumor tissue was much higer than that of NLT OR=5.82 (95%CI: 3.74-9.06, P0.05 and plasma OR=1.41 (95%CI: 0.90-2.20, P>0.05. Conclusion Hypermethylation of FHIT gene promoter region was found more frequent in cancer tissue than that of NLT which may demonstrated association between lung cancer risk and FHIT gene promoter methylation.

  9. Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

    Science.gov (United States)

    Lee, Mon-Juan; Chen, Yuhsin; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chiang, Lan-Hsin; Chen, Tzu-Yu; Wang, Gwo-Jaw

    2013-12-01

    Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines. PMID:23794266

  10. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Law, Alice Y.S. [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong); Wong, Chris K.C., E-mail: ckcwong@hkbu.edu.hk [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong)

    2010-02-01

    Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1{alpha} dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

  11. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

    International Nuclear Information System (INIS)

    Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1α dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

  12. In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells

    OpenAIRE

    Mittal, Mukul K; Myers, Jeremy N.; Misra, Smita; Bailey, Charvann K.; Chaudhuri, Gautam

    2008-01-01

    The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628 bp fragment (−613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site...

  13. Relationship between Expression of the Human Alpha-Fetoprotein Gene and DNA Methylation Status of the Promoter Region

    Institute of Scientific and Technical Information of China (English)

    Lijun Chen; Wei Wang; Qiuyue Jin; Ruimin Wang; Wenliang Hu

    2006-01-01

    OBJECTIVE DNA methylation has been regarded as an important epigenetic signature reflecting the transcription state of DNA in cells. This study was to conducted to assess the relationship between human alpha-fetoprotein (AFP) gene expression and the DNA methylation status of the promoter region in three different cells, namely two human hepatocellular carcinoma (HCC) cell lines and normal human fibroblasts.METHODS Transcription of the AFP gene was verified by RT-PCR. After bisulphate treatment of DNA, the methods of MSP and BSP were used to analyze the methylation density and status within single DNA strands of two closely spaced CpG dinucleotides of the promoter region in the different cells.RESULTS RT-PCR analysis indicated that the expression of the AFP gene in HepG2 cells was significantly higher than in SMMC-7721 cells,and that the AFP gene was not expressed in normal human fibroblasts.By MSP and BSP we observed that the promoter region was demethylated in the AFP-high-expressing cell lines, and that the sites of -2,494 bp and -2,431 bp in the AFP genomic sequence can be used as detection sites for early tumorous diagnosis.CONCLUSION These results indicate that the DNA methylation state of the promoter region has a negative correlation with AFP gene expression.

  14. THE ABERRANT PROMOTER HYPERMETHYLATION PATTERN OF THE ANTI - ANGIOGENIC TSP1 GENE IN EPITHELIAL OVARIAN CARCINOMA: AN INDIAN STUDY

    Directory of Open Access Journals (Sweden)

    Ramesh

    2015-06-01

    Full Text Available PURPOSE: The promoter hypermethylation patterns of Thrombospodin - 1 gene in 50 EOC patients were studied and the methylation pattern was correlated with various clinic pathological parameters. METHODS: The promoter hypermethylation pattern of the TSP - 1 gene was assessed using nested PCR and Methylation specific PCR. STATISTICAL ANALYSIS: All the available data was statistically analyzed using the Chi square test or Fisher Exact Test on the SPSS software version 22.0 and a value <0.0 5 was considered statistically significant. RESULTS: Forty of the fifty ovarian carcinoma samples reported positive for methylation corresponding to a methylation frequency of 80%. A methylation frequency of 89.2%, 83.3% and 42.8% was observed in malignant , Low malignant potential (borderline and benign sample cohorts. CONCLUSION: From the results drawn from this study, it clearly shows that the anti angiogenic protein TSP - 1 is extensively hypermethylated in ovarian carcinoma and that it accumulates over t he progression of the disease from benign to malignant. As previous reports suggest that there is no evidence of mutation of this gene, promoter hypermethylation may be a crucial factor for the down regulation of the gene. Further by clubbing together the promoter hypermethylation pattern of TSP - 1 gene with hypermethylation patterns of other TSG may provide a better insight into the application of using methylation profiles of TSG as a biomarker in the detection of ovarian carcinoma.

  15. Adenovirus E1A gene autorepression: revertants of an E1A promoter mutation encode altered E1A proteins

    Energy Technology Data Exchange (ETDEWEB)

    Larsen, P.L.; Tibbetts, C.

    1987-12-01

    Revertants have been isolated from Ad3hr15, a mutant of human adenovirus type 3 that carries a defective E1A promoter. Transcription of these revertant E1A genes is restored - from nil for Ad3hr15 mutant to levels exceeding that of the wild-type virus. The mutant Ad3hr15 virus and the revertants all have an aberrant E1A promoter that contains two short tandem duplications of viral DNA sequence. The E1A gene-coding region of the mutant is the same as that for wild-type adenovirus type 3, whereas the revertants are characterized by short in-frame deletions within the 5' exon region of their E1A genes. Location of these reverting, second-site deletions is discussed in relation to E1A gene autoregulation and the evolved diversity of E1A-related oncogenic potential among different human adenoviruses.

  16. Mutational analysis of the promoter and the coding region of the 5-HT1A gene

    Energy Technology Data Exchange (ETDEWEB)

    Erdmann, J.; Noethen, M.M.; Shimron-Abarbanell, D. [Univ. of Bonn (Germany)] [and others

    1994-09-01

    Disturbances of serotonergic pathways have been implicated in many neuropsychiatric disorders. Serotonin (5HT) receptors can be subdivided into at least three major families (5HT1, 5HT2, and 5HT3). Five human 5HT1 receptor subtypes have been cloned, namely 1A, 1D{alpha}, 1D{beta}, 1E, and 1F. Of these, the 5HT1A receptor is the best characterized subtype. In the present study we sought to identify genetic variation in the 5HT1A receptor gene which through alteration of protein function or level of expression might contribute to the genetics of neuropsychiatric diseases. The coding region and the 5{prime} promoter region of the 5HT1A gene from 159 unrelated subjects (45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette`s syndrome, as well as 25 controls) were analyzed using SSCA. SSCA revealed the presence of two mutations both located in the coding region of the 5HT1A receptor gene. The first mutation is a rare silent C{r_arrow}T substitution at nucleotide position 549. The second mutation is characterized by a base pair substitution (A{r_arrow}G) at the first position of codon 28 and results in an amino acid exchange (Ile{r_arrow}Val). Since Val28 was found only in a single schizophrenic patient and in none of the other patients or controls, we decided to extend our samples and to use a restriction assay for screening a further 74 schizophrenic, 95 bipolar affective, and 49 patients with Tourette`s syndrome, as well as 185 controls, for the presence of the mutation. In total, the mutation was found in 2 schizophrenic patients, in 3 bipolars, in 1 Tourette patient, and in 5 controls. To our knowledge the Ile-28-Val substitution reported here is the first natural occuring molecular variant which has been identified for a serotonin receptor so far.

  17. Polyplex micelles with thermoresponsive heterogeneous coronas for prolonged blood retention and promoted gene transfection.

    Science.gov (United States)

    Li, Yang; Li, Junjie; Chen, Biao; Chen, Qixian; Zhang, Guoying; Liu, Shiyong; Ge, Zhishen

    2014-08-11

    Adequate retention in blood circulation is a prerequisite for construction of gene delivery carriers for systemic applications. The stability of gene carriers in the bloodstream requires them to effectively resist protein adsorption and maintain small size in the bloodstream avoiding dissociation, aggregation, and nuclease digestion under salty and proteinous medium. Herein, a mixture of two block catiomers consisting of the same cationic block, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), but varying shell-forming blocks, poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEO2MA), and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), was used to complex with plasmid DNA (pDNA) to fabricate polyplex micelles with mixed shells (MPMs) at 20 °C. The thermoresponsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 to 37 °C, which results in a distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of the MPMs. Subsequent study verified that this transition promoted further condensation of pDNA, thereby giving rise to improved complex and colloidal stability. The proposed system has shown remarkable stability in salty and proteinous solution and superior tolerance to nuclease degradation. As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET) block copolymer, in vivo circulation experiments in the bloodstream further confirmed that the retention time of MPMs was prolonged significantly. Moreover, the proposed system exhibited remarkably high cell transfection activity especially at low N/P ratios and negligible cytotoxicity and thus portends promising utility for systemic gene therapy applications. PMID:24978294

  18. Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins.

    Science.gov (United States)

    Elboudwarej, Emon; Cole, Michael; Briggs, Farren B S; Fouts, Alexandra; Fain, Pamela R; Quach, Hong; Quach, Diana; Sinclair, Elizabeth; Criswell, Lindsey A; Lane, Julie A; Steck, Andrea K; Barcellos, Lisa F; Noble, Janelle A

    2016-04-01

    Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%-5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%-16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T

  19. Bax function in the absence of mitochondria in the primitive protozoan Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Adrian B Hehl

    Full Text Available Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria.

  20. Bax function in the absence of mitochondria in the primitive protozoan Giardia lamblia.

    Science.gov (United States)

    Hehl, Adrian B; Regos, Attila; Schraner, Elisabeth; Schneider, André

    2007-01-01

    Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes) that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria. PMID:17534438

  1. Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia

    Institute of Scientific and Technical Information of China (English)

    Sheng-Mian Li; Shu-Kun Yao; Nobuyoshi Yamamura; Toshitsugu Nakamura

    2003-01-01

    AIM: To compare the difference of expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia, and to analyze the role of Bcl-2 and Bax proteins in the progression from dysplasia to carcinoma and to evaluate the correlation of Bcl-2/Bax protein expression with the biological behaviors.METHODS: Expressions of Bcl-2 and Bax were examined immunohistochemically in 27 cases of extrahepatic biliary tract carcinomas (bile duct carcinoma: n=21, carcinoma of ampulla of Vater: n=6), and 10 cases of atypical dysplasia.Five cases of normal biliary epithelial tissues were used as controls. A semiquantitative scoring system was used to assess the Bcl-2 and Bax reactivity.RESULTS: The expression of Bd-2 was observed in 10 out of 27 (37.0 %) invasive carcinomas, 1 out of 10 clysplasias, none out of 5 normal epithelial tissues. Bax expression rate was 74.1% (20/27) in invasive carcinoma, 30 % (3/10) in dysplasia,and 40 % (2/5) in normal biliary epithelium. Bcl-2 and Bax activities were more intense in carcinoma than in dysplasia,with no significant difference in Bcl-2 expression (P=0.1:10),and significant difference in Bax expression (P=0.038). Level of Bax expression was higher in invasive carcinoma than in dysplasia and normal tissue (P=0.012). Bcl-2 expression was correlated to Bax expression (P=0.0059). However, Bcl-2/Bax expression had no correlation with histological subtype,grade of differentiation, or level of invasion.CONCLUSION: Increased Bcl-2/Bax expression from dysplasia to invasive tumors supports the view that this is the usual route for the development of extrahepatic biliary tract carcinoma. Bcl-2/Bax may be involved, at least in part,in the apoptotic activity in extrahepatic biliary carcinoma.

  2. FANTOM4 EdgeExpressDB: an integrated database of promoters, genes, microRNAs, expression dynamics and regulatory interactions

    OpenAIRE

    Severin, J.; Waterhouse, A. M.; Kawaji, H.; Lassmann, T.; van Nimwegen, E; Balwierz, P. J.; de Hoon, M. J. L.; Hume, D A; Carninci, P.; Hayashizaki, Y.; Suzuki, H.; Daub, C.O.; Forrest, A. R. R.

    2009-01-01

    EdgeExpressDB is a novel database and set of interfaces for interpreting biological networks and comparing large high-throughput expression datasets that requires minimal development for new data types and search patterns. The FANTOM4 EdgeExpress database http://fantom.gsc.riken.jp/4/edgeexpress summarizes gene expression patterns in the context of alternative promoter structures and regulatory transcription factors and microRNAs using intuitive gene-centric and sub-network views. This is an ...

  3. Interleukin 6 induces a liver-specific nuclear protein that binds to the promoter of acute-phase genes.

    OpenAIRE

    Poli, V.; Cortese, R

    1989-01-01

    Interleukin 6 (IL-6) is responsible for a variety of biological effects related to the activation of defenses against infection or inflammation, including the immune response and the acute-phase reaction. Its mechanism of action is unknown. It has recently been shown to induce transcription of several genes encoding acute-phase proteins. Here we describe the identification of an IL-6 responsive element (IL-6RE) present in the promoter of the human hemopexin gene. This element is necessary and...

  4. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

    Institute of Scientific and Technical Information of China (English)

    Bonsu Ku; Chengyu Liang; Jae U Jung; Byung-Ha Oh

    2011-01-01

    Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic Bc1-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague.Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, hut was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-XL, MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could he expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.

  5. Gene expression and promoter analysis of a novel tomato aldo-keto reductase in response to environmental stresses.

    Science.gov (United States)

    Suekawa, Marina; Fujikawa, Yukichi; Inada, Shuhei; Murano, Asako; Esaka, Muneharu

    2016-08-01

    The functional role of an uncharacterized tomato (Solanum lycopersicum) aldo-keto reductase 4B, denoted as SlAKR4B, was investigated. The gene expression of tomato SlAKR4B was detected at a high level in the senescent leaves and the ripening fruits of tomato. Although d-galacturonic acid reductase activities tended to be higher in tomato SlAKR4B-overexpressing transgenic tobacco BY-2 cell lines than those in control cell lines, SlAKR4B gene expression was not well correlated with l-ascorbic acid content among the cell lines. The analysis of the transgenic cell lines showed that tomato SlAKR4B has enzyme activities toward d-galacturonic acid as well as glyceraldehyde and glyoxal, suggesting that the SlAKR4B gene encodes a functional enzyme in tomato. Gene expression of SlAKR4B was induced by NaCl, H2O2, and plant hormones such as salicylic acid and jasmonic acid, suggesting that SlAKR4B is involved in the stress response. The transient expression assay using protoplasts showed the promoter activity of the SlAKR4B gene was as high as that of the cauliflower mosaic virus 35S promoter. Also, the promoter region of the SlAKR4B gene was suggested to contain cis-element(s) for abiotic stress-inducible expression. PMID:27337067

  6. Generation and evaluation of an IPTG-regulated version of Vav-gene promoter for mouse transgenesis.

    Directory of Open Access Journals (Sweden)

    Francesca Grespi

    Full Text Available Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner.

  7. Promoter region of interleukin-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells.

    OpenAIRE

    Siebenlist, U; Durand, D B; Bressler, P; Holbrook, N J; Norris, C A; Kamoun, M.; Kant, J A; Crabtree, G R

    1986-01-01

    The chromatin structure of the interleukin-2 (IL-2) gene was probed by DNase I treatment of isolated nuclei. The 5' region of the IL-2 gene contains three regions of hypersensitivity to DNase I. When peripheral blood T cells or Jurkat T cells are stimulated with mitogens, IL-2 message is induced, and the promoter region of the IL-2 gene develops an additional hypersensitive site. This suggests that a DNA sequence close to the transcriptional start site is involved in the transduction of the e...

  8. Insulin-like growth factor I gene promoter polymorphism, collagen type II alpha1 (COL2A1) gene, and the prevalence of radiographic osteoarthritis: the Rotterdam Study

    OpenAIRE

    Zhai, Guangju; Duijn, Cock; Rivadeneira Ramirez, Fernando; Houwing-Duistermaat, Jeanine; Meulenbelt, Ingrid; Bijkerk, Casper; Meurs, Joyce; Uitterlinden, André; Pols, Huib; Slagboom, Eline; Hofman, Albert

    2004-01-01

    textabstractOBJECTIVE: To examine the role of an IGF-I gene promoter polymorphism in the prevalence of radiographic osteoarthritis (ROA), and study its interaction with the COL2A1 gene. METHODS: Individuals genotyped for IGF-I (n = 1546) and COL2A1 gene polymorphisms (n = 808) were selected from a random sample (n = 1583) derived from the Rotterdam study. The presence of ROA was defined as a Kellgren score of 2 or more in at least one of four joints (knee, hip, hand, and spine). Genotype spec...

  9. Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein: A potential for developing hepatitis C virus targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Shan-Shan Mao; Qiong-Qiong He; Yuan Zi; Ji-Fang Wen; De-Yun Feng

    2009-01-01

    AIM: To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed.RESULTS: L02/core cell line stably expressing HCV-core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

  10. SERPINB5 and AKAP12 -- Expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma

    International Nuclear Information System (INIS)

    Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly

  11. Control of Glycosylation-Related Genes by DNA Methylation: the Intriguing Case of the B3GALT5 Gene and Its Distinct Promoters

    Directory of Open Access Journals (Sweden)

    Marco Trinchera

    2014-08-01

    Full Text Available Glycosylation is a metabolic pathway consisting of the enzymatic modification of proteins and lipids through the stepwise addition of sugars that gives rise to glycoconjugates. To determine the full complement of glycoconjugates that cells produce (the glycome, a variety of genes are involved, many of which are regulated by DNA methylation. The aim of the present review is to briefly describe some relevant examples of glycosylation-related genes whose DNA methylation has been implicated in their regulation and to focus on the intriguing case of a glycosyltransferase gene (B3GALT5. Aberrant promoter methylation is frequently at the basis of their modulation in cancer, but in the case of B3GALT5, at least two promoters are involved in regulation, and a complex interplay is reported to occur between transcription factors, chromatin remodelling and DNA methylation of typical CpG islands or even of other CpG dinucleotides. Transcription of the B3GALT5 gene underwent a particular evolutionary fate, so that promoter hypermethylation, acting on one transcript, and hypomethylation of other sequences, acting on the other, cooperate on one gene to obtain full cancer-associated silencing. The findings may also help in unravelling the complex origin of serum CA19.9 antigen circulating in some patients.

  12. Enhanced EJ Cell Killing of 125I Radiation by Combining with Cytosine Deaminase Gene Therapy Regulated by Synthetic Radio-Responsive Promoter

    OpenAIRE

    Li, Ling; Zhang, Chun-Li; Kang, Lei; Wang, Rong-fu; Yan, Ping; Zhao, Qian; Yin, Lei; Guo, Feng-qin

    2015-01-01

    Abstract Aim: To investigate the enhancing effect of radionuclide therapy by the therapeutic gene placed under the control of radio-responsive promoter. Methods: The recombinant lentivirus E8-codA-GFP, including a synthetic radiation-sensitive promoter E8, cytosine deaminase (CD) gene, and green fluorescent protein gene, was constructed. The gene expression activated by 125I radiation was assessed by observation of green fluorescence. The ability of converting 5-fluorocytosine (5-FC) to 5-flu...

  13. Developmental regulation and complex organization of the promoter of the non-coding hsr gene of Drosophila melanogaster

    Indian Academy of Sciences (India)

    S C Lakhotia; T K Rajendra; K V Prasanth

    2001-03-01

    The nucleus-limited large non-coding hsrω-n RNA product of the 93D or the hsrω gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsrω05241 allele due to insertion of a P-LacZ-rosy+ transposon at — 130 bp position of the hsrω promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsrω promoter upstream of the LacZ reporter. The hsrω gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsrω05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsrω transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsrω RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsrω gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.

  14. A 350 bp region of the proximal promoter of Rds drives cell-type specific gene expression

    OpenAIRE

    Cai, Xue; Conley, Shannon M.; Cheng, Tong; Al-Ubaidi, Muayyad R.; Naash, Muna I.

    2010-01-01

    RDS (retinal degeneration slow) is a photoreceptor-specific tetraspanin protein required for the biogenesis and maintenance of rod and cone outer segments. Mutations in the Rds gene are associated with multiple forms of rod- and cone-dominant retinal degeneration. To gain more insight into the mechanisms underlying the regulation of this gene the identification of regulatory sequences within the promoter of Rds was undertaken. A 3.5kb fragment of the 5′ flanking region of the mouse Rds gene w...

  15. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    International Nuclear Information System (INIS)

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  16. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    Energy Technology Data Exchange (ETDEWEB)

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn, E-mail: LoneB.Madsen@agrsci.dk

    2013-08-23

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  17. Design and characterization of a dual-mode promoter with activation and repression capability for tuning gene expression in yeast.

    Science.gov (United States)

    Mazumder, Mostafizur; McMillen, David R

    2014-08-01

    Modularity in controlling gene expression artificially is becoming an essential aspect of synthetic biology. Artificial transcriptional control of gene expression is one of the most well-developed methods for the design of novel synthetic regulatory networks. Such networks are intended to help understand natural cellular phenomena and to enable new biotechnological applications. Promoter sequence manipulation with cis-regulatory elements is a key approach to control gene expression transcriptionally. Here, we have designed a promoter that can be both activated and repressed, as a contribution to the library of synthetic biological 'parts'. Starting with the minimal cytochrome C (minCYC) promoter in yeast, we incorporated five steroid hormone responsive elements (SHREs) and one lac operator site, respectively, upstream and downstream of the TATA box. This allows activation through the testosterone-responsive androgen receptor, and repression through the LacI repressor. Exposure to varying concentrations of testosterone (to vary activation) and IPTG (to vary repression) demonstrated the ability to tune the promoter's output curve over a wide range. By integrating activating and repressing signals, the promoter permits a useful form of signal integration, and we are optimistic that it will serve as a component in future regulatory networks, including feedback controllers. PMID:25056312

  18. Fusion of the promoter region of rRNA operon rrnB to lac Z gene.

    OpenAIRE

    Glaser, G; Kobi, S.; Oppenheim, A B

    1980-01-01

    A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.

  19. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site

    DEFF Research Database (Denmark)

    Schulze, A; Zerfass, K; Spitkovsky, D;

    1995-01-01

    Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is...

  20. Identification and genetic effect of a variable duplication in the promoter region of the cattle ADIPOQ gene

    Science.gov (United States)

    The ADIPOQ gene of cattle, is located in the vicinity of the quantitative trait locus (QTL) wich effects marbling, the rib eye muscle area and fat thickness on BTA1. In our study, a novel variable duplication (NW_003103812.1:g.9232067_9232133 dup) in the bovine ADIPOQ promoter region was identified ...

  1. Adiponectin gene ADIPOQ SNP associations with serum adiponectin in two female populations and effects of SNPs on promoter activity

    NARCIS (Netherlands)

    Kyriakou, Theodosios; Collins, Laura J.; Spencer-Jones, Nicola J.; Malcolm, Claire; Wang, Xiaoling; Snieder, Harold; Swaminathan, Ramasamyiyer; Burling, Keith A.; Hart, Deborah J.; Spector, Tim D.; O'Dell, Sandra D.

    2008-01-01

    Adiponectin is an insulin sensitiser in muscle and liver, and low serum levels characterise obesity and insulin resistance. Eight tagging single nucleotide polymorphisms (tSNPs) in the ADIPOQ gene and promoter were selected, and association with serum adiponectin was tested, in two independent sampl

  2. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  3. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    International Nuclear Information System (INIS)

    Highlights: → A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. → The promoter was characterized with radiation-inducibility and tumor-specificity. → Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. → Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  4. Expression of the human amylase genes: Recent origin of a salivary amylase promoter from an actin pseudogene

    Energy Technology Data Exchange (ETDEWEB)

    Samuelson, L.C.; Gumucio, D.L.; Meisler, M.H. (Univ. of Michigan, Ann Arbor (USA)); Wiebauer, K. (Friedrich Miescher Institut, Basel (Switzerland))

    1988-09-12

    The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. The authors have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5{prime} regions of the five human amylase genes contain a processed {gamma}-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3{prime} untranslated region of {gamma}-actin. In addition, insertion of an endogenous retrovirus has interrupted the {gamma}-actin pseudogene in four of the five amylase genes.

  5. scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.

    Science.gov (United States)

    Roark, M; Sturtevant, M A; Emery, J; Vaessin, H; Grell, E; Bier, E

    1995-10-01

    The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices. PMID:7557390

  6. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter.

    Science.gov (United States)

    Omidvar, V; Siti Nor Akmar, A; Marziah, M; Maheran, A A

    2008-09-01

    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms. PMID:18563415

  7. Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters.

    Science.gov (United States)

    Cowan, Peter J; Shinkel, Trixie A; Fisicaro, Nella; Godwin, James W; Bernabéu, Carmelo; Almendro, Nuria; Rius, Carlos; Lonie, Andrew J; Nottle, Mark B; Wigley, Peter L; Paizis, Kathy; Pearse, Martin J; d'Apice, Anthony J F

    2003-05-01

    It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs. PMID:12694542

  8. Isolation and characterization of an oil palm constitutive promoter derived from a translationally control tumor protein (TCTP) gene.

    Science.gov (United States)

    Masura, Subhi Siti; Parveez, Ghulam Kadir Ahmad; Ti, Leslie Low Eng

    2011-07-01

    We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system. PMID:21549610

  9. Interleukin 10.G microsatellite in the promoter region of the interleukin-10 gene in severe sepsis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background The highly polymorphic interleukin 10.G (IL10.G) microsatellite located in the promoter region of the interleukin-10 (IL-10) gene exerts a positive transcriptional regulatory effect on IL-10 gene expression and correlates with the in vitro IL-10 secretion. This study was conducted to investigate whether IL10.G microsatellite is associated with the incidence and /or the outcome of severe sepsis.Methods One hundred and fifteen patients with severe sepsis who had been treated at the intensive care unit of the university hospital were studied. One hundred and forty-one healthy individuals served as controls. IL10.G microsatellite genotyping was performed with the following two methods: fluorescent based polymerase chain reaction (PCR) techniques and silver staining of the amplified DNA fragment in polyacrylamide gel. Alleles were defined according to the size of the amplified DNA product.Results Ten alleles and 36 genotypes were detected both in the patients with severe sepsis and in the healthy controls. Allele IL10.G9 and allele IL10.G13 were the commonest alleles with the frequencies of 32.6% and 21.3% respectively in the patients with severe sepsis, and 34% and 27% respectively in the healthy controls. The allele frequencies of IL10.G microsatellite were neither different between the patients with severe sepsis and the healthy controls (P > 0.05), nor between survivors and non-survivors (P > 0.05). However, the frequency of one common allele IL10.G13 was slightly lower in the patients with severe sepsis than in the healthy controls (21.3% vs 27%, P > 0.05), and the frequency of allele IL10.G9 was slightly higher in the non-survivors than in the survivors (37.1% vs 28.1%, P > 0.05).Conclusion IL10.G microsatellite may neither contribute to the susceptibility to severe sepsis nor to the fatal outcome of severe sepsis.

  10. Human herpesvirus 6 major immediate early promoter has strong activity in T cells and is useful for heterologous gene expression

    Directory of Open Access Journals (Sweden)

    Yamanishi Koichi

    2011-01-01

    Full Text Available Abstract Background Human herpesvirus-6 (HHV-6 is a beta-herpesvirus. HHV-6 infects and replicates in T cells. The HHV-6-encoded major immediate early gene (MIE is expressed at the immediate-early infection phase. Human cytomegalovirus major immediate early promoter (CMV MIEp is commercially available for the expression of various heterologous genes. Here we identified the HHV-6 MIE promoter (MIEp and compared its activity with that of CMV MIEp in various cell lines. Methods The HHV-6 MIEp and some HHV-6 MIEp variants were amplified by PCR from HHV-6B strain HST. These fragments and CMV MIEp were subcloned into the pGL-3 luciferase reporter plasmid and subjected to luciferase reporter assay. In addition, to investigate whether the HHV-6 MIEp could be used as the promoter for expression of foreign genes in a recombinant varicella-zoster virus, we inserted HHV-6 MIEp-DsRed expression casette into the varicella-zoster virus genome. Results HHV-6 MIEp showed strong activity in T cells compared with CMV MIEp, and the presence of intron 1 of the MIE gene increased its activity. The NF-κB-binding site, which lies within the R3 repeat, was critical for this activity. Moreover, the HHV-6 MIEp drove heterologous gene expression in recombinant varicella-zoster virus-infected cells. Conclusions These data suggest that HHV-6 MIEp functions more strongly than CMV MIEp in various T-cell lines.

  11. Molecular cloning and characterization of GbMECT and GbMECP gene promoters from Ginkgo biloba.

    Science.gov (United States)

    Cheng, S Y; Li, L L; Yuan, H H; Xu, F; Cheng, H

    2015-01-01

    Ginkgolides are key pharmaceutical components in Ginkgo biloba. Using the cDNA sequence of the MECP and MECT genes to design primers, we obtained the promoters of these genes from Ginkgo genomic DNA using the genome walking method. The two promoters were 744 and 982 bp in length, respectively. The cis-elements of the GbMECPs and GbMECT promoters were predicted and analyzed using the plant cis-acting regulatory element database. We found major cis-elements in the sequence of the GbMECT and GbMECPs promoters. The GbMECP promoter contains six TATA boxes and eight CAAT boxes. The GbMECT contains five TATA boxes and seven CAAT boxes. Furthermore, some cis-elements in the promoters of GbMECPs and GbMECT included hormone and light-regulated elements, UB-B-induced elements, and stress-related dehydration-responsive elements. Expression analysis results showed that the MECP gene is mainly involved in responses to CCC (cycocel) and UV-B, and that MECT is mainly involved in responses to wounding treatment. These results also showed that the expression model was consistent with the cis-elements present. During the annual growth cycle, the level of GbMECPs was significantly correlated with terpene lactones accumulation in leaves. A fitted quadratic curve showed the best model for correlating GbMECPs with terpene lactones in leaves. These results will help us to understand the transcriptional regulatory mechanisms involved in key gene expression and ginkgolide accumulation in G. biloba. PMID:26634474

  12. Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer

    International Nuclear Information System (INIS)

    In normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context. Comparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay. Comparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion. These observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis

  13. Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR.

    Science.gov (United States)

    D'Orazio, S E; Thomas, V; Collins, C M

    1996-08-01

    The Proteus mirabilis and plasmid-encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC-like transcriptional activator. Previously, it was shown that the plasmid-encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea- and UreR-dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid-encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid-encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5 alpha background, as determined by primer-extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR-binding sites within the plasmid-encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid-encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid-encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea. PMID:8866486

  14. Novel characterisation of the gene encoding conglutinin reveals that previously characterised promoter corresponds to the CL-43 promoter

    DEFF Research Database (Denmark)

    Hansen, Soren; Moeller, Vivi; Holm, Dorte; Vitved, Lars; Bendixen, Christian; Skjødt, Karsten; Holmskov, Uffe

    Conglutinin, a collectin found in bovine serum, is an opsonin that binds to glycoconjugates on the surface of microorganisms or on deposited iC3b, and acts in concert with phagocytes to establish a first-line of immune defense. We have isolated a genomic conglutinin phage clone and found that the 5...... CL-43. Comparison of potential cis-regulatory elements in relation to the functional studies indicates that the two genes are regulated by different mechanisms....

  15. Nonredundant Role of Bax and Bak in Bid-Mediated Apoptosis

    OpenAIRE

    Cartron, Pierre-François; Juin, Philippe; Oliver, Lisa; Martin, Stéphane; Meflah, Khaled; Vallette, François M.

    2003-01-01

    Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM)...

  16. Injury-specific promoters enhance herpes simplex virus mediated gene therapy for treating neuropathic pain in rodents

    Science.gov (United States)

    Smith, Sherika N.; Paige, Candler; Velazquez, Kandy T.; Smith, Terika P.; Raja, Srinivasa N.; Wilson, Steven P.; Sweitzer, Sarah M.

    2015-01-01

    Chronic neuropathic pain is often difficult to treat with current pain medications. Gene therapy is currently being explored as a therapeutic approach for the treatment of neuropathic and cancer pain. In this study we sought to use an injury-specific promoter to deliver the mu opioid receptor (MOR) transgene such that expression would only occur during the injured state in response to release of injury-specific galanin. To determine whether an injury specific promoter can produce neuron-specific MOR expression and enhanced antinociception we compared animals infected with a galanin promoter virus (galMOR) or a human cytomegalovirus (CMV) promoter virus. (cmvMOR). In behavioral assays, we found an earlier onset and a larger magnitude of antinociception in animals infected with galMOR compared to cmvMOR. Immunohistochemistry of dorsal root ganglion (DRG) neurons revealed a significant increase in MOR positive staining in cmvMOR and galMOR treated mice. Spinal cord sections from galMOR treated mice showed a greater increase in density but not area of MOR positive staining. These results suggest that using injury-specific promoters to drive gene expression in primary afferent neurons can influence the onset and magnitude of antinociception in a rodent model of neuropathic pain and can be used to upregulate MOR expression in populations of neurons that are potentially injury specific. Perspective An injury specific promoter (galMOR) was used to drive MOR expression in a population- and injury- specific manner. GalMOR increased antinociception and density of MOR staining in spinal cord. This article presents evidence that promoter selection is an important component in successful gene expression in an injury- and population-specific manner. PMID:25576797

  17. Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma

    OpenAIRE

    Sun Wenjie; Yu Haijun; Liu Zhengchun; Hou Jinxuan; Xu Yu; Xiong Jie; Liao Zhengkai; Zhou Fuxiang; Xie Conghua; Zhou Yunfeng

    2011-01-01

    Abstract Background Gene therapy is a promising therapeutic approach for cancer. Targeted expression of desired therapeutic proteins within the tumor is the best approach to reduce toxicity and improve survival. This study is to establish a more effective and less toxic gene therapy of cancer. Methods Combined gene therapy strategy with recombinant adenovirus expressing horseradish peroxidase (HRP) mediated by human telomerase reverse transcriptase (hTERT) promoter (AdhTERTHRP) and murine int...

  18. The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia

    International Nuclear Information System (INIS)

    Background and purpose: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. Material and methods: Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. Results: ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. Conclusions: We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome

  19. Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta

    Science.gov (United States)

    Parobchak, Nataliya; Rosen, Alex; Vetrano, Anna M.; Srinivasan, Aarthi; Wang, Bingbing; Rosen, Todd

    2016-01-01

    Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth. PMID:26751383

  20. REN: a novel, developmentally regulated gene that promotes neural cell differentiation.

    Science.gov (United States)

    Gallo, Rita; Zazzeroni, Francesca; Alesse, Edoardo; Mincione, Claudia; Borello, Ugo; Buanne, Pasquale; D'Eugenio, Roberta; Mackay, Andrew R; Argenti, Beatrice; Gradini, Roberto; Russo, Matteo A; Maroder, Marella; Cossu, Giulio; Frati, Luigi; Screpanti, Isabella; Gulino, Alberto

    2002-08-19

    Expansion and fate choice of pluripotent stem cells along the neuroectodermal lineage is regulated by a number of signals, including EGF, retinoic acid, and NGF, which also control the proliferation and differentiation of central nervous system (CNS) and peripheral nervous system (PNS) neural progenitor cells. We report here the identification of a novel gene, REN, upregulated by neurogenic signals (retinoic acid, EGF, and NGF) in pluripotent embryonal stem (ES) cells and neural progenitor cell lines in association with neurotypic differentiation. Consistent with a role in neural promotion, REN overexpression induced neuronal differentiation as well as growth arrest and p27Kip1 expression in CNS and PNS neural progenitor cell lines, and its inhibition impaired retinoic acid induction of neurogenin-1 and NeuroD expression. REN expression is developmentally regulated, initially detected in the neural fold epithelium of the mouse embryo during gastrulation, and subsequently throughout the ventral neural tube, the outer layer of the ventricular encephalic neuroepithelium and in neural crest derivatives including dorsal root ganglia. We propose that REN represents a novel component of the neurogenic signaling cascade induced by retinoic acid, EGF, and NGF, and is both a marker and a regulator of neuronal differentiation. PMID:12186855

  1. A COMMON VARIATION IN THE PROMOTER REGION OF INTERLEUKIN-6 GENE SHOWS ASSOCIATION WITH EXERCISE PERFORMANCE

    Directory of Open Access Journals (Sweden)

    Antti Huuskonen

    2009-06-01

    Full Text Available Skeletal muscle-derived interleukin-6 (IL-6 is a pleiotropic cytokine which regulates body metabolism during strenuous physical exercise. OBJECTIVE: The effect of a potentially functional single nucleotide polymorphism (SNP -174G/C of the IL6 gene (rs1800795 promoter was examined on maximal oxygen uptake (VO2max, body mass index (BMI and plasma IL-6 levels in response to physical training. Fifty four male military conscripts were studied for 8 weeks during their basic training. At weeks 1, 5 and 8, VO2max and anthropometrics were measured, and blood samples collected before and after acute aerobic exercise. Acute exercise increased plasma IL-6 in subjects with genotype CG. Moreover, during the 8-week training period, a tendency for increased plasma IL-6 was observed in subjects with this genotype. VO2max values increased in all genotype groups, but subjects with genotype CG made the greatest gains in VO2max. Training significantly decreased BMI only in subjects with genotype CG. Our findings suggest that the allele C may have an effect on plasma IL-6 response to acute exercise in healthy male subjects. Exercise training has a favourable effect on VO2max and BMI, with the most prominent effects in subjects with genotype CG. Thus we conclude that this SNP may account for individual response to exercise training.

  2. Mono-(2-Ethylhexyl Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

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    Ximi K Wang

    Full Text Available Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH and cyclooxygenase-2 (COX-2 are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52 signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl-phthalate (MEHP, increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth.

  3. Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence.

    Science.gov (United States)

    Patel, Priyanka L; Suram, Anitha; Mirani, Neena; Bischof, Oliver; Herbig, Utz

    2016-08-23

    Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc-dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression. PMID:27503890

  4. Significant association between polymorphism of the erythropoietin gene promoter and myelodysplastic syndrome

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    O'Brien Susan

    2010-11-01

    Full Text Available Abstract Background Myelodysplastic syndrome (MDS may be induced by certain mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP rs1617640 in the erythropoietin (EPO promoter has been shown to be associated with decreased EPO expression. We examined the association of rs1617640 genotype with MDS. Methods We genotyped the EPO rS1617640 SNP in 189 patients with MDS, 257 with acute myeloid leukemia (AML, 106 with acute lymphoblastic leukemia, 97 with chronic lymphocytic leukemia, 353 with chronic myeloid leukemia, and 95 healthy controls. Results The G/G genotype was significantly more common in MDS patients (47/187; 25.1% than in controls (6/95; 6.3% or in patients with other leukemias (101/813; 12.4% (all P P = 0.03. Time to neutrophils recovery after therapy was significantly longer in MDS patients with the G/G genotype (P = 0.02. Conclusions These findings suggest a strong association between the rs1617640 G/G genotype and MDS. Further studies are warranted to investigate the utility of screening for this marker in individuals exposed to environmental toxins or chemotherapy.

  5. A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

    Science.gov (United States)

    Kumar, Santosh; Bhatia, Sabhyata

    2016-01-01

    Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5' flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5'UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8-21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5'-UTR significantly enhances the level of gene expression. We termed this phenomenon as "microsatellite mediated enhancement" (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids. PMID:27623355

  6. Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells.

    Science.gov (United States)

    González-Mariscal, J A; Fernández-Trujillo, M A; Alonso, M C; García-Rosado, E; Álvarez, M C; Béjar, J

    2016-03-01

    The understanding of virus-host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the

  7. Isolation and sequencing of a putative promoter region of the murine G protein beta 1 subunit (GNB1) gene.

    Science.gov (United States)

    Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Wang, Xiao-Bing; Hembree, Cambria M; Goodman, Nancy L; Uhl, George R

    2002-02-01

    The expression of the heterotrimeric GTP-binding protein beta 1 subunit gene (GNB1) is regulated by psychostimulants such as cocaine and amphetamines. Since the up-regulation appears to be one of the candidate processes of sensitization, it is necessary to elucidate the cellular and molecular mechanism of the GNB1 gene regulation for a better understanding the establishment of sensitization. In the present study, we describe the isolation and nucleotide sequence analysis of the GNB1 gene promoter region. We have isolated approximately 10 kb of the 5'-flanking region of the mouse of GNB1 gene and found potential elements involved in putative transcriptional control of the GNB1, such as AP1, AP2, Sp1, cyclic AMP response element, and nuclear factor kappa B recognition sites, within the sequences 0.3 kb upstream from the putative transcription start site. This region was highly rich in G + C content, but lacked TATA or CATT boxes. Comparing the nucleotide sequence of the cDNA clone with the human genome databases using the BLAST program a region containing putative exon 1 and promoter of the human GNB1 gene in chromosome 1 was found. The cloning and sequence analysis of an extensive portion of the 5'-flanking regulatory region of the GNB1 gene provides new insights into the factors involved in the regulation by psychostimulants of GNB1 expression. PMID:12180136

  8. Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon.

    Directory of Open Access Journals (Sweden)

    Ting Min

    Full Text Available A hypoxic environment is generally undesirable for most plants and stimulates anaerobic metabolism. It is a beneficial treatment, however, for the removal of astringency from persimmon to improve the fruit quality after harvest. High soluble tannins (SCTs content is one of most important causes of astringency. High CO2 (95% treatment effectively reduced SCTs in both "Mopan" and "Gongcheng-shuishi" persimmon fruit by causing increases in acetaldehyde. Using RNA-seq and realtime PCR, twelve ethylene response factor genes (DkERF11-22 were isolated and characterized, to determine those responsive to high CO2 treatment. Only two genes, DkERF19 and DkERF22, showed trans-activation effects on the promoters of deastringency-related genes pyruvate decarboxylase genes (DkPDC2 and DkPDC3 and the transcript levels of these genes was enhanced by hypoxia. Moreover, DkERF19 and the previously isolated DkERF9 had additive effects on activating the DkPDC2 promoter. Taken together, these results provide further evidence that transcriptome changes in the level of DkERF mRNAs regulate deastringency-related genes and their role in the mechanism of persimmon fruit deastringency is discussed.

  9. Vitamin D-mediated modifications in protein-DNA interactions at two promoter elements of the osterocalcin gene

    International Nuclear Information System (INIS)

    By the combined use of DNase I footprinting, electrophoretic mobility-shift assay, and methylation interference analysis, the authors have identified a series of sequence-specific protein-DNA interactions in the 5' flanking region of the rat osteocalcin gene. Stimulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3, a physiologic mediator of this bone-specific gene in vitro and in vivo, is associated with modifications in the binding of ROS 17/2.8 cell nuclear factors to two promoter segments that up-regulate transcription. One segment located between -462 and -437 exhibits a vitamin D-dependent increase in sequence-specific binding of nuclear factors. This element (CTGGGTGAATGAGGACATTACT-GACC), identified at single nucleotide resolution, contains a region of hyphenated dyad symmetry and shares sequence homology with consensus steroid-responsive elements and with the sequence that has been identified as the vitamin D receptor binding site in the human osteocalcin gene. They have also observed that vitamin D stimulation of osteocalcin gene expression results in a 5-fold increase in protein binding to the region of the osteocalcin box, a 24-nucleotide segment in the proximal promoter with a CCAAT motif as the central core. The results demonstrate protein-DNA interactions in a vitamin D-responsive element and in a second sequence, the osteocalcin box, both of which are involved in the physiologic regulation of the osteocalcin gene in response to 1,25-dihydroxyvitamin D3

  10. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

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    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  11. Study of promoter and structural gene sequence of whiB7 in MDR and XDR forms of Mycobacterium tuberculosis.

    Science.gov (United States)

    Arjomandzadegan, M; Sadrnia, M; Surkova, L K; Titov, L P

    2011-06-01

    Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCR for sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets of primers proved that there were no differences between sequence of susceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design of an inhibitor for the WhiB7 protein in all clinical forms in further studies. PMID:22224334

  12. Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma. The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry. mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells. Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS

  13. Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter.

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    Anastasia L Bodnar

    Full Text Available High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27 kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27 kDa gamma zein than the unselected control populations. These higher levels of the 27 kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression.

  14. Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

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    E.V. Adorno

    2008-02-01

    Full Text Available The fetal hemoglobin (HbF levels and ßS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2% patients had CAR/Ben genotype; 36 (28.8% Ben/Ben; 18 (14.4% CAR/CAR; 2 (1.6% CAR/Atypical; 2 (1.6% Ben/Cam; 1 (0.8% CAR/Cam; 1 (0.8% CAR/Arab-Indian, and 1 (0.8% Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

  15. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  16. Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis-elements and trans-acting factors

    DEFF Research Database (Denmark)

    Metz, B A; Welters, P; Hoffmann, H J; Jensen, E O; Schell, J

    1988-01-01

    The primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania rostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb...... proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal...

  17. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

    International Nuclear Information System (INIS)

    Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%). Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it

  18. Isolation and characterization of the hamster gadd153 gene. Activation of promoter activity by agents that damage DNA

    Energy Technology Data Exchange (ETDEWEB)

    Luethy, J.D.; Fargnoli, J.; Park, J.S.; Fornace, A.J. Jr.; Holbrook, N.J. (National Institute on Aging, Baltimore, MD (USA))

    1990-09-25

    A group of five cDNA clones, representing the gadd genes, were recently isolated from Chinese hamster ovary (CHO) cells as genes induced upon growth arrest and after DNA damage. We have isolated and characterized one of these genes, gadd153. The gene spans five kilobases and contains four exons. The 5'-flanking region of the gene, within 420 base pairs of the transcription initiation site, contains a number of cis elements associated with transcriptional regulation in other genes. These include a Hogness box, ATAAAA, an inverted GCCAAT box; seven SP1 transcription factor binding sites, and an AP-1 site. This region is rich in G + C content (greater than 70%) and contains an unusually long stretch of alternating CpG residues. The 800-base pair region immediately upstream of the transcription start site can drive expression of the bacterial chloramphenicol acetyltransferase (CAT) gene, but only in its endogenous orientation, in three different cell lines: HeLa, CHO, and Jurkat. The gadd153 promoter is strongly activated by methyl methanesulfonate, hydrogen peroxide, and UV irradiation, but not by growth arrest signals. This suggests that separate and very different regulatory pathways are involved in the induction of the gadd153 gene by growth cessation and DNA damage.

  19. Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Manganelli Riccardo

    2005-01-01

    Full Text Available Abstract Background In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. Results The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat, was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the

  20. Triple helix formation with the promoter of human alpha1(I) procollagen gene by an antiparallel triplex-forming oligodeoxyribonucleotide.

    OpenAIRE

    Nakanishi, M.; K. T. Weber; Guntaka, R V

    1998-01-01

    The promoters of alpha1(I) procollagen genes of vertebrates contain two contiguous stretches of polypyrimidine/polypurine sequences, referred to as C1 (-140 to -170) and C2 (-171 to -200). Antiparallel triplex-forming upstream oligonucleotides form efficient triplexes with C1. The C1 tract of human differs from rodent alpha1(I) promoters by 7 nt which are mainly A-->G transitions. Human triplex-forming oligodeoxyribonucleotide (TFO) formed stable triplexes efficiently with a K d of approximat...

  1. O 6 -methylguanine DNA methyltransferase gene promoter methylation in high-grade gliomas: A review of current status

    Directory of Open Access Journals (Sweden)

    Vaishali Suri

    2011-01-01

    Full Text Available Assessment of promoter methylation of the O 6 -methylguanine DNA methyltransferase (MGMT gene has recently gained importance in molecular profiling of high-grade gliomas. It has emerged not only as an important prognostic marker but also as a predictive marker for response to temozolomide in patients with newly diagnosed glioblastoma. Further, recent studies indicate that MGMT promoter methylation has strong prognostic relevance even in anaplastic (grade III gliomas, irrespective of therapy (chemotherapy or radiotherapy. This article provides an overview of its use as a predictive and prognostic biomarker, as well as the methods employed for its assessment and use in therapeutic decision making.

  2. Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum

    OpenAIRE

    Giacani, Lorenzo; Lukehart, Sheila; Centurion-Lara, Arturo

    2007-01-01

    In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with th...

  3. Upregulation of metastasis-associated gene 2 promotes cell proliferation and invasion in nasopharyngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Wu MH

    2016-03-01

    Full Text Available Minhua Wu,1,2,* Xiaoxia Ye,2,* Xubin Deng,3,* Yanxia Wu,4 Xiaofang Li,4 Lin Zhang11Department of Histology and Embryology, Southern Medical University, Guangzhou, 2Department of Histology and Embryology, Guangdong Medical University, Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University, Guangzhou, 4Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’s Republic of China*These authors contributed equally to this workAims: Metastasis-associated gene 2 (MTA2 is reported to play an important role in tumor progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC. The aim of the study was to explore the expression and function of MTA2 in NPC.Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochemistry and Western blotting. Relationship between MTA2 expression and clinicopathological features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony formation assay. Cell migration ability was evaluated by wound healing and transwell invasion assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was determined by nude mouse xenograft models. Expression of several Akt pathway proteins was detected by Western blotting.Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, CNE2, and HNE1. MTA2 expression was related to clinical stage and lymph node metastasis of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion suppressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression

  4. Possible association of the 5-HTTLPR serotonin transporter promoter gene polymorphism with premature ejaculation in a Turkish population

    Institute of Scientific and Technical Information of China (English)

    Emin Ozbek; Ali I.Tasci; Volkan Tugcu; Yusuf O.Ilbey; Abdulmuttalip Simsek; Levent Ozcan; Emre C.Polat; Vedat Koksal

    2009-01-01

    We evaluated the genotypes of the serotonin transporter gene (5-HTT) in patients with premature ejaculation (PE) to determine the role of genetic factors in the etiopathogenesis of PE and possibly to identify the patient subgroups. A total of 70 PE patients and 70 controls were included in this study. All men were heterosexual, had no other disorders and were either married or in a stable relationship. PE was defined as ejaculation that occurred within 1 min of vaginal intromission. Genomic DNA from patients and controls was analyzed using polymerase chain reaction, and allelic variations of the promoter region of the serotonin transporter gene (5-HTTLPR) were determined. The 5-HTTLPR (serotonin transporter promoter gene) genotypes in PE patients vs. Controls were distributed as follows: L/L 16% vs. 17%, L/S 30% vs. 53% and S/S 54% vs. 28%. We examined the haplotype analysis for three polymorphisms of the 5-HTTLPR gene: LL, LS and SS. The appropriateness of the allele frequencies in the 5-HTTLPR gene was analyzed by the Hardy-Weinberg equilibrium using the χ2-test. The short (S) allele of the 5-HTTLPR gene was significantly more frequent in PE patients than in controls (P<0.05). We suggest that the 5-HTTLPR gene plays a role in the pathophysiology of all primary PE cases. Further studies are needed to evaluate the relationship between 5-HTTLPR gene polymorphism and patient subgroup (such as primary and secondary PE) responses to selective serotonin reuptake inhibitors as well as ethnic differences.

  5. Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation: indicators of tumor staging and metastasis in adenocarcinomatous sporadic colorectal cancer in Indian population.

    Directory of Open Access Journals (Sweden)

    Rupal Sinha

    Full Text Available OBJECTIVE: Colorectal cancer (CRC development involves underlying modifications at genetic/epigenetic level. This study evaluated the role of Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation together/independently in sporadic CRC in Indian population and correlation with clinicopathological variables of the disease. METHODS: One hundred and twenty four consecutive surgically resected tissues (62 tumor and equal number of normal adjacent controls of primary sporadic CRC were included and patient details including demographic characteristics, lifestyle/food or drinking habits, clinical and histopathological profiles were recorded. Polymerase chain reaction - Restriction fragment length polymorphism and direct sequencing for Kras gene mutation and Methylation Specific-PCR for RASSF1A, FHIT and MGMT genes was performed. RESULTS: Kras gene mutation at codon 12 & 13 and methylated RASSF1A, FHIT and MGMT gene was observed in 47%, 19%, 47%, 37% and 47% cases, respectively. Alcohol intake and smoking were significantly associated with presence of Kras mutation (codon 12 and MGMT methylation (p-value <0.049. Tumor stage and metastasis correlated with presence of mutant Kras codon 12 (p-values 0.018, 0.044 and methylated RASSF1A (p-values 0.034, 0.044, FHIT (p-values 0.001, 0.047 and MGMT (p-values 0.018, 0.044 genes. Combinatorial effect of gene mutation/methylation was also observed (p-value <0.025. Overall, tumor stage 3, moderately differentiated tumors, presence of lymphatic invasion and absence of metastasis was more frequently observed in tumors with mutated Kras and/or methylated RASSF1A, FHIT and MGMT genes. CONCLUSION: Synergistic interrelationship between these genes in sporadic CRC may be used as diagnostic/prognostic markers in assessing the overall pathological status of CRC.

  6. Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

    Science.gov (United States)

    Chao, Chun-Nun; Lin, Mien-Chun; Fang, Chiung-Yao; Chen, Pei-Lain; Chang, Deching; Shen, Cheng-Huang; Wang, Meilin

    2016-01-01

    Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma. PMID:27322500

  7. Myristic Acid (MA) Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

    Institute of Scientific and Technical Information of China (English)

    LU Nai-sheng; ZHANG Yong-liang; JIANG Qing-yan; SHU Gang; XIE Qiu-ping; ZHU Xiao-tong; GAO Ping; ZHOU Gui-xuan; WANG Song-bo; WANG Li-na; XI Qian-yun

    2014-01-01

    Intramuscular fat (IMF) content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid (MA) on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle (LDM) and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ(PPARγ) and adipose-related genes, such as glucose transporter 1 (GLUT1), lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4/aP2), fatty acid translocase (FAT), acetyl-CoA carboxylaseα(ACCα), adipose triglyceride lipase (ATGL) and fatty acid synthase (FASN). However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α(C/EBPα) or hormone sensitive lipase (HSL). The expression of pyruvate dehydrogenase kinase 4 (PDK4) was increased by MA during the early stages of differentiation (day 1-3). In addition, MA also increased the absolute content of C14 (P<0.001) and saturated fatty acids (SFA) (P<0.05) to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.

  8. beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter.

    OpenAIRE

    Altiok, S; Groner, B

    1994-01-01

    beta-Casein gene expression in mammary epithelial cells is under the control of the lactogenic hormones, glucocorticoids, insulin, and prolactin. The hormonal control affects gene transcription, and several regulatory elements in the beta-casein gene promoter between positions -80 and -221 have previously been identified. A region located in the promoter between positions -170 and -221 contains overlapping sequences for negative and positive regulatory elements. A sequence-specific single-str...

  9. BimL involvement in Bax activation during UV irradiation-induced apoptosis

    International Nuclear Information System (INIS)

    Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but translocates to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors in response to apoptotic stimuli. However, the molecular mechanism of Bax activation is not fully understood. We show here the role of BimL in Bax activation during UV irradiation-induced apoptosis. In this study, GFP-BimL plasmid was constructed. The dynamic interaction between BimL and Bax during UV irradiation-induced apoptosis was observed using fluorescence resonance energy transfer (FRET) technique. Our experimental results showed that BimL translocation to mitochondria occurred before Bax translocation, and that BimL activated Bax indirectly. Moreover, inhibition of c-Jun N-terminal protein kinase (JNK) activation blocked BimL translocation, delayed and attenuated Bax translocation and subsequent apoptosis. These results demonstrate that BimL is involved in UV irradiation-induced apoptosis by indirectly activating Bax

  10. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

    OpenAIRE

    Tatsuya eKon; Nobuyuki eYoshikawa

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the CaMV 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1...

  11. The role of the P1BS element containing promoter-driven genes in Pi transport and homeostasis in plants

    DEFF Research Database (Denmark)

    Sobkowiak, Lukasz; Bielewicz, Dawid; Malecka, Ewelina M.;

    2012-01-01

    Inorganic phosphate (Pi) is an easily accessible form of phosphorus for plants. Plant Pi uptake is usually limited however by slow Pi diffusion through the soil which strongly adsorps phosphate species. Plants have developed mechanisms to increase Pi availability. There are also abiotic (phosphate...... level) and biotic (e.g.,mycorrhizal) factors regulating the expression of Pi-responsive genes. Transcription factors binding to the promoters of Pi-responsive genes activate different pathways of Pi transport, distribution, and homeostasis maintenance. Pi metabolism involves not only functional proteins...

  12. Promoter methylation and expression levels of selected hematopoietic genes in pediatric B-cell acute lymphoblastic leukemia

    OpenAIRE

    Musialik, Ewa; Bujko, Mateusz; Kober, Paulina; Agnieszka WYPYCH; Gawle-Krawczyk, Karolina; Matysiak, Michal; Siedlecki, Janusz Aleksander

    2015-01-01

    Background Precursor B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common neoplasm in children and is characterized by genetic and epigenetic aberrations in hematopoietic transcription factor (TF) genes. This study evaluated promoter DNA methylation and aberrant expression levels of early- and late-acting hematopoietic TF genes homeobox A4 and A5 (HOXA4 and HOXA5), Meis homeobox 1 (MEIS1), T-cell acute lymphocytic leukemia 1 (TAL1), and interferon regulatory factors 4 and 8 (IR...

  13. Effect and Regulatory Mechanism of Clock Gene Per1 on Biological Behaviors of Human Oral Squamous Carcinoma Cell.

    Science.gov (United States)

    Han-Xue, L I; Kai, Yang; Xiao-Juan, F U; Qin, Zhao

    2016-04-10

    Objective To investigate the effect and regulatory mechanism of clock gene Per1 on the proliferation,apoptosis,migration,and invasion of human oral squamous carcinoma SCC15 cells. Methods RNA interference was used to knock down Per1 gene in human oral squamous cell carcinoma SCC15 cell line. Changes of cell proliferation and apoptosis were analyzed by flow cytometry. Transwell assay was carried out to assess cell migration and invasion. Real-time polymerase chain reaction was used to detect the mRNA expressions of Ki-67,murine double minute 2(MDM2),c-Myc,p53,Bax,Bcl-2,metalloproteinase (MMP)2,MMP9,and vascular endothelial growth factor (VEGF). Results shRNA-mediated knockdown of Per1 promoted the proliferation,migration and invasion capacity,and inhibited cell apoptosis capacity of SCC15 cells (all PKi-67,MDM2,Bcl-2,MMP2,and MMP9 and decreased the mRNA expressions of c-Myc,p53,and Bax (all P0.05). Conclusions Clock gene Perl can regulate important tumor-related genes downstream such as Ki-67,MDM2,c-Myc,p53,Bax,Bcl-2,MMP2,and MMP9,and the aberrant expression of Per1 can affect tumor cell proliferation,apoptosis,migration and invasion. An in-depth study of Per1 may further clarify the mechanism of tumorigenesis and tumor development and thus provides new effective molecular targets for cancer treatment. PMID:27181891

  14. Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO generation and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.

  15. Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

    Directory of Open Access Journals (Sweden)

    González-Manchón Consuelo

    2006-05-01

    Full Text Available Abstract Background Podocalyxin (podxl is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. Results The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. Conclusion Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s and that DNA-methylation of the CpG promoter islands

  16. Cloning and characterization of interferon stimulated genes Viperin and ISG15,and their promoters from snakehead Channa argus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    By suppression subtractive hybridization,rapid amplification of cDNA ends and gene walking methods,interferon stimulated genes (ISGs),Viperin and ISG15,and their promoters have been cloned and characterized from snakehead Channa argus.The Viperin cDNA was found to be 1474 nt and contain an open reading frame(ORF)of 1059 nt that translates into a putative peptide of 352amino acid(aa).The putative peptide of Viperin shows high identity to that in teleosts and mamals except for the N-terminal 70 aa.The ISG15 cDNA was found to be 758 nt and contain an ORF of 468 nt that translates into a putative peptide of 155 aa.The putative peptide of ISG15 is composed of two tandem repeats of ubiquitin-like(UBL)domains,and a canonical conjugation motif(LRGG)at C-terminal.Viperin and ISG15 promoter regions were characterized by the presence of interferon stimulating response elements(ISRE)and γ-IFN activation sites (GAS).ISRE is a feature of IFN-induced gene promoter and partially overlaps interferon regulatory factor (IRF)1 and IRF2 recognition sites.GAS is responsible for the γ-IFN mediated transcription.One conserved site for NF-kB was found in the promoter region of Viperin.This is the first report of conservative binding motif for NF-kB in accordance with the consensus sequence(GGGRNNYYCC)among teleost ISG promoters.Moreover,there were also TATA,CAAT and Spl transcription factor sites in Viperin and ISG15 promoters.In 5'untranslated region (UTR),snakehead ISG15 gene contains a single intron,which differs from Viperin gene.The transcripts of Vipeirn and ISG15 mRNA were mainly expressed in head kidney,posterior kidney,spleen and gill.The expression levels in liver were found to increase obviously in response to induction by IFN-inducer poly I:C.

  17. Reiterated Targeting Peptides on the Nanoparticle Surface Significantly Promote Targeted Vascular Endothelial Growth Factor Gene Delivery to Stem Cells.

    Science.gov (United States)

    Wang, Dong-Dong; Yang, Mingying; Zhu, Ye; Mao, Chuanbin

    2015-12-14

    Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells. PMID:26588028

  18. Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Mang-Li Zhang; Sen Lu; Lin Zhou; Shu-Sen Zheng

    2008-01-01

    BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTuⅠ) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (MspⅠ/HpaⅡ) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as veriifed by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

  19. Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphism

    OpenAIRE

    Martins, R.; Morais, A.; Dias, A; Soares, I; Rolão, C; Ducla-Soares, J; Braga, L.; Seixas, T.; Nunes, B.; Olim, G; Romão, L; Lavinha, J; Faustino, P

    2008-01-01

    Elevated erythrocyte destruction in sickle cell disease (SCD) results in chronic hyperbilirubinaemia and, in a subset of patients, cholelithiasis occurs. We investigated whether the (TA)n promoter polymorphism in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) may modify bilirubin metabolism, influencing bilirubinaemia, predisposition to cholelithiasis and subsequent cholecystectomy, in a group of 153 young SCD patients (mean age 12.0 +/- 9.0 years) predominantly of Bantu beta S haplotype. ...

  20. The gamma-glutamyl transpeptidase gene is transcribed from a different promoter in rat hepatocytes and biliary cells.

    OpenAIRE

    Brouillet, A.; Holic, N.; Chobert, M. N.; Laperche, Y

    1998-01-01

    Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adul...

  1. Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1 Gene in Primary Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Naoko Minatani

    Full Text Available Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1 gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004. Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007. Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

  2. An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

    OpenAIRE

    Larsen, Camilla; Franch-Marro, Xavier; Hartenstein, Volker; Alexandre, Cyrille; Vincent, Jean-Paul

    2006-01-01

    Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic male...

  3. Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts

    OpenAIRE

    Dron, Michel; Clouse, Steven D.; Dixon, Richard A.; Lawton, Michael A; Lamb, Christopher J.

    1988-01-01

    To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5′ flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled...

  4. Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

    Science.gov (United States)

    Tatulian, Suren A.; Garg, Pranav; Nemec, Kathleen N.; Chen, Bo; Khaled, Annette R.

    2015-01-01

    Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVL-TASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an α/β-type secondary structure within membranes. Both β-strands and α-helices are significantly (by 30–60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8–9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an “α/β-ring” structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi. PMID:23110300

  5. Dynamical analysis on gene activity in the presence of repressors and an interfering promoter.

    Science.gov (United States)

    Nakanishi, Hiizu; Mitarai, Namiko; Sneppen, Kim

    2008-11-01

    Transcription is regulated through interplay among transcription factors, an RNA polymerase (RNAP), and a promoter. Even for a simple repressive transcription factor that disturbs promoter activity at initial binding of RNAP, its repression level is not determined solely by the dissociation constant of transcription factor but is sensitive to timescales of processes in RNAP. We first analyze the promoter activity under strong repression by a slow binding repressor, in which case transcription events occur in bursts, followed by long quiescent periods while a repressor binds to the operator; the number of transcription events, bursting, and quiescent times are estimated by reaction rates. We then examine interference effect from an opposing promoter, using the correlation function of initiation events for a single promoter. The interference is shown to de-repress the promoter because RNAPs from the opposing promoter most likely encounter the repressor and remove it in case of strong repression. This de-repression mechanism should be especially prominent for the promoters that facilitate fast formation of open complex with the repressor whose binding rate is slower than approximately 1/s. Finally, we discuss possibility of this mechanism for high activity of promoter PR in the hyp-mutant of lambda-phage. PMID:18658208

  6. Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.

    Science.gov (United States)

    Sahu, Soumyadip; Ganguly, Rituparna; Raman, Priya

    2016-08-01

    We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, p