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Sample records for activity promotes dna

  1. Prediction of fine-tuned promoter activity from DNA sequence

    Siwo, Geoffrey; Rider, Andrew; Tan, Asako; Pinapati, Richard; Emrich, Scott; Chawla, Nitesh; Ferdig, Michael

    2016-01-01

    The quantitative prediction of transcriptional activity of genes using promoter sequence is fundamental to the engineering of biological systems for industrial purposes and understanding the natural variation in gene expression. To catalyze the development of new algorithms for this purpose, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) organized a community challenge seeking predictive models of promoter activity given normalized promoter activity data for 90 ribosomal protein promoters driving expression of a fluorescent reporter gene. By developing an unbiased modeling approach that performs an iterative search for predictive DNA sequence features using the frequencies of various k-mers, inferred DNA mechanical properties and spatial positions of promoter sequences, we achieved the best performer status in this challenge. The specific predictive features used in the model included the frequency of the nucleotide G, the length of polymeric tracts of T and TA, the frequencies of 6 distinct trinucleotides and 12 tetranucleotides, and the predicted protein deformability of the DNA sequence. Our method accurately predicted the activity of 20 natural variants of ribosomal protein promoters (Spearman correlation r = 0.73) as compared to 33 laboratory-mutated variants of the promoters (r = 0.57) in a test set that was hidden from participants. Notably, our model differed substantially from the rest in 2 main ways: i) it did not explicitly utilize transcription factor binding information implying that subtle DNA sequence features are highly associated with gene expression, and ii) it was entirely based on features extracted exclusively from the 100 bp region upstream from the translational start site demonstrating that this region encodes much of the overall promoter activity. The findings from this study have important implications for the engineering of predictable gene expression systems and the evolution of gene expression in naturally occurring

  2. Regularities in the E.coli promoters composition in connection with the DNA strands interaction and promoter activity

    BEREZHNOY Andrey Yu; SHCKORBATOV Yuriy G.; HISANORI Kiryu

    2006-01-01

    The energy of interaction between DNA strands in promoters is of great functional importance. Visualization of the energy of DNA strands distribution in promoter sequences was achieved. The separation of promoters in groups by their energetic properties enables evaluation of the dependence of promoter strength on the energetic properties. The analysis of groups (clusters)of promoters distributed by the energy of DNA strands interaction in -55, -35, -10 and +6 sequences indicates their connection with the transcriptional activity.

  3. nifH Promoter Activity Is Regulated by DNA Supercoiling in Sinorhizobium meliloti

    Yan-Jie LIU; Biao HU; Jia-Bi ZHU; Shan-Jiong SHEN; Guan-Qiao YU

    2005-01-01

    In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown cells, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoilingdependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.

  4. Lamin A Is an Endogenous SIRT6 Activator and Promotes SIRT6-Mediated DNA Repair

    Shrestha Ghosh

    2015-11-01

    Full Text Available The nuclear lamins are essential for various molecular events in the nucleus, such as chromatin organization, DNA replication, and provision of mechanical support. A specific point mutation in the LMNA gene creates a truncated prelamin A termed progerin, causing Hutchinson-Gilford progeria syndrome (HGPS. SIRT6 deficiency leads to defective genomic maintenance and accelerated aging similar to HGPS, suggesting a potential link between lamin A and SIRT6. Here, we report that lamin A is an endogenous activator of SIRT6 and facilitates chromatin localization of SIRT6 upon DNA damage. Lamin A promotes SIRT6-dependent DNA-PKcs (DNA-PK catalytic subunit recruitment to chromatin, CtIP deacetylation, and PARP1 mono-ADP ribosylation in response to DNA damage. The presence of progerin jeopardizes SIRT6 activation and compromises SIRT6-mediated molecular events in response to DNA damage. These data reveal a critical role for lamin A in regulating SIRT6 activities, suggesting that defects in SIRT6 functions contribute to impaired DNA repair and accelerated aging in HGPS.

  5. Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

    Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

    2013-09-20

    Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. PMID:23902794

  6. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

    Jakob Madsen Pedersen

    Full Text Available To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

  7. Use of liposome-mediated DNA transfection to determine promoter activity in smooth muscle cells.

    Fabunmi, R P

    1999-01-01

    The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosidase, chloramphenicol acetyl transferase, and green flourescent protein are ideal to study promoter activities as their gene products are not endogenous to smooth muscle cells (SMC) and their expression can be readily detected using convenient assays (1). Among these genes, a popular choice is the firefly luciferase, as its expression can be easily detected in cells using a highly sensitive chemiluminescent assay (2). The firefly luciferase catalyses a rapid, ATP-dependent oxidation of the substrate, luciferin, which then emits light. Reactions catalyzed by firefly luciferase are: [Formula: see text]. PMID:21341022

  8. Isolation and characterization of the hamster gadd153 gene. Activation of promoter activity by agents that damage DNA

    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.

  9. Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

    Windelinckx Saskia

    2009-06-01

    Full Text Available Abstract Background Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc+ gene and low temperature-responsive luciferase activation was monitored in real time. Results Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26°C followed by a gradual decrease to 8°C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed. Conclusion This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate

  10. Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae.

    Margarida Carrolo

    Full Text Available Streptococcus pneumoniae (pneumococcus is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.

  11. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- XTM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-XTM genome, which was transformed into E. coli to get recombinant Adeno-XTM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 1011 TCID50/ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-XTM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

  12. Activation of the Pleiotropic Drug Resistance Pathway Can Promote Mitochondrial DNA Retention by Fusion-Defective Mitochondria in Saccharomyces cerevisiae

    Dunn, Cory, D.; Mutlu, Nebibe; Garipler, Gorkem; Akdogan, Emel

    2014-01-01

    1 Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae Nebibe Mutlu1, Görkem Garipler, Emel Akdoğan and Cory D. Dunn Department of Molecular Biology and Genetics Koç University Sarıyer, İstanbul, 34450 Turkey 1 Present address: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, 48109, U.S.A. NCBI Sequence...

  13. Active DNA Demethylation Mediated by DNA Glycosylases

    Zhu, Jian-Kang

    2009-01-01

    Active DNA demethylation is involved in many vital developmental and physiological processes of plants and animals. Recent genetic and biochemical studies in Arabidopsis have demonstrated that a subfamily of DNA glycosylases function to promote DNA demethylation through a base excision-repair pathway. These specialized bifunctional DNA glycosylases remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, resulting in a gap that is then filled with an unmethylated ...

  14. DNA signals at isoform promoters.

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  15. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  16. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    Kiran, Shashi; Oddi, Vineesha [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences, Delhi 110070 (India)

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  17. Placental DNA methylation of peroxisome-proliferator-activated receptor-γ co-activator-1α promoter is associated with maternal gestational glucose level.

    Xie, Xuemei; Gao, Hongjie; Zeng, Wanjiang; Chen, Suhua; Feng, Ling; Deng, Dongrui; Qiao, Fu-yuan; Liao, Lihong; McCormick, Kenneth; Ning, Qin; Luo, Xiaoping

    2015-08-01

    Intrauterine exposure to hyperglycaemia may increase the risk of later-life metabolic disorders. Although the underlying mechanism is not fully understood, epigenetic dysregulation in fetal programming has been implicated. With regard to energy homoeostasis, PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α, encoded by the PPARGC1A gene) plays a regulatory role in several biochemical processes. We hypothesized that maternal gestational glucose levels would positively correlate with DNA methylation of the PPARGC1A promoter in placental tissue. We undertook a cross-sectional study of 58 mothers who underwent uncomplicated Caesarean delivery in a university hospital. Maternal gestational glucose concentration was determined after a 75-g OGTT (oral glucose tolerance test) at 24-28 weeks of gestation. Placenta tissue and cord blood were collected immediately after delivery. Genomic DNA was extracted and thereafter bisulfite conversion was performed. After PCR amplification, the DNA methylation of the PPARGC1A promoter was quantified using a pyrosequencing technique. The protein level of PGC-1α was evaluated by Western blotting. For all participants as a whole, including the GDM (gestational diabetes mellitus) and normoglycaemia groups, the maternal gestational glucose level was positively correlated with placental DNA methylation, and negatively correlated with cord blood DNA methylation of the PPARGC1A promoter in a CpG site-specific manner. In the GDM group alone, the placental CpG site-specific methylation of the PPARGC1A promoter strongly correlated with gestational 2-h post-OGTT glycaemia. Epigenetic alteration of the PPAGRC1A promoter may be one of the potential mechanisms underlying the metabolic programming in offspring exposed to intrauterine hyperglycaemia. PMID:25875376

  18. In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

    Ajit Kumar Passari

    Full Text Available Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA ranging between 10-32 μg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 μg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 μg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM and chitinase (chiC were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34 and Leifsonia xyli (BPSAC24 were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L. under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from

  19. Computational analysis of promoters and DNA-protein interactions

    Tomovic, Andrija

    2009-01-01

    The investigation of promoter activity and DNA-protein interactions is very important for understanding many crucial cellular processes, including transcription, recombination and replication. Promoter activity and DNA-protein interactions can be studied in the lab (in vitro or in vivo) or using computational methods (in silico). Computational approaches for analysing promoters and DNA-protein interactions have become more powerful as more and more complete genome sequences, 3D...

  20. Conjugative DNA Transfer Induces the Bacterial SOS Response and Promotes Antibiotic Resistance Development through Integron Activation

    Baharoglu, Zeynep; Bikard, David; Mazel, Didier

    2010-01-01

    Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response contr...

  1. Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation.

    Zeynep Baharoglu

    2010-10-01

    Full Text Available Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

  2. In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants

    Ajit Kumar Passari; Vineet Kumar Mishra; Vijai Kumar Gupta; Mukesh Kumar Yadav; Ratul Saikia; Bhim Pratap Singh

    2015-01-01

    Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using...

  3. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager; Andreasen, Lotte; Mundbjerg, Kamilla; Kruhøffer, Mogens; Brinch, Marie; Schierup, Mikkel Heide; Bjergbæk, Lotte; Andersen, Anni Hangaard

    -depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and...... elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during...

  4. O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

    Bobola, Michael S.; Alnoor, Mohammad; Chen, John Y.-S.; Kolstoe, Douglas D.; Silbergeld, Daniel L.; Rostomily, Robert C.; Blank, A.; Chamberlain, Marc C.; Silber, John R.

    2014-01-01

    Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. PMID:25558448

  5. Finding human promoter groups based on DNA physical properties

    Zeng, Jia; Cao, Xiao-Qin; Zhao, Hongya; Yan, Hong

    2009-10-01

    DNA rigidity is an important physical property originating from the DNA three-dimensional structure. Although the general DNA rigidity patterns in human promoters have been investigated, their distinct roles in transcription are largely unknown. In this paper, we discover four highly distinct human promoter groups based on similarity of their rigidity profiles. First, we find that all promoter groups conserve relatively rigid DNAs at the canonical TATA box [a consensus TATA(A/T)A(A/T) sequence] position, which are important physical signals in binding transcription factors. Second, we find that the genes activated by each group of promoters share significant biological functions based on their gene ontology annotations. Finally, we find that these human promoter groups correlate with the tissue-specific gene expression.

  6. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  7. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  8. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation

  9. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line.

    Song, Deye; Ni, Jiangdong; Xie, Hongming; Ding, Muliang; Wang, Jun

    2014-05-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  10. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line

    SONG, DEYE; NI, JIANGDONG; XIE, HONGMING; DING, MULIANG; WANG, JUN

    2014-01-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  11. DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene

    Giuseppina M. Carbone; McGuffie, Eileen; Napoli, Sara; Flanagan, Courtney E.; Dembech, Chiara; Negri, Umberto; Arcamone, Federico; Capobianco, Massimo L.; Catapano, Carlo V

    2004-01-01

    Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of ...

  12. UV Damage in DNA Promotes Nucleosome Unwrapping*

    Duan, Ming-Rui; Smerdon, Michael J.

    2010-01-01

    The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA dam...

  13. Recruitment of TATA-Binding Protein–TAFI Complex SL1 to the Human Ribosomal DNA Promoter Is Mediated by the Carboxy-Terminal Activation Domain of Upstream Binding Factor (UBF) and Is Regulated by UBF Phosphorylation

    Tuan, JoAnn C.; Zhai, Weiguo; Comai, Lucio

    1999-01-01

    Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any seque...

  14. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

    Lukas, C; Kramer, E R; Peters, J M;

    2000-01-01

    Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with...

  15. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    Kukshal, Vandna; Kim, In-Kwon; Gregory L. Hura; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with ...

  16. Catalytic DNA with phosphatase activity

    Chandrasekar, Jagadeeswaran; Silverman, Scott K.

    2013-01-01

    Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn2+-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase ac...

  17. Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation.

    Umebayashi, Mayumi; Sumita, Yoshinori; Kawai, Yousuke; Watanabe, Sumiko; Asahina, Izumi

    2015-01-01

    To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and β-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic

  18. Osmotic pressure: resisting or promoting DNA ejection from phage

    Jeembaeva, Meerim; Larsson, Frida; Evilevitch, Alex

    2008-01-01

    Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I on the course of ejection. We argue in this work by combination of experimental techniques (osmotic suppression without DNaseI monitored by UV absorbance, pulse-field electrophoresis, and cryo-EM visualization) and simple scaling modeling that intact genome (i.e. undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resisting it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well...

  19. Roundup® exposure promotes gills and liver impairments, DNA damage and inhibition of brain cholinergic activity in the Amazon teleost fish Colossoma macropomum.

    Braz-Mota, Susana; Sadauskas-Henrique, Helen; Duarte, Rafael M; Val, Adalberto L; Almeida-Val, Vera M F

    2015-09-01

    Roundup Original® (RD) is a glyphosate-based herbicide used to control weeds in agriculture. Contamination of Amazon waters has increased as a consequence of anthropogenic pressure, including the use of herbicides as RD. The central goal of this study was to evaluate the toxic effects of RD on juveniles of tambaqui (Colossoma macropomum). Our findings show that biomarkers in tambaqui are organ specific and dependent on RD concentration. Alterations in gills structural and respiratory epithelium were followed by changes in hematological parameters such as concentration of hemoglobin, particularly in fish exposed to the higher concentration tested (75% of RD LC50 96 h). In addition, both RD concentrations affected the biotransformation process in gills of tambaqui negatively. Instead, liver responses suggest that a production of reactive oxygen species (ROS) occurred in fish exposed to RD, particularly in the animals exposed to 75% RD, as seen by imbalances in biotransformation and antioxidant systems. The increased DNA damage observed in red blood cells of tambaqui exposed to RD is in agreement with this hypothesis. Finally, both tested sub-lethal concentrations of RD markedly inhibited the cholinesterase activity in fish brain. Thus, we can suggest that RD is potentially toxic to tambaqui and possibly to other tropical fish species. PMID:25898390

  20. Human neuronal tau promoting the melting temperature of DNA

    2000-01-01

    The hyperchromic effect of ultraviolet spectroscopy shows that adding recombinant human neuronal tau to the solution of calf thymus DNA will promote the melting temperature (Tm) from 67℃ to 81℃. Similar result has been detected when adding tau to plasmid pBluescript-Ⅱ SK, by raising Tm from 75℃ to 85℃. The kinetics of thermal denaturation of DNA with tau is much slower than that of control. It suggests that tau may stabilize the double helix conformation of DNA.

  1. Heterogeneity in the properties of burst-forming units of erythroid lineage in sickle cell anemia: DNA synthesis and burst-promoting activity production is related to peripheral hemoglobin F levels

    Circulating 14-day erythroid progenitors (BFU-E) from 28 sickle cell anemia (SS) patients with hemoglobin F (HbF) levels ranging from 2% to 16% were studied to determine their sensitivity to [3H] thymidine kill and burst-promoting activity (BPA)-like factor production. We find that the proportion of BFU-E sensitive to 3H-dT kill, and hence active in DNA synthesis, was inversely correlated with the percent of peripheral HbF when light density (LD) mononuclear cells were used for plating. Regression analysis showed that the correlation between HbF level and BFU-E kill was highly significant (r = .88; P less than .00003). We confirmed the BPA-like factor(s) production by LD mononuclear cells of SS patients, and found, in addition, that this phenomenon is restricted to the population of SS patients with HbF levels lower than 9%. Circulating BFU-E of patients with high HbF levels are not sensitive to 3H-dT, and their mononuclear cells do not release BPA-like factor. In summary, SS patients exhibit differences in the capacity of their mononuclear cells to produce BPA activity according to their peripheral HbF level, as well as to the DNA synthesis-state of their circulating BFU-E. We conclude that erythroid progenitors differ among SS patients in relation to their peripheral HbF level

  2. International energy-promotion-activities

    NONE

    1995-09-01

    Comprehensive promotion of energy and environmental measures are demanded in order to realize improvement in energy demand/supply structures in developing countries where increase in energy demand is anticipated. To achieve this goal, technical transfer related to energy saving technologies and clean coal as well as international energy promotion activities are implemented in China and Indonesia since fiscal 1993. In the field of energy saving, model operations are performed to improve efficiency in such energy consuming fields as steel making, power generation, and oil refining, in addition to cooperation in structuring databases and establishing master plans. In the clean coal field, model operations are conducted to reduce environmental load in coal utilizing areas, in addition to cooperation in establishing master plans for coal utilization. This paper describes feasibility studies on environmentally harmonious coal utilization systems in developing countries, assistance to introduction thereof, and joint verification operations. To rationalize international energy usage, basic surveys on energy utilization efficiency improvement and model operations are carried out mainly in the Asia-Pacific countries.

  3. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  4. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

    Sen, Doyel; Patel, Gayatri; Patel, Smita S

    2016-05-19

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE-a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  5. Species dependence of the major late promoter in adenovirus type 12 DNA.

    Weyer, U; Doerfler, W

    1985-01-01

    In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the exp...

  6. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    Ganna Bilousova

    2005-12-01

    Full Text Available Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.

  7. The Bacillus subtilis DnaD and DnaB Proteins Exhibit Different DNA Remodelling Activities

    Zhang, Wenke; Carneiro, Maria J. V. M.; Turner, Ian J.; ALLEN, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2005-01-01

    Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a “square-like” tetramer with a hole in the ...

  8. TDP1 promotes assembly of non-homologous end joining protein complexes on DNA.

    Heo, Jinho; Li, Jing; Summerlin, Matthew; Hays, Annette; Katyal, Sachin; McKinnon, Peter J; Nitiss, Karin C; Nitiss, John L; Hanakahi, Leslyn A

    2015-06-01

    The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. PMID:25841101

  9. Rep provides a second motor at the replisome to promote duplication of protein-bound DNA.

    Guy, Colin P; Atkinson, John; Gupta, Milind K; Mahdi, Akeel A; Gwynn, Emma J; Rudolph, Christian J; Moon, Peter B; van Knippenberg, Ingeborg C; Cadman, Chris J; Dillingham, Mark S; Lloyd, Robert G; McGlynn, Peter

    2009-11-25

    Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA. PMID:19941825

  10. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  11. 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

    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.

  12. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. PMID:23598277

  13. Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

    Giladi, H; Goldenberg, D; Koby, S; Oppenheim, A B

    1995-01-01

    The response of the early phage lambda PL promoter to temperature was investigated. Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature. The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures. IHF protein binds DNA in vi...

  14. Promoting 21st-Century Skills in the Science Classroom by Adapting Cookbook Lab Activities: The Case of DNA Extraction of Wheat Germ

    Alozie, Nonye M.; Grueber, David J.; Dereski, Mary O.

    2012-01-01

    How can science instruction engage students in 21st-century skills and inquiry-based learning, even when doing simple labs in the classroom? We collaborated with teachers in professional development workshops to transform "cookbook" activities into engaging laboratory experiences. We show how to change the common classroom activity of DNA…

  15. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

    Riballo, Enriqueta; Woodbine, Lisa; Stiff, Thomas; Walker, Sarah A; Goodarzi, Aaron A; Jeggo, Penny A

    2009-02-01

    XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex. PMID:19056826

  16. Biological activity of SV40 DNA

    This thesis deals with a study on the biological activity of SV40 DNA. The transforming activity of SV40 DNA and DNA fragments is investigated in order to define as precisely as possible the area of the viral genome that is involved in the transformation. The infectivity of SV40 DNA is used to study the defective repair mechanisms of radiation damages of human xeroderma pigmentosum cells. (C.F.)

  17. Promoting and avoiding recombination: contrasting activities of the Escherichia coli RuvABC Holliday junction resolvase and RecG DNA translocase.

    Zhang, Jing; Mahdi, Akeel A; Briggs, Geoffrey S; Lloyd, Robert G

    2010-05-01

    RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC. PMID:20157002

  18. Promotion marketing activities in universities

    Guseva I.B.; Ledentcova E.A.

    2014-01-01

    The article discusses the need for promotion of educational services through such means of marketing communications as advertising and personal selling , able to satisfy user requests. The results of market research - questioning school graduates of Perm, which was carried out in order to create an effective advertising campaign to attract entrants. Experience can be used in the advertising campaign universities in Russia , in particular , Perm State National Research University.

  19. FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress.

    Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Sung, Patrick; Schildkraut, Carl L; Schildkraut, Carl; Pagano, Michele

    2013-01-21

    Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress. PMID:23319600

  20. THE PROMOTIONAL ACTIVITY IN THE TOURISTIC SECTOR

    Costel Iliuta Negricea

    2008-05-01

    Full Text Available The promotion as one of the components of the marketing mix, laying stress, în this regard,on its role în the deployment of the tourism companies’ activity, the structure of the promotional activity în thetouristic sector as well as the use of the promotional strategies în the attainment of the development targets ofthe tourism companies.So, în the paper there have been mentioned the three levels at which it is made the touristic promotionîn Romania, respectively nationally, by the Ministry of the Tourism, under whose subordination it is theTourism National Authority, the second level is the regional/local one, concerning the activity carried out bythe Centers/Offices of Touristic Information from a series of localities, and the last level refers to the microone, respectively at the level of the tourism companies, which promote their offer individually (the most often.The important role of the promotion în the deployment of the activity of the tourism companies isbeing highlighted by the fact that this makes the connection between the activity of an organization and itscustomers (effective or potential, and, în the touristic field, the content of the promotional activity is stronglystressed by the features of this type of services and of the system of creation and delivery, as well as of thepurchasing behaviour.

  1. Evaluation of Results from Sales Promotion Activities

    Olimpia Ban

    2007-01-01

    An essential element of the sales promotion strategy and not only is the evaluation of the results obtained from the activities performed. Due to their nature and applicability, the evaluation of the sales promotion is much easier to be achieved, but it raises some problems. Using a hypothetical example, we have tried to develop a "classic" evaluation model of the specialty literature.

  2. Public Relations as Promotional Activity

    Almira CURRI-MEMETI

    2011-11-01

    Full Text Available Public relations give opportunity to the organization to present its image and personality to its own “public”- users, supporters, sponsors, donors, local community and other public.It is about transferring the message to the public, but that is a twoway street. You must communicate with your public, but at the same time you must give opportunity to the public to communicate easier with you. The real public relations include dialog – you should listen to the others, to see things through their perspective. This elaborate is made with the purpose to be useful for every organization, not for the sensational promotion of its achievements, but to become more critical towards its work. Seeing the organization in the way that the other see it, you can become better and sure that you are giving to your users the best service possible.

  3. Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends.

    Tsai, Chun J; Kim, Sunny A; Chu, Gilbert

    2007-05-01

    Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation or V(D)J recombination of the immunoglobulin genes. The breaks often leave mismatched or nonligatable ends, and NHEJ must repair the breaks with high efficiency and minimal nucleotide loss. Here, the NHEJ proteins Ku, DNA-dependent protein kinase catalytic subunit, XRCC4/Ligase IV, and Cernunnos/XRCC4-like factor joined mismatched and noncohesive DNA ends in the absence of processing factors. Depending on the mismatch, Cernunnos stimulated joining 8- to 150-fold. For substrates with a blunt end and a 3' overhanging end, Ku, XRCC4/Ligase IV, and Cernunnos ligated the 3' overhanging hydroxyl group to the 5' phosphate of the blunt end, leaving the other strand unjoined. This activity provides a mechanism for retaining 3' overhang sequences, as observed during V(D)J recombination in vivo. Thus, Cernunnos/XRCC4-like factor promotes a mismatched end (MEnd) DNA ligase activity to facilitate joining and to preserve DNA sequence. Furthermore, MEnd ligase activity may have applications in recombinant DNA technology. PMID:17470781

  4. Rep Provides a Second Motor at the Replisome to Promote Duplication of Protein-Bound DNA

    Guy, Colin P.; Atkinson, John; Gupta, Milind K.; Mahdi, Akeel A.; Gwynn, Emma J.; Rudolph, Christian J.; Moon, Peter B.; van Knippenberg, Ingeborg C.; Cadman, Chris J.; Dillingham, Mark S.; Lloyd, Robert G.; McGlynn, Peter

    2009-01-01

    Summary Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vi...

  5. Promoting physical activity in socially vulnerable groups

    Herens, M.C.

    2016-01-01

    Background:  In the Netherlands, inequalities in physical activity behaviour go hand in hand with socioeconomic inequalities in health. To promote physical activity effectively and equitably, participatory community-based physical activity interventions seem promising and are s

  6. Regulation of ALF promoter activity in Xenopus oocytes.

    Dan Li

    Full Text Available BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

  7. Promoting Physical Activity among Underserved Populations.

    Mendoza-Vasconez, Andrea S; Linke, Sarah; Muñoz, Mario; Pekmezi, Dori; Ainsworth, Cole; Cano, Mayra; Williams, Victoria; Marcus, Bess H; Larsen, Britta A

    2016-01-01

    Underserved populations, including racial/ethnic minorities, individuals with low socioeconomic status, and individuals with physical disabilities, are less likely to engage in sufficient moderate to vigorous physical activity (MVPA) and are thus at increased risk of morbidity and mortality. These populations face unique challenges to engaging in MVPA. Learning how to overcome these challenges is a necessary first step in achieving health equity through health promotion research. In this review of the literature, we discuss issues and strategies that have been used to promote MVPA among individuals from underserved populations, focusing on recruitment, intervention delivery, and the use of technology in interventions. Physical activity promotion research among these vulnerable populations is scarce. Nevertheless, there is preliminary evidence of efficacy in the use of certain recruitment and intervention strategies including tailoring, cultural adaptation, incorporation of new technologies, and multilevel and community-based approaches for physical activity promotion among different underserved populations. PMID:27399827

  8. CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

    Y. P. Wang; Kolb, A; M. Buck; Wen, J.; O'Gara, F.; Buc, H

    1998-01-01

    The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the sta...

  9. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    La, Honggui

    2011-09-06

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

  10. INTRANUCLEAR MATRIX METALLOPROTEINASES PROMOTE DNA DAMAGE AND APOPTOSIS INDUCED BY OXYGEN–GLUCOSE DEPRIVATION IN NEURONS

    HILL, J. W.; PODDAR, R.; THOMPSON, J. F.; ROSENBERG, G. A.; YANG, Y.

    2016-01-01

    Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood–brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24 h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons

  11. Promoting physical activity in socially vulnerable groups

    Herens, M.C.

    2016-01-01

    Background:  In the Netherlands, inequalities in physical activity behaviour go hand in hand with socioeconomic inequalities in health. To promote physical activity effectively and equitably, participatory community-based physical activity interventions seem promising and are supported by the Dutch government’s policy. Although many strategies have been developed to increase physical activity levels in general and in socially vulnerable groups in particular, most evaluations show o...

  12. The Evolution of Physical Activity Promotion

    Richards, Elizabeth

    2015-01-01

    Overview: A physically active lifestyle has numerous physical and mental health benefits for patients of all ages. Despite these significant benefits, a majority of Americans do not meet current physical activity guidelines. Health care providers, especially nurses, play a vital role in physical activity promotion. Over the past several decades, exercise and physical activity guidelines have evolved from a focus on structured, vigorous exercise to a focus on moderate-intensity “lifestyle” phy...

  13. NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection

    Wei, Meili; Wang, Lu; Wu, Tao; Xi, Jun; Han, Yuze; Yang, Xingxiang; Zhang, Ding; Fang, Qiang

    2016-01-01

    Mycobacterium tuberculosis (Mtb) infection activates the NLRP3 inflammasome in macrophages and dendritic cells. Much attention has been paid to the mechanisms for regulation of NLRP3 against Mtb. However, whether epigenetic mechanisms participated in NLRP3 activation is still little known. Here we showed that NLRP3 activation was regulated by DNA methylation modification. Mtb infection promoted NLRP3 activation and inflammatory cytokines expression. NLRP3 promoter was cloned and subsequently identified by Dual-Luciferase Reporter System. The results showed that NLRP3 promoter activity was decreased after methylation by DNA methylase Sss I in vitro. Meanwhile, DNA methyltransferases inhibitor DAC could upregulate the expression of NLRP3. Furthermore, promoter region of NLRP3 gene was demethylated after Mtb H37Rv strain infection. These data revealed that DNA methylation was involved in NLRP3 inflammasome activation during Mtb infection and provided a new insight into the relationship between host and pathogens. PMID:27366746

  14. Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity.

    Kaiserman, H B; Benbow, R. M.

    1987-01-01

    We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rathe...

  15. Evaluation of Results from Sales Promotion Activities

    Olimpia Ban

    2007-02-01

    Full Text Available An essential element of the sales promotion strategy and not only is the evaluation of the results obtained from the activities performed. Due to their nature and applicability, the evaluation of the sales promotion is much easier to be achieved, but it raises some problems. Using a hypothetical example, we have tried to develop a "classic" evaluation model of the specialty literature.

  16. TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

    Meller, Stephan; Domizio, Jeremy Di; Voo, Kui S; Friedrich, Heike C; Chamilos, Georgios; Ganguly, Dipyaman; Conrad, Curdin; Gregorio, Josh; Roy, Didier Le; Roger, Thierry; Ladbury, John E; Homey, Bernhard; Watowich, Stanley; Modlin, Robert L; Kontoyiannis, Dimitrios P; Liu, Yong-Jun; Arold, Stefan T; Gilliet, Michel

    2016-01-01

    Interleukin 17–producing helper T cells (TH17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human TH17 cell–derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, TH17 cell–derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26–DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of TH17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death. PMID:26168081

  17. Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links*

    Kumari, Anuradha; Minko, Irina G.; Smith, Rebecca L.; Lloyd, R. Stephen; McCullough, Amanda K.

    2010-01-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3′ to 5′ direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD int...

  18. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

    Chih-Jie Shen

    2014-01-01

    Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

  19. Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects

    Switzeny Olivier J

    2012-10-01

    Full Text Available Abstract Background Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1 gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS, we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2 and impaired fasting glucose (IFG. Methods In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student’s two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount. Results The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate. Conclusion This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a

  20. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with 32P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of 32P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined

  1. Effects of DNA strand breaks on transcription by RNA polymerase III: insights into the role of TFIIIB and the polarity of promoter opening

    Kassavetis, George A.; Grove, Anne; Geiduschek, E.Peter

    2002-01-01

    Certain deletion mutants of the Brf1 and Bdp1 subunits of transcription factor (TF) IIIB retain the ability to recruit RNA polymerase (pol) III to its promoters, but fail to support promoter opening: deletions within an internal Bdp1 segment interfere with initiation of DNA strand separation, and an N-terminal Brf1 deletion blocks propagation of promoter opening past the transcriptional start site. The ability of DNA strand breaks to restore pol III transcription activity to these defective T...

  2. cis Determinants of Promoter Threshold and Activation Timescale

    Anders S. Hansen

    2015-08-01

    Full Text Available Although the relationship between DNA cis-regulatory sequences and gene expression has been extensively studied at steady state, how cis-regulatory sequences affect the dynamics of gene induction is not known. The dynamics of gene induction can be described by the promoter activation timescale (AcTime and amplitude threshold (AmpThr. Combining high-throughput microfluidics with quantitative time-lapse microscopy, we control the activation dynamics of the budding yeast transcription factor, Msn2, and reveal how cis-regulatory motifs in 20 promoter variants of the Msn2-target-gene SIP18 affect AcTime and AmpThr. By modulating Msn2 binding sites, we can decouple AmpThr from AcTime and switch the SIP18 promoter class from high AmpThr and slow AcTime to low AmpThr and either fast or slow AcTime. We present a model that quantitatively explains gene-induction dynamics on the basis of the Msn2-binding-site number, TATA box location, and promoter nucleosome organization. Overall, we elucidate the cis-regulatory logic underlying promoter decoding of TF dynamics.

  3. ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3

    Subramaniam, Srinivasa; Zirrgiebel, Ute; von Bohlen und Halbach, Oliver; Strelau, Jens; Laliberté, Christine; Kaplan, David R.; Unsicker, Klaus

    2004-01-01

    Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the “death” phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)–, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-n...

  4. SUMO-1 possesses DNA binding activity

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  5. Gene activation by induced DNA rearrangements

    A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10(-7), whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10(6) and 67 per 10(6) cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33 degrees C) for varying periods of time (1-5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5 degrees C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9-52 per 10(6) cells over 1-5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome

  6. Alpha-Lipoic acid counteracts the promoted oxidative DNA damage in the liver of septic rats

    Viral, parasitic infections and chemical carcinogens are among the etiological factors of liver cancer. It seems important to study the initiating and promoting agents to evaluate the etiology and prevention of such life threatening disease. Intestine-derived bacteria product, lipopolysaccharide (LPS), is mainly detoxified by the liver. It has shown to induce a state of oxidative DNA damage is not fully investigated. Increased oxidative DNA damage and rate of cell proliferation may initiate or even promote cancer. In the present work, the capability of LPS to induce 8-hydroxydeoxyguanosine (8-HDG), a specific DNA adduct for oxidative DNA damage, in rat livers is tested. Furthermore, a possible protective effect of alpha lipoic acid (ALA) is also assessed. Investigated parameters are liver contents of glutathione (GSH), lipid peroxides (MDA), nitric oxide (NO) and 8-HDG in the liver-extracted DNA. Serum activities of ALT, AST and GGT as liver-function markers as well as IL2 are assessed. Moreover, liver histology is examined. LPS was given doses of 1, 3, 5, 7 and 9 mg/kg once i.p. while, the rat mortality was examined 24 hours later. ALA was given in doses of 50, 100 and 200 mg/kg once i.p. 3h before LPS is found to be 5mg/kg. LPS increased the level of 8-HDG, MDA and NO in the liver. It also induced acute liver necrosis and inflammatory cell infiltration as shown in liver-histopathology and in the significant increase in the activities of ALT, AST and GGT. LPS increased the serum level of IL2 as well. The dose 200mg/kg of ALA revealed a 100% protection against LPS-induced lethality. It also, prevented the LPS-induced increase in 8-HDG in liver extracted DNA, the liver contents of MDA and NO. ALA also rescued the LPS-induced GSH depletion. It corrected the liver function as shown by the prevention of increases in the activity of ALT, AST and GGT with a remarkable improvement in the liver histology. Moreover, it prevented the increase in serum level of IL2. These

  7. Protein found to promote DNA repair, prevent cancer

    2008-01-01

    @@ An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair, researchers at University of Texas reported on-line in the Proceedings of the National Academies of Science.

  8. The murine biglycan: Complete cDNA cloning, genomic organization, promoter function, and expression

    Wegrowski, Y.; Pillarisetti, J.; Danielson, K.G.; Iozzo, R.V. [Thomas Jefferson Univ., Philadelphia, PA (United States); Suzuki, S. [Univ. of Southern California, Los Angeles, CA (United States)

    1995-11-01

    Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5{prime} flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large > 4.5-kb intron between exons 1 and 2. The mouse Bgn gene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-a-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerous cis-acting elements including binding sites for SP-1, AP-1, and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 hp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5{prime} deletion constructs showed that the distal promoter of the Bgn gene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of the Bgn gene. 56 refs., 7 figs., 1 tab.

  9. Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA.

    Roy, Basab; Talukder, Poulami; Kang, Hyun-Jin; Tsuen, Shujian S; Alam, Mohammad P; Hurley, Laurence H; Hecht, Sidney M

    2016-08-31

    The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of gene expression via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein-DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA with RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein-DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-aminobenzo[g]quinazoline-2-one (Cf) in lieu of cytidine22 in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element. PMID:27483029

  10. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

    Orzalli, Megan H.; Conwell, Sara E.; Berrios, Christian; DeCaprio, James A.; Knipe, David M.

    2013-01-01

    Cells have evolved mechanisms to silence foreign DNA to prevent the expression of foreign genes within them. In mammalian cells, this involves the assembly of heterochromatin on foreign DNAs such as viral or transfected DNA. Herpesviruses have evolved strategies to counteract these host mechanisms to express their own genes. Herein we demonstrate that the nuclear DNA sensor IFN-inducible protein 16 (IFI16) is involved in the host silencing response to foreign DNA. IFI16 promotes the assembly ...

  11. Tumour promoter activity in Malaysian Euphorbiaceae.

    Norhanom, A W; Yadav, M

    1995-04-01

    Herbal medication has been practised by the rural Malaysian Malays for a long time. However, the long-term side-effects have never been studied. In the present study, 48 species of Euphorbiaceae were screened for tumour-promoter activity by means of an in vitro assay using a human lymphoblastoid cell line harbouring the Epstein-Barr virus (EBV) genome. Twenty-seven per cent (13 out of 48) of the species tested were found to be positive, and in four species, namely Breynia coronata Hk.f, Codiaeum variegatum (L) Bl, Euphorbia atoto and Exocoecaria agallocha, EBV-inducing activity was observed when the plant extracts were tested at low concentrations of between 0.2 and 1.2 micrograms ml-1 in cell culture. This observation warrants attention from the regular users of these plants because regular use of plants with tumour-promoting activity could well be an aetiological factor for the promotion of tumours among rural Malaysian Malays. PMID:7710943

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

    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.

  13. Identification of Escherichia coli DNA helicase IV with the use of a DNA helicase activity gel.

    Trieu, V N; McCarthy, D

    1989-01-01

    A DNA helicase activity gel was developed based on the assumption that DNA helicases could unwind double-stranded DNA in a polyacrylamide matrix. The production of single-stranded DNA was detected by staining the activity gel with acridine orange and visualizing the gel under long-wave UV light. The products of DNA helicase activities appeared as red bands within a green fluorescent background. A novel DNA helicase, called helicase IV, was detected in crude extracts of Escherichia coli with t...

  14. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint

  15. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  16. THE CONTROL AND EVALUATION OF PROMOTIONAL ACTIVITIES

    Felicia Sabou

    2012-01-01

    Full Text Available The paper focused on importance and benefits of control and evaluation of marketing activities. The control of efficiency review the assessment of the resources for marketing activity, checking also the efficiency of the human resources, advertising, promotion activities and distribution activities. In the analyse of human resources the most important ratio are: the average of costumers visits on a day, the number of custom order received from 100 visits, the number of new customers from a period, the number of lost customers from a period, the marketing human expenditures from all the sales.The strategic control is made to check if the objectives and the company strategy are adapted to the marketing environment.

  17. Using evaluation strategically to promote active learning

    Münster, Marie

    Rationale: The challenge presented here is how to utilise evaluation to promote active learning. The method used is constructive alignment (Biggs & Tang, 2007) of learning objectives, learning and evaluation along with further considerations including which competences are promoted, the time...... for discussions and organising the course with group work allows for plenty of that. Furthermore, as group work is how many companies organise work today – the ability to cooperate well in groups is assessed to be an important competence for engineering students to achieve. The course is taught using...... the principle of inductive learning (Prince & Felder, 2006) with the students being presented with the case from the beginning and subsequently achieving the tools to perform the projects. This is both frustrating and motivating for the students as they know why they need to have the tools, but they feel...

  18. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  19. Loyalty Card Promotional Activity in Budget Hotel

    Teng, Fei

    2010-01-01

    Loyalty card is one of the most commonly used promotional activities in business. Thus far, there are some research has been done on luxury hotel, but very few researches are on budget hotel. So, the purpose of the thesis is finding out the Swedish customers’ attitude and behavior towards budget hotel’s loyalty card; getting to know what factors influence Swedish customers’ response towards the loyalty card and budget hotels. In the thesis, the main research problem is “How do Swedish custome...

  20. Tumour promoter activity in Malaysian Euphorbiaceae.

    Norhanom, A W; Yadav, M

    1995-01-01

    Herbal medication has been practised by the rural Malaysian Malays for a long time. However, the long-term side-effects have never been studied. In the present study, 48 species of Euphorbiaceae were screened for tumour-promoter activity by means of an in vitro assay using a human lymphoblastoid cell line harbouring the Epstein-Barr virus (EBV) genome. Twenty-seven per cent (13 out of 48) of the species tested were found to be positive, and in four species, namely Breynia coronata Hk.f, Codia...

  1. DNA structure in human RNA polymerase II promoters

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves;

    1998-01-01

    in a region upstream of the transcriptional start point and significantly higher downstream. Investigation of the sequence composition in the two regions shows that the bendability profile originates from the sequential structure of the DNA, rather than the general nucleotide composition...... in sequence and bendability, which is in phase with the DNA helical pitch. The periodic bendability profile shows bending peaks roughly at every 10bp with stronger bending at 20bp intervals. These observations suggest that DNA in the region downstream of the transcriptional start point is able to...

  2. The suppression and promotion of DNA charge inversion by mixing counterions.

    Qiu, Shixue; Wang, Yanwei; Cao, Bozhi; Guo, Zilong; Chen, Yang; Yang, Guangcan

    2015-05-28

    In the preset study, we report the suppression and promotion of DNA charge inversion by mixing a quadrivalent counterion (spermine) with mono-, di- and trivalent counterions by dynamic light scattering (DLS) and single molecule electrophoresis (SME) methods. We find that the electrophoretic mobility of DNA in spermine solution decreases in the presence of monovalent sodium ions and divalent magnesium ions. It means that the charge neutralization of DNA by the quadrivalent counterion is suppressed when adding extra mono- or divalent counterions. More specifically, at a high concentration of spermine, the positive mobility can switch back to a negative value by adding mono- and divalent counterions. Thus, charge neutralization and inversion of DNA by quadrivalent counterions is suppressed in the mono- and divalent ion solution. However, the scenario changes dramatically when we add trivalent ions into the solution of DNA and spermine. In this case, the charge neutralization and inversion of DNA is promoted rather than suppressed by mixing with trivalent ions. The negative electrophoretic mobility can be promoted to a positive value, which corresponds to the charge inversion, by trivalent counterions. Thus trivalent and quadrivalent counterions work cooperatively in DNA charge neutralization and inversion. This promotion also occurs when highly positively charged chitosan is introduced into the solution. We explain the observation by the counterion complexation that is related to DNA condensation, which is supported by the images of atomic force microscopy (AFM). PMID:25913809

  3. Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

    Günther, Claudia; Kind, Barbara; Reijns, Martin A.M.; Berndt, Nicole; Martinez-Bueno, Manuel; Wolf, Christine; Tüngler, Victoria; Chara, Osvaldo; Lee, Young Ae; Hübner, Norbert; Bicknell, Louise; Blum, Sophia; Krug, Claudia; Schmidt, Franziska; Kretschmer, Stefanie; Koss, Sarah; Astell, Katy R.; Ramantani, Georgia; Bauerfeind, Anja; Morris, David L.; Cunninghame Graham, Deborah S.; Bubeck, Doryen; Leitch, Andrea; Ralston, Stuart H.; Blackburn, Elizabeth A.; Gahr, Manfred; Witte, Torsten; Vyse, Timothy J.; Melchers, Inga; Mangold, Elisabeth; Nöthen, Markus M.; Aringer, Martin; Kuhn, Annegret; Lüthke, Kirsten; Unger, Leonore; Bley, Annette; Lorenzi, Alice; Isaacs, John D.; Alexopoulou, Dimitra; Conrad, Karsten; Dahl, Andreas; Roers, Axel; Alarcon-Riquelme, Marta E.; Jackson, Andrew P.; Lee-Kirsch, Min Ae

    2014-01-01

    Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2–associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage–associated pathways in the initiation of autoimmunity. PMID:25500883

  4. DNA-based control of protein activity.

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  5. TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

    Meller, Stephan

    2015-07-13

    Interleukin 17-producing helper T cells (TH 17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human TH17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, TH17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of TH17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death. © 2015 Nature America, Inc.

  6. EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma.

    Lu, Jingchen; Tang, Min; Li, Hongde; Xu, Zhijie; Weng, Xinxian; Li, Jiangjiang; Yu, Xinfang; Zhao, Luqing; Liu, Hongwei; Hu, Yongbin; Tan, Zheqiong; Yang, Lifang; Zhong, Meizuo; Zhou, Jian; Fan, Jia; Bode, Ann M; Yi, Wei; Gao, Jinghe; Sun, Lunquan; Cao, Ya

    2016-09-28

    We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy. PMID:27255972

  7. PROMOTION OF ACTIVE MEASURES AND EMPLOYMENT STIMULATION

    LAVINIA ELISABETA POPP

    2012-01-01

    Full Text Available Researches in the field of the labour market has allowed the identification of certain specific mechanisms for employment promotion; at present, on the Romanian labour market we find passive policies, concretised in financial aids paid to the unemployed, along with active policies, constituting the most efficient social protection activity addressed to the unemployed (they aim at counterbalancing the inefficiencies determined by the granting of financial allowances, help population to find a job by actions of information, professional training and contributing to the encouragement of the labour force mobility. The paper refers to some theoretical considerations related to the influence factors of employment stimulation, as well as to the unemployment – correlated adequate measures synapse. The applied research comprises the analysis of statistic documents; the method used is the case study, i.e. the activity of employment stimulation carried on by the County Agency for Employment Caraş-Severin, in the period 2004-2012. The conclusions highlight the impact of the activity of the institutions involved in the system of social protection and security within the labour market.

  8. Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical

    Yavin, Eylon; Boal, Amie K.; Stemp, Eric D. A.; Boon, Elizabeth M; Livingston, Alison L.; O'Shea, Valerie L.; David, Sheila S.; Barton, Jacqueline K.

    2005-01-01

    DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S](2+) cluster of MutY to [4Fe-4S](3+) and its decomposition product [3Fe-4S](1+). Flash/quench experiments monitored by EPR spec...

  9. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.

  10. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

    2014-11-15

    -κB activation inhibited CCL20 expression in mtBALB cybrids and decreased their migratory capabilities. Thus, acquired mtDNA mutations may promote tumorigenic phenotypes through up-regulation of chemokine CCL20.

  11. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    -κB activation inhibited CCL20 expression in mtBALB cybrids and decreased their migratory capabilities. Thus, acquired mtDNA mutations may promote tumorigenic phenotypes through up-regulation of chemokine CCL20

  12. On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

    Belintsev, B N; Zavriev, S.K.; Shemyakin, M.F.

    1980-01-01

    Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is disc...

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

    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.

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

    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.

  15. Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting.

    Lee, Se Hee; Kim, Jeong Myeong; Lee, Hyo Jung; Jeon, Che Ok

    2011-02-01

    We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers. PMID:21259288

  16. Cell Type-Specific Activation of the Cytomegalovirus Promoter by Dimethylsulfoxide and 5-Aza-2′-deoxycytidine

    Radhakrishnan, Prakash; Basma, Hesham; Klinkebiel, David; Christman, Judith; Cheng, Pi-Wan

    2008-01-01

    The cytomegalovirus promoter is a very potent promoter commonly used for driving the expression of transgenes, though it gradually becomes silenced in stably transfected cells. We examined the methylation status of the cytomegalovirus promoter in two different cell lines and characterized its mechanisms of activation by dimethylsulfoxide and 5-Aza-2′-deoxycytidine. The cytomegalovirus promoter stably transfected into Chinese hamster ovary cells is suppressed by DNA methylation-independent mec...

  17. AMP-activated protein kinase-regulated activation of the PGC-1alpha promoter in skeletal muscle cells.

    Isabella Irrcher

    Full Text Available The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.

  18. RNA Pol II promotes transcription of centromeric satellite DNA in beetles.

    Zeljka Pezer

    Full Text Available Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera. This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80% are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.

  19. AGU Activities to Promote Undergraduate Research

    Grove, K.; Johnson, R.; Giesler, J.

    2001-05-01

    A primary goal of the AGU Committee on Education and Human Resources (CEHR) is to significantly increase the participation of undergraduate students at AGU meetings. Involving students in scientific meetings at this level of their education helps them to better prepare for graduate school and for a career in the geophysical sciences. Ongoing CEHR activities to promote undergraduate participation include: (1) sponsoring technical sessions to showcase undergraduate research; (2) sponsoring sessions about careers and other topics of special interest to students; (3) sponsoring workshops to inform faculty about doing research with undergraduates; (4) sponsoring meeting events to partner graduate student mentors with first-time undergraduate attendees; (5) working with sections to create situations where undergraduates and section scientists can interact; (6) creating a guide for first-time meeting attendees; (7) sponsoring an Academic Recruiting Forum at meetings to connect undergraduates with geophysical graduate programs; (8) running a Career Center at meetings to connect students and employers; (9) raising funds for more travel grants to provide more student support to attend meetings; (10) developing a listserve to inform AGU members about opportunities to do research with undergraduates and to involve more members in mentoring activities; and (11) collecting data, such as career outcomes and demographic characteristics of recent Ph.D. recipients, that are of interest to students.

  20. Effect of alternative temozolomide schedules on glioblastoma O 6-methylguanine-DNA methyltransferase activity and survival

    Robinson, C G; Palomo, J M; Rahmathulla, G; McGraw, M; Donze, J; L. Liu; Vogelbaum, M A

    2010-01-01

    Background: O 6-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity. Methods: Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, ...

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

    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

  2. Effect of gamma-irradiated DNA on DNA polymerase activity: a possible mechanism for cell killing

    A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. The mechanism for DNA polymerase inhibition was investigated. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme. The inhibition of DNA polymerase occurs prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation. As in vitro assay for DNA polymerase α and β in irradiated HeLa cells was developed. The activities of both polymerases decreased as the dose received by the cells increased. Both DNA polymerases were found to recover by 2 hr postirradiation. Since DNA repair capability is intimately connected with cell survival, the observed diminution in DNA polymerase activity, following low doses of radiation, could be highly significant

  3. Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways.

    Gietl, Andreas; Holzmeister, Phil; Blombach, Fabian; Schulz, Sarah; von Voithenberg, Lena Voith; Lamb, Don C; Werner, Finn; Tinnefeld, Philip; Grohmann, Dina

    2014-06-01

    During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex. PMID:24744242

  4. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    Alwine, J C

    1985-05-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. PMID:2987671

  5. Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

    Gietl, Andreas; Holzmeister, Phil; Blombach, Fabian; Schulz, Sarah; von Voithenberg, Lena Voith; Lamb, Don C; Werner, Finn; Tinnefeld, Philip; Grohmann, Dina

    2014-01-01

    During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP–DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly differen...

  6. The Vibrio harveyi master quorum-sensing regulator, LuxR, a TetR-type protein is both an activator and a repressor: DNA recognition and binding specificity at target promoters

    Pompeani, Audra J; Irgon, Joseph J; Berger, Michael F; Bulyk, Martha L; Wingreen, Ned S; Bassler, Bonnie L

    2008-01-01

    Quorum sensing is the process of cell-to-cell communication by which bacteria communicate via secreted signal molecules called autoinducers. As cell population density increases, the accumulation of autoinducers leads to co-ordinated changes in gene expression across the bacterial community. The marine bacterium, Vibrio harveyi, uses three autoinducers to achieve intra-species, intra-genera and inter-species cell–cell communication. The detection of these autoinducers ultimately leads to the production of LuxR, the quorum-sensing master regulator that controls expression of the genes in the quorum-sensing regulon. LuxR is a member of the TetR protein superfamily; however, unlike other TetR repressors that typically repress their own gene expression and that of an adjacent operon, LuxR is capable of activating and repressing a large number of genes. Here, we used protein binding microarrays and a two-layered bioinformatics approach to show that LuxR binds a 21 bp consensus operator with dyad symmetry. In vitro and in vivo analyses of two promoters directly regulated by LuxR allowed us to identify those bases that are critical for LuxR binding. Together, the in silico and biochemical results enabled us to scan the genome and identify novel targets of LuxR in V. harveyi and thus expand the understanding of the quorum-sensing regulon. PMID:18681939

  7. The role of CopG mediated DNA bending on the regulation of the σ54-dependent promoters in E. coli

    2006-01-01

    In order to investigate the role of DNA bending on the regulation of σ54-dependent promoters, we introduced the CopG binding site between the enhancer-like element and the core promoter of glnAp2, nifLp and glnHp2, without changing the distance in between. The expression activities of these homologous promoters were either activated or repressed by the CopG-induced DNA bending in E. coli. In this case, similar regulatory pattern (either activated or repressed) could be observed, when the bending centers from CopG are in integral DNA helixes interval, while opposite regulatory pattern could be observed, when the bending centers from CopG are in integral plus a half DNA helixes interval. These results suggested that CopG-induced DNA bending can exert regulatory effects on the transcription of σ54-dependent promoters probably by altering the relative DNA helix phase of 54 RNA polymerase and NtrC.

  8. Genetic variation in the proximal promoter of ABC and SLC superfamilies: liver and kidney specific expression and promoter activity predict variation.

    Stephanie E Hesselson

    Full Text Available Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC and Solute Carrier (SLC superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

  9. Promoter DNA methylation regulates progranulin expression and is altered in FTLD

    Banzhaf-Strathmann, Julia; Claus, Rainer; Mücke, Oliver; Rentzsch, Kristin; van der Zee, Julie; Engelborghs, Sebastiaan; De Deyn, Peter P.; Cruts, Marc; Van Broeckhoven, Christine; Plass, Christoph; Edbauer, Dieter

    2013-01-01

    Background Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of neurodegenerative diseases associated with personality changes and progressive dementia. Loss-of-function mutations in the growth factor progranulin (GRN) cause autosomal dominant FTLD, but so far the pathomechanism of sporadic FTLD is unclear. Results We analyzed whether DNA methylation in the GRN core promoter restricts GRN expression and, thus, might promote FTLD in the absence of GRN mutations. GRN expression ...

  10. Modulation of UvrD helicase activity by covalent DNA-protein cross-links.

    Kumari, Anuradha; Minko, Irina G; Smith, Rebecca L; Lloyd, R Stephen; McCullough, Amanda K

    2010-07-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links. PMID:20444702

  11. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A; Kumar, Sheetal; Kalab, Petr

    2016-04-15

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  12. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-01

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. PMID:26814232

  13. Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53

    Osborn, Alexander J.; Elledge, Stephen J.

    2003-01-01

    When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication f...

  14. Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

    Rivas-Marín, Elena; Floriano, Belén; Santero, Eduardo

    2016-01-01

    Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators. PMID:27087658

  15. The isolation of transcription factors from lambda gt11 cDNA expression libraries: human steroid 5 alpha-reductase 1 has sequence-specific DNA binding activity.

    Gaston, K; Fried, M

    1992-01-01

    The Surf-1/Surf-2 bi-directional promoter contains binding sites for at least three transcription factors (Su1, Su2, and Su3). By screening a lambda gt11 HeLa cell cDNA expression library with a concatenated Su2 factor binding site, we isolated a cDNA which encodes a protein with sequence-specific DNA binding activity. Gel retardation assays showed that the cloned factor binds specifically to the Su2 factor binding site present in the human Surf-1/Surf-2 promoter but not to an Su2 site contai...

  16. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

    Orzalli, Megan H.; Conwell, Sara E.; Berrios, Christian; DeCaprio, James A.; Knipe, David M.

    2013-01-01

    Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate–early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses. PMID:24198334

  17. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA.

    Orzalli, Megan H; Conwell, Sara E; Berrios, Christian; DeCaprio, James A; Knipe, David M

    2013-11-19

    Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate-early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses. PMID:24198334

  18. Hair Growth Promotion Activity and Its Mechanism of Polygonum multiflorum

    Yunfei Li; Mingnuan Han; Pei Lin; Yanran He; Jie Yu; Ronghua Zhao

    2015-01-01

    Polygonum multiflorum Radix (PMR) has long history in hair growth promotion and hair coloring in clinical applications. However, several crucial problems in its clinic usage and mechanisms are still unsolved or lack scientific evidences. In this research, C57BL/6J mice were used to investigate hair growth promotion activity and possible mechanism of PMR and Polygonum multiflorum Radix Preparata (PMRP). Hair growth promotion activities were investigated by hair length, hair covered skin ratio,...

  19. Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

    LEI,Ke-Lin; HOU,Han-Na; LIU,Yi; YE,Xue-Cheng; SHEN,Ping

    2007-01-01

    Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase (CAT) gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.

  20. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  1. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    GuillaumeRiviere

    2014-04-01

    Full Text Available DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster’s developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5’-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment.

  2. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan; M. Bompiani, Kristin; Hansen, Majken Nørgaard; Gothelf, Kurt Vesterager; Sullenger, Bruce A; LaBean, Thomas H.

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation o...

  3. Promoter proximal polyadenylation sites reduce transcription activity

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  4. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  5. Purification of total DNA extracted from activated sludge.

    Shan, Guobin; Jin, Wenbiao; Lam, Edward K H; Xing, Xinhui

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible. PMID:18572527

  6. Purification of total DNA extracted from activated sludge

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

  7. A novel DNA joining activity catalyzed by T4 DNA ligase.

    Western, L M; Rose, S. J.

    1991-01-01

    The use of T4 and E. coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules. We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site. Enzyme concentration, loop size, substrate specificity, and ...

  8. Integrity and Biological Activity of DNA after UV Exposure

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  9. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

    Fabio Lapenta

    Full Text Available DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

  10. Activity-promoting gaming systems in exercise and rehabilitation

    Matthew J. D. Taylor, PhD

    2011-12-01

    Full Text Available Commercial activity-promoting gaming systems provide a potentially attractive means to facilitate exercise and rehabilitation. The Nintendo Wii, Sony EyeToy, Dance Dance Revolution, and Xbox Kinect are examples of gaming systems that use the movement of the player to control gameplay. Activity-promoting gaming systems can be used as a tool to increase activity levels in otherwise sedentary gamers and also be an effective tool to aid rehabilitation in clinical settings. Therefore, the aim of this current work is to review the growing area of activity-promoting gaming in the context of exercise, injury, and rehabilitatio

  11. Active DNA demethylation by oxidation and repair

    Zhizhong Gong; Jian-Kang Zhu

    2011-01-01

    DNA methylation and demethylation are increasingly recognized as important epigenetic factors in both plants and animals.DNA methylation,which is catalyzed by DNA methyltransferases (DNMTs),is a relatively stable and heritable modification that controls gene expression,cellular differentiation,genomic imprinting,paramutation,transposon movement,X-inactivation,and embryogenesis [1].The methylation of cytosine to 5-methylcytosine (5mC) is an important example of DNA modification in animals and plants.This highlight concerns DNA demethylation mechanisms in mammals and whether they are similar to that in plants.

  12. Invariant distribution of promoter activities in Escherichia coli.

    Alon Zaslaver

    2009-10-01

    Full Text Available Cells need to allocate their limited resources to express a wide range of genes. To understand how Escherichia coli partitions its transcriptional resources between its different promoters, we employ a robotic assay using a comprehensive reporter strain library for E. coli to measure promoter activity on a genomic scale at high-temporal resolution and accuracy. This allows continuous tracking of promoter activity as cells change their growth rate from exponential to stationary phase in different media. We find a heavy-tailed distribution of promoter activities, with promoter activities spanning several orders of magnitude. While the shape of the distribution is almost completely independent of the growth conditions, the identity of the promoters expressed at different levels does depend on them. Translation machinery genes, however, keep the same relative expression levels in the distribution across conditions, and their fractional promoter activity tracks growth rate tightly. We present a simple optimization model for resource allocation which suggests that the observed invariant distributions might maximize growth rate. These invariant features of the distribution of promoter activities may suggest design constraints that shape the allocation of transcriptional resources.

  13. Activation of the DNA Damage Response by RNA Viruses

    Ryan, Ellis L.; Robert Hollingworth; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively ...

  14. A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining

    Pawelczak, Katherine S; Turchi, John J.

    2008-01-01

    DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-...

  15. Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis.

    Wang, Xindan; Le, Tung B K; Lajoie, Bryan R; Dekker, Job; Laub, Michael T; Rudner, David Z

    2015-08-01

    SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis. PMID:26253537

  16. Cooperation between core promoter elements influences transcriptional activity in vivo.

    Colgan, J.; Manley, J L

    1995-01-01

    Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effe...

  17. Activity-promoting gaming systems in exercise and rehabilitation

    Matthew J. D. Taylor, PhD; Darren McCormick, BSc; Teshk Shawis, MBChB, FRCP; Rebecca Impson, MSc; Murray Griffin, PhD

    2011-01-01

    Commercial activity-promoting gaming systems provide a potentially attractive means to facilitate exercise and rehabilitation. The Nintendo Wii, Sony EyeToy, Dance Dance Revolution, and Xbox Kinect are examples of gaming systems that use the movement of the player to control gameplay. Activity-promoting gaming systems can be used as a tool to increase activity levels in otherwise sedentary gamers and also be an effective tool to aid rehabilitation in clinical settings. Therefore, the aim of t...

  18. Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences.

    Baum, E Z; Buttner, M J; Lin, L S; Rothstein, D. M.

    1989-01-01

    We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, P1a, P1b, P1c, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters P1a...

  19. DNA polymerase activity and radiation-induced unscheduled synthesis of DNA at the nuclear matrix

    It is shown that both DNA polymerase α and β are involved in DNA synthesis at the nuclear matrix. DNA polymerase β is more firmly attached to the nuclear matrix of normal than of regenerating liver cells. In the nuclear matrix of UV- and gamma-irradiated cells of Zajdela hepatoma a higher level of hydroxyurea-resistant DNA synthesis has been observed in the initial 1.5-5 min of postradiation incubation if compared to that of total nuclear DNA. However 1-β-D-arabinofuranosylcytosine-resistant radiation-induced synthesis of DNA is similar in both the nuclear matrix and the whole nuclei of these cells. Poly(ADP-ribose)synthetase activity is shown to be associated with the nuclear matrix. Inhibition of this activity results in increase of the hydroxyurea-resistant synthesis of DNA at nuclear matrix. (author)

  20. Promoting Physical Activity in Primary Care: Overcoming the barriers

    Singer, Joel; Lindsay, Elizabeth A.; Wilson, Douglas M.C.

    1991-01-01

    The principle barriers preventing health care professionals from promoting physical activity include an incomplete understanding of the evidence linking physical activity and health, difficulty in translating research findings into a feasible and efficacious clinical intervention, resistance to adopting a preventive orientation, and concerns about the risks of physical activity. Low level activities likely provide benefit with little risk.

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

    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

  2. PKA-mediated phosphorylation of ATR promotes recruitment of XPA to UV-induced DNA damage.

    Jarrett, Stuart G; Wolf Horrell, Erin M; Christian, Perry A; Vanover, Jillian C; Boulanger, Mary C; Zou, Yue; D'Orazio, John A

    2014-06-19

    The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals. PMID:24950377

  3. Effect of γ-irradiated DNA on the activity of DNA polymerase

    A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation

  4. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan;

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

  5. Coevolution of teaching activity promotes cooperation

    Evolutionary games are studied where the teaching activity of players can evolve in time. Initially all players following either the cooperative or defecting strategy are distributed on a square lattice. The rate of strategy adoption is determined by the payoff difference and a teaching activity characterizing the donor's capability to enforce its strategy on the opponent. Each successful strategy adoption process is accompanied by an increase in the donor's teaching activity. By applying an optimum value of the increment, this simple mechanism spontaneously creates relevant inhomogeneities in the teaching activities that support the maintenance of cooperation for both the prisoner's dilemma and the snowdrift game

  6. Physical Activity Promotion in Call Centres: Employers' Perspectives

    Renton, Sheila J.; Lightfoot, Nancy E.; Maar, Marion A.

    2011-01-01

    This study followed a predominantly qualitative approach to explore the perspectives of employers in Sudbury, Ontario, Canada, call centres (CCs) regarding physical activity (PA) promotion in workplaces, by identifying current practices and employers' motivation to promote PA, as well as perceived facilitators and barriers. In-depth interviews…

  7. Brazilian physical activity guidelines as a strategy for health promotion

    Emerson Sebastião; Andiara Schwingel; Wojtek Chodzko-Zajko

    2014-01-01

    Public health actions endorsed by the federal government, for instance, health promotion initiatives, usually have greater impact at population level compared to other types of initiatives. This commentary aims to instigate debate on the importance and necessity of producing federally endorsed brazilian physical activity guidelines as a strategy for health promotion.

  8. Promoter and nonspecific DNA binding by the T7 RNA polymerase.

    Smeekens, S.P.; Romano, L J

    1986-01-01

    T7 RNA polymerase plays an important role in both the transcription and replication of bacteriophage T7. In this study we have used a nitrocellulose filter binding assay to examine the binding properties of the T7 RNA polymerase with T7 promoters cloned into plasmid DNAs. Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA. On the other hand, nonpromoter interior-site ...

  9. Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

    Thornton, Tina M; Delgado, Pilar; Chen, Liang; Salas, Beatriz; Krementsov, Dimitry; Fernandez, Miriam; Vernia, Santiago; Davis, Roger J; Heimann, Ruth; Teuscher, Cory; Krangel, Michael S; Ramiro, Almudena R; Rincón, Mercedes

    2016-01-01

    Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response. PMID:26822034

  10. Promoting Physical Activity during Early Childhood

    Vidoni, Carla; Ignico, Arlene

    2011-01-01

    The prevalence of obesity in children and adolescents from low-income families in the USA has become a significant concern over the last 20 years. One of the major contributors to this problem is the lack of physical activity. The purpose of this paper is to describe initiatives designed to: (1) engage young children in physical activity during…

  11. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdkl activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chkl and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  12. miR-29 Represses the Activities of DNA Methyltransferases and DNA Demethylases

    Izuho Hatada

    2013-07-01

    Full Text Available Members of the microRNA-29 (miR-29 family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1 and thymine DNA glycosylase (TDG. Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

  13. Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII

    D'Addario, Claudio; Dell'Osso, Bernardo; Palazzo, Maria Carlotta; Benatti, Beatrice; Lietti, Licia; Cattaneo, Elisabetta; Galimberti, Daniela; Fenoglio, Chiara; Cortini, Francesca; Scarpini, Elio; Arosio, Beatrice; Di Francesco, Andrea; Di Benedetto, Manuela; Romualdi, Patrizia; Candeletti, Sanzio

    2012-01-01

    The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expr...

  14. Improving health through policies that promote active travel

    de Nazelle, Audrey; Nieuwenhuijsen, Mark J; Antó, Josep M;

    2011-01-01

    Substantial policy changes to control obesity, limit chronic disease, and reduce air pollution emissions, including greenhouse gasses, have been recommended. Transportation and planning policies that promote active travel by walking and cycling can contribute to these goals, potentially yielding...

  15. Kinesin KIFC1 actively transports bare double-stranded DNA

    Farina, Francesca; Pierobon, Paolo; Delevoye, Cédric; Monnet, Jordan; Dingli, Florent; Loew, Damarys; Quanz, Maria; Dutreix, Marie; Cappello, Giovanni

    2013-01-01

    During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton fi...

  16. Coevolution of teaching activity promotes cooperation

    Szolnoki, Attila [Research Institute for Technical Physics and Materials Science, PO Box 49, H-1525 Budapest (Hungary); Perc, Matjaz [Department of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroska cesta 160, SI-2000 Maribor (Slovenia)], E-mail: szolnoki@mfa.kfki.hu, E-mail: matjaz.perc@uni-mb.si

    2008-04-15

    Evolutionary games are studied where the teaching activity of players can evolve in time. Initially all players following either the cooperative or defecting strategy are distributed on a square lattice. The rate of strategy adoption is determined by the payoff difference and a teaching activity characterizing the donor's capability to enforce its strategy on the opponent. Each successful strategy adoption process is accompanied by an increase in the donor's teaching activity. By applying an optimum value of the increment, this simple mechanism spontaneously creates relevant inhomogeneities in the teaching activities that support the maintenance of cooperation for both the prisoner's dilemma and the snowdrift game.

  17. DNA methylation dynamics in the rat EGF gene promoter after partial hepatectomy

    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.

  18. APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

    Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

  19. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  20. FANCD2 Maintains Fork Stability in BRCA1/2-Deficient Tumors and Promotes Alternative End-Joining DNA Repair.

    Kais, Zeina; Rondinelli, Beatrice; Holmes, Amie; O'Leary, Colin; Kozono, David; D'Andrea, Alan D; Ceccaldi, Raphael

    2016-06-14

    BRCA1/2 proteins function in homologous recombination (HR)-mediated DNA repair and cooperate with Fanconi anemia (FA) proteins to maintain genomic integrity through replication fork stabilization. Loss of BRCA1/2 proteins results in DNA repair deficiency and replicative stress, leading to genomic instability and enhanced sensitivity to DNA-damaging agents. Recent studies have shown that BRCA1/2-deficient tumors upregulate Polθ-mediated alternative end-joining (alt-EJ) repair as a survival mechanism. Whether other mechanisms maintain genomic integrity upon loss of BRCA1/2 proteins is currently unknown. Here we show that BRCA1/2-deficient tumors also upregulate FANCD2 activity. FANCD2 is required for fork protection and fork restart in BRCA1/2-deficient tumors. Moreover, FANCD2 promotes Polθ recruitment at sites of damage and alt-EJ repair. Finally, loss of FANCD2 in BRCA1/2-deficient tumors enhances cell death. These results reveal a synthetic lethal relationship between FANCD2 and BRCA1/2, and they identify FANCD2 as a central player orchestrating DNA repair pathway choice at the replication fork. PMID:27264184

  1. Promoting Physical Activity through Goal Setting Strategies

    Martinez, Ray

    2004-01-01

    Physical educators are used to setting specific goals for students within a given unit. Here, the author emphasizes that they should also encourage students to set their own goals. Goal setting engages students in the learning process and allows them to develop the skills that support an active lifestyle. The author presents goal setting…

  2. Extraction DNA from Activated Sludge-Comparing with Soil Sample

    谢冰; 奚旦立; 陈季华

    2003-01-01

    DNA directly extraction from activated sludge and soil sample with enzyme lyses methods was investigated in this paper. DNA yield from activated sludge was 3.0 mg/g. VLSS, and 28.2-43.8 μg/g soil respectively. The resulting DNA is suitable for PCR.By studied methods, higher quality and quantity of sludge DNA could be obtained rapidly and inexpensively from large number of samples, and the PCR product obtained from this protocol was not affected by contaminated higher concentration of heavy metals.

  3. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products...

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

    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. Separation of DNA-dependent polymerate activities in Micrococcus radiodurans

    DNA polymerase activities in Micrococcus radiodurans were separated into two fractions after purification more than 2000 fold. They differ in pH optimum and residual activities in the absence of a full deoxyribonucleoside triphosphates complement. NAD partly inhibited one of the activities. Both activities were eluted as a single peak on gel filtration and sedimented at the same rate on glycerol gradient centrifugation. Molecular weight 140000 was calculated from Stokes radius and sedimentation constant. Deoxyribonuclease activity was detected on one of the polymerase activities which preferentially degraded double-stranded DNA. Priming activity of nicked DNA was reduced by γ-radiation. These results have been related to the possible roles in repair synthesis in vivo or DNA synthesis in permeable cells of M. radiodurans

  6. Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6-methylguanine-DNA methyltransferase.

    Xiao-Yang Wang

    Full Text Available Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1 is a member of the basic helix-loop-helix (bHLH transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7 and O(6-methylguanine-DNA methyltransferase (MGMT. Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

  7. Can musical activities promote healthy ageing?

    Park, A-La

    2015-01-01

    Background: As most of the baby boomer generation have now reached retirement age, there are increasing demands for long-term care services. Depression and psychological distress can be highly prevalent at advanced ages. Regardless of chronological age, it is important to have a decent quality of life as a human being by improving resilience. The present study aims to briefly look at the current evidence on the effects of musical activities on quality of life in older adults. Methods: A liter...

  8. Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

    Narayanan, Shalini; Beckham, Simone A; Davies, John K; Roujeinikova, Anna

    2014-12-01

    DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli. PMID:25156536

  9. Endonuclease activity in E. coli against photoalkylated DNA

    Photoalkylation, the ultraviolet irradiation (wavelength > 305 nm) of DNA in the presence of isopropanol and the free radical photoinitiator di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanine, 8-(2-hydroxy-2-propyl)adenine and thymine dimers. The authors have found a endonuclease activity in E. coli directed against photoalkylated DNA. This activity converts photoalkylated superhelical PM2 phage DNA to the nicked form, as measured by a nitrocellulose filter binding assay. Enzyme activities were compared between crude extracts of mutant strain BW9051 (xth-), lacking in exonuclease III activity, and strain BW434 (xth-, nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the E. coli strain lacking only exonuclease III. This was paralleled by a similar finding of deficient activity in the double mutant against partially depurinated PM2 DNA. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. These studies suggest that the enzyme activity against photoalkylated DNA is endonuclease III and that the substrate photoproduct is neither a 8-(2-hydroxy-2-propyl)purine nor a thymine dimer

  10. Development of transcriptional fusions to assess Leptospira interrogans promoter activity.

    Gustavo M Cerqueira

    Full Text Available BACKGROUND: Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. METHODOLOGY AND PRINCIPAL FINDINGS: A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA and Sphingomyelinase 2 (sph2 promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. CONCLUSIONS: The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

  11. Biological activity of cloned mammary tumor virus DNA fragments that bind purified glucocorticoid receptor protein in vitro

    To test whether high-affinity receptor:DNA interactions can be correlated with receptor effects on promoter function in vivo, we have mapped in greater detail the receptor-binding regions on murine mammary tumor virus DNA, using both nitrocellulose-filter binding and electron microscopy. Recombinant plasmids bearing these receptor-binding domains have been transfected into cultured cells, and the expression of the plasmid sequences has been monitored for hormonal regulation. The results are considered in terms of a speculative proposal that the glucocorticoid receptor may effect changes in promoter activity via specific alteration of chromatin and/or DNA structure. 37 references, 6 figures, 2 tables

  12. Synergistic transcription activation: a dual role for CRP in the activation of an Escherichia coli promoter depending on MalT and CRP

    Richet, Evelyne

    2000-01-01

    Activation of the Escherichia coli malEp promoter relies on the formation of a higher order structure involving cooperative binding of MalT to promoter-proximal and promoter-distal sites as well as CRP binding to three sites located in between. MalT is the primary activator and one function of CRP is to facilitate cooperative binding of MalT to its cognate sites by bending the intervening DNA. It is shown here that CRP also participates directly in malEp activation. This function is carried o...

  13. Honey, I Shrunk the DNA : DNA Length as a Probe for Nucleic-Acid Enzyme Activity

    Oijen, Antoine M. van

    2007-01-01

    The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and d

  14. Activation and Regulation of DNA-Driven Immune Responses

    Paludan, Søren R

    2015-01-01

    The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally,...

  15. Archaeal promoter architecture and mechanism of gene activation.

    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

  16. Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes*

    McDougall, William M.; Okany, Chinelo; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription. PMID:21737457

  17. Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

    Yuichiro Shimizu

    2010-01-01

    Full Text Available We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG, which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

  18. Different human TFIIIB activities direct RNA polymerase III transcription from TATA-containing and TATA-less promoters

    Schramm, Laura; Pendergrast, P. Shannon; Sun, Yuling; Hernandez, Nouria

    2000-01-01

    Transcription initiation at RNA polymerase III promoters requires transcription factor IIIB (TFIIIB), an activity that binds to RNA polymerase III promoters, generally through protein–protein contacts with DNA binding factors, and directly recruits RNA polymerase III. Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide β″. Although human homologs for two of the TFIIIB subunits, the TATA box–binding prot...

  19. APLF promotes the assembly and activity of non-homologous end joining protein complexes.

    Grundy, Gabrielle J; Rulten, Stuart L; Zeng, Zhihong; Arribas-Bosacoma, Raquel; Iles, Natasha; Manley, Katie; Oliver, Antony; Caldecott, Keith W

    2013-01-01

    Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway. PMID:23178593

  20. Promoting Physical Activity Among Overweight Young African American Women

    2014-01-15

    This podcast is an interview with Nefertiti Durant, MD, MPH, from the University of Alabama at Birmingham about promoting physical activity among overweight and obese young African American Women using Internet-based tools.  Created: 1/15/2014 by Preventing Chronic Disease (PCD), National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP).   Date Released: 1/15/2014.

  1. Promotion as a Tool in Sustaining the Destination Marketing Activities

    Ivo Mulec

    2010-01-01

    Promoting the tourism destination in the right and best possible way is today one of vital marketing activities of all Destination Management Organizations. Only successful promotion can entice and attract potential travelers to visit the destination. The number of new destinations is increasing every year and some of them are quite similar. Market segmentation is one of the starting points for devising marketing strategy. Only by presenting the destination to the right segment of potential c...

  2. Evaluation of hair growth promoting activity of Phyllanthus niruri

    Satish Patel; Vikas Sharma; Nagendra Chauhan; Mayank Thakur; Vinod Dixit

    2015-01-01

    Objective: This study was designed to investigate the potential Phyllanthus niruri (P. niruri ) extracts in promotion of hair growth. Materials and Methods: Here, we studied the hair growth promoting activity of petroleum ether extract of P. niruri following its topical administration. Alopecia was induced in albino rats by subcutaneous administration of testosterone for 21 days. Evaluation of hair loss inhibition was done by concurrent administration of extract and monitoring parameters like...

  3. Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.

    Sozhamannan, S; Chattoraj, D K

    1993-01-01

    Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have conf...

  4. O-methylguanine-DNA methyltransferase (MGMT mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation.

    Simone Kreth

    Full Text Available BACKGROUND: We analyzed prospectively whether MGMT (O(6-methylguanine-DNA methyltransferase mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. METHODOLOGY/PRINCIPAL FINDINGS: ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10 were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA database (N = 229 glioblastomas. Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001; the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001; however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6 did significantly worse than those with low transcriptional activity (p<0.01. Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6 did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001. Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT m

  5. Hair Growth Promotion Activity and Its Mechanism of Polygonum multiflorum

    Yunfei Li

    2015-01-01

    Full Text Available Polygonum multiflorum Radix (PMR has long history in hair growth promotion and hair coloring in clinical applications. However, several crucial problems in its clinic usage and mechanisms are still unsolved or lack scientific evidences. In this research, C57BL/6J mice were used to investigate hair growth promotion activity and possible mechanism of PMR and Polygonum multiflorum Radix Preparata (PMRP. Hair growth promotion activities were investigated by hair length, hair covered skin ratio, the number of follicles, and hair color. Regulation effects of several cytokines involved in the hair growth procedure were tested, such as fibroblast growth factor (FGF-7, Sonic Hedgehog (SHH, β-catenin, insulin-like growth factor-1 (IGF-1, and hepatocyte growth factor (HGF. Oral PMR groups had higher hair covered skin ratio (100 ± 0.00% than oral PMRP groups (48%~88%. However, topical usage of PMRP had about 90% hair covered skin ratio. Both oral administration of PMR and topically given PMRP showed hair growth promotion activities. PMR was considered to be more suitable for oral administration, while PMRP showed greater effects in external use. The hair growth promotion effect of oral PMR was most probably mediated by the expression of FGF-7, while topical PMRP promoted hair growth by the stimulation of SHH expression.

  6. A DNA enzyme with N-glycosylase activity

    Sheppard, Terry L.; Ordoukhanian, Phillip; Joyce, Gerald F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 106-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of...

  7. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane

    Aamann, Maria Diget; Sorensen, Martin M; Hvitby, Christina Poulsen;

    2010-01-01

    Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role...... in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress....... Moreover, our results suggest that CSB plays a significant role in mitochondrial base excision repair (BER) regulation. In particular, we find reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil BER incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlates with...

  8. Commercial activities and the promotion of health in schools.

    Raine, Gary

    2013-11-01

    Many companies nowadays consider schools to be an important setting for marketing to children. However, important concerns can be raised from a health promotion perspective about the potential negative impact of commercial activities on the health and well-being of pupils. As this discussion paper will demonstrate, some commercial activities raise concerns in relation to physical health and obesity, not only by potentially undermining formal curriculum messages, but also through the active promotion of specific products, particularly those high in fat, sugar or salt. Nonetheless, the issues raised by commercial activities are not solely limited to effects on physical health. By allowing commercial activities, schools risk instilling in pupils consumer-orientated values. This is significant as such values have been linked to the development of poor health and well-being. Furthermore, the presence in schools of commercial activities will also militate against informed decision-making and be disempowering. There is also evidence that business-sponsored teaching materials can contain biased and misleading information. The potential negative impacts of commercial activities are inconsistent with goals in relation to the promotion of health and the principles of health-promoting schools. PMID:23135869

  9. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter

  10. Inactivation efficiencies of radical reactions with biologically active DNA

    Lafleur, M. V. M.; Retèl, J.; Loman, H.

    Dilute aqueous solutions of biologically active θX174 DNA may serve as a simplified model system of the cell. Damage to the DNA after irradiation with γ-rays, may be ascribed to reactions with .OH, .H and e -aq or secondary radicals, arising from reactions of water radicals with added scavengers. Conversion of primary (water) radicals into secondary (scavenger) radicals leads to a considerable protection of the DNA, which, however, would have been larger if these secondary radicals did not contribute to DNA inactivation. The inactivation yield due to isopropanol or formate (secondary) radicals depends on dose rate as well as DNA concentration. Furthermore the inactivation efficiencies of the reactions of both the primary and the secondary radicals with single-stranded DNA could be established.

  11. Inactivation efficiencies of radical reactions with biologically active DNA

    Dilute aqueous solutions of biologically active ΦX174 DNA may serve as a simplified model system of the cell. Damage to the DNA after irradiation with γ-rays, may be ascribed to reactions with radical OH, radical H and esub(aq)- or secondary radicals, arising from reactions of water radicals with added scavengers. Conversion of primary (water) radicals into secondary (scavenger) radicals leads to a considerable protection of the DNA, which however, would have been larger if these secondary radicals did not contribute to DNA inactivation. The inactivation yield due to isopropanol or formate (secondary) radicals depends on dose rate as well as DNA concentration. Furthermore the inactivation efficiencies of the reactions of both the primary and the secondary radicals with single-stranded DNA could be established. (author)

  12. hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

    Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells. Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter. We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity. By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls. Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive

  13. The Dynamic Character of the BCL2 Promoter i-Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure

    Kendrick, Samantha; Kang, Hyun-Jin; Alam, Mohammad P.; Madathil, Manikandadas M.; Agrawal, Prashansa; Gokhale, Vijay; Yang, Danzhou; Hecht, Sidney M.; Hurley, Laurence H

    2014-01-01

    It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that...

  14. THE ACTIVITY OF ARABIDOSPIS DLL PROMOTER IN TRANSGENIC TOBACCO PLANTS UNDER WATER STRESS CONDITIONS

    Zuzana Polóniová

    2014-02-01

    Full Text Available In this work we used the Cre/loxP recombination system to study the activity of the Arabidopsis DLL promoter under water stress treatment. For this, the T-DNA containing the Cre/loxP self-excision recombination cassette was introduced into tobacco genome via A. tumefaciens LBA 4404. The expression of the cre gene was regulated by the DLL promoter. On activity of the DLL the Cre recombinase was expected to remove Cre/loxP cassette. Transgenic nature of regenerated transgenic T0 tobacco plantlets was proved by GUS and PCR analyses. The selected 10 transgenic T0 plants were subjected to the water stress analyses under in vitro as well as under in vivo conditions. The osmotic stress experiments were performed with 10 % PEG and 100 mmol.l-1 mannitol (individually. The activity of the DLL was evaluated after 24 hours. For drought stress experiments, the watering was withheld for 10 days. The activity of the DLL was monitored using PCR approach. Under given abiotic stress conditions, no activity of the DLL was observed. The DLL promoter remained stable. It points out the DLL as the promoter with precise control of the gene expression with wide usability in plant biotechnology.

  15. A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive.

    Galadriel Hovel-Miner

    2016-05-01

    Full Text Available African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs and toward the rest of

  16. A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive

    Hovel-Miner, Galadriel; Mugnier, Monica R.; Goldwater, Benjamin; Cross, George A. M.; Papavasiliou, F. Nina

    2016-01-01

    African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive. PMID

  17. PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening.

    Osterwald, Sarah; Deeg, Katharina I; Chung, Inn; Parisotto, Daniel; Wörz, Stefan; Rohr, Karl; Erfle, Holger; Rippe, Karsten

    2015-05-15

    The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation. PMID:25908860

  18. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

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

    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

  20. Transcriptional activity of acetylcholinesterase gene is regulated by DNA methylation during C2C12 myogenesis.

    Lau, Kei M; Gong, Amy G W; Xu, Miranda L; Lam, Candy T W; Zhang, Laura M L; Bi, Cathy W C; Cui, D; Cheng, Anthony W M; Dong, Tina T X; Tsim, Karl W K; Lin, Huangquan

    2016-07-01

    The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation. PMID:27021952

  1. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  2. Replicase-based plasmid DNA shows anti-tumor activity

    Weiss Richard

    2011-03-01

    Full Text Available Abstract Background Double stranded RNA (dsRNA has multiple anti-tumor mechanisms. Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it became clear that intracellular dsRNA is more effective than extracellular dsRNA on promoting apoptosis and orchestrating adaptive immune responses. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. Methods The anti-tumor activity of a plasmid (pSIN-β that encodes the sindbis RNA replicase genes (nsp1-4 was evaluated in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells and compared to a traditional pCMV-β plasmid. Results In cell culture, transfection of tumor cells with pSIN-β generated dsRNA. In mice with model tumors, pSIN-β more effectively delayed tumor growth than pCMV-β, and in some cases, eradicated the tumors. Conclusion RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth.

  3. Replicase-based plasmid DNA shows anti-tumor activity

    Double stranded RNA (dsRNA) has multiple anti-tumor mechanisms. Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it became clear that intracellular dsRNA is more effective than extracellular dsRNA on promoting apoptosis and orchestrating adaptive immune responses. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. The anti-tumor activity of a plasmid (pSIN-β) that encodes the sindbis RNA replicase genes (nsp1-4) was evaluated in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells) and compared to a traditional pCMV-β plasmid. In cell culture, transfection of tumor cells with pSIN-β generated dsRNA. In mice with model tumors, pSIN-β more effectively delayed tumor growth than pCMV-β, and in some cases, eradicated the tumors. RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth

  4. Bifidobacteria DNA Induces Murine Macrophages Activation in vitro

    Yalin Li; Xun Qu; Hua Yang; Li Kang; Yingping Xu; Bo Bai; Wengang Song

    2005-01-01

    Previous studies have shown that oligodeoxynucleotides containing unmethylated CpG motifs were used as adjuvants for immunoregulation and immune response. This study was to explore the activation effects of Bifidobacteria DNA containing unmethylated CpG motifs (CpG DNA) on murine macrophage J774A.1 cells. The genomic DNA of Bifidobacteria was extracted and purified, and the methylation degree of CpG motifs was tested.The phagocytic ability of the macrophages was detected by flow cytometry. The cytokines (IL-1β, IL-6, IL-12p40 and TNF-α) levels in the culture supernatants of Bifidobacteria DNA treated J774A.1 cells were assayed by ELISA. The content of nitric oxide (NO) was detected by Griess reagent. After treated with Bifidobacteria DNA for 24h,Nile Red stain increased in J774A.1 macrophage, which suggested that the lipid metabolism increased in the macrophages. The phagocytic ability and levels of NO and cytokines of IL-1β, IL-6, IL-12p40 and TNF-α were significantly higher than PBS group and CT DNA group. The results indicated that Bifidobacteria DNA could activate murine macrophages J774A.1, which could provide scientific basis for the research and application of microorganism DNA preparation.

  5. Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

    Grönholm Juha

    2012-10-01

    Full Text Available Abstract Background STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood. Results Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1. Conclusions Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

  6. TBP-TAF Complex SL1 Directs RNA Polymerase I Pre-initiation Complex Formation and Stabilizes Upstream Binding Factor at the rDNA Promoter*||

    Friedrich, J. Karsten; Panov, Kostya I.; Cabart, Pavel; Russell, Jackie; Zomerdijk, Joost C. B. M.

    2005-01-01

    Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports o...

  7. Conserved hypothetical BB0462 protein enhances the transcription activity of oppAV promoter

    2008-01-01

    Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids.A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure.The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E.coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter,but does not affect those of oppAⅠ,Ⅱ,Ⅲ and Ⅳ promoters.Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene.All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

  8. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    Highlights: ► DNA methylation is dynamic and flexible and changes rapidly upon cell activation. ► DNA methylation controls the inducible gene expression in a given cell type. ► Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  9. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    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.

  10. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    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

  11. It Takes a Virtual Community: Promoting Collaboration through Student Activities

    Battista, Ludmila; Forrey, Carol; Stevenson, Carolyn

    2008-01-01

    Distance education provides many nontraditional students with the opportunity to pursue a college education not possible through traditional brick and mortar education. Although not meeting face-to-face, student activities help promote a stronger connection between the classroom and university community. This paper will discuss strategies for…

  12. Promoting Educational Equity through School Libraries: Equity Activity Book.

    Arizona State Univ., Tempe.

    The activities suggested in this workbook for participants in a continuing education program for inservice school media specialists are designed to develop the trainee's skills in identifying instances of sexism and sex stereotyping in education, and in promoting sex fairness in the library. Exercises and tests on the first module are concerned…

  13. Internet as a Medium of Pharmaceutical Companies Promotional Activities

    Darko Pantelic

    2009-01-01

    Pharmaceuticals market is highly regulated, and it can be stated that prescription (legal or ethical) drugs have a status of substances in controlled circulation. Promotional activities are also under strict legislation, further burdened with ethical consideration and public scrutiny. Internet as liberal and hard to control medium brings entirely new sets of solutions and/or problems to pharmaceuticals market(ers).

  14. How do general practitioners in Denmark promote physical activity?

    Jørgensen, Tanja K; Nordentoft, Merete; Krogh, Jesper

    2012-01-01

    The primary objective of this study was to quantify the frequency of advice given on type, frequency, duration, and intensity of exercise during physical activity (PA) promoting sessions by general practitioners. Second, to find GP characteristics associated with high quality of PA counselling....

  15. Health Promotion Guidance Activity of Youth Sports Clubs

    Kokko, Sami; Kannas, Lasse; Villberg, Jari; Ormshaw, Michael

    2011-01-01

    Purpose: This paper aims to clarify the extent to which youth sports clubs guide their coaches to recognise health promotion as a part of the coaching practice. The guidance activity of clubs is seen parallel to internal organisational communication. Design/methodology/approach: A survey of 93 (from 120, 78 per cent) youth sports clubs in Finland…

  16. Healthy and Active Ageing: Social Capital in Health Promotion

    Koutsogeorgou, Eleni; Davies, John Kenneth; Aranda, Kay; Zissi, Anastasia; Chatzikou, Maria; Cerniauskaite, Milda; Quintas, Rui; Raggi, Alberto; Leonardi, Matilde

    2014-01-01

    Objectives: This paper examines the context of health promotion actions that are focused on/contributing to strengthening social capital by increasing community participation, reciprocal trust and support as the means to achieve better health and more active ageing. Method: The methodology employed was a literature review/research synthesis, and a…

  17. Analysis of Significance of Unite Examination of AFP and DNA Polymorphism of P3 Promoter of IGF-ⅡGene

    LUO Su; ZHANG Feng-chun; SUN Chang-jiang; LIU Cheng-bai; ZHUANG Jiang-xing; ZHANG Jin

    2004-01-01

    The DNA of P3 promoter region of IGF-Ⅱ gene was obtained by means of PCR technique. The examination of DNA polymorphism by restriction endonuclease BstE Ⅱ and the examination of AFP by bioluminescence immunoassay technique were carried out. The results have a significant difference(P<0.005). But the positive rate of AFP is higher than that of DNA polymorphism. The experimental result shows that the change of the DNA polymorphism of IGF-Ⅱis not the only carcinogenic factor. The suggested unite examination is the best method for the diagnosis of the primary hepatocellular carcinoma.

  18. Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription.

    McAndrew, M; Graham, S; Hartmann, C; Clayton, C

    1998-09-01

    In trypanosomes, most genes are arranged in polycistronic transcription units. Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation. Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting. PMID:9709032

  19. In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

    Cicaré, J; Caille, A; Zumoffen, C; Ghersevich, S; Bahamondes, L; Munuce, M J

    2015-10-01

    The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity. PMID:25233794

  20. Creative professional activity: an additional platform for promotion of faculty.

    Levinson, Wendy; Rothman, Arthur I; Phillipson, Eliot

    2006-06-01

    Academic promotion has traditionally been based on research and teaching, but faculty members' contributions to the profession may not be fully captured in those dimensions. Faculty members may influence the practice of medicine and improve the care of patients yet not obtain traditional measures of achievement through publications, grants, or teaching awards. With this problem in mind, at the University of Toronto Faculty of Medicine, the promotions committee developed and implemented a promotions criterion called Creative Professional Activity (CPA) to recognize and reward a variety of types of academic endeavors that have a demonstrable impact on medical practice and care. CPA comprises three activities: professional innovation, exemplary practice, and contributions to the development of the discipline. In this article, the authors define CPA, provide illustrative case examples, describe how faculty members document CPA, and report the use of this promotions criterion in the Department of Medicine over the last decade. The challenges of implementing CPA as a promotion criterion are described. CPA is consistent with the Department of Medicine's goal of achieving excellence through original research, education, or creative work that advances the care of patients. PMID:16728810

  1. Invariant distribution of promoter activities in Escherichia coli.

    Alon Zaslaver; Shai Kaplan; Anat Bren; Adrian Jinich; Avi Mayo; Erez Dekel; Uri Alon; Shalev Itzkovitz

    2009-01-01

    Author Summary Cells respond to a changing environment by regulating the activity of genes. Here, we sought to understand how E. coli cells distribute their limited transcriptional resources among their target genes, and how this allocation varies with growth rate and growth conditions. To achieve this, we assayed the expression of a comprehensive library of transcriptional reporter strains under different conditions. High-temporal resolution measurements of promoter activities were obtained ...

  2. Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference.

    Grandin, Mélodie; Mathot, Pauline; Devailly, Guillaume; Bidet, Yannick; Ghantous, Akram; Favrot, Clementine; Gibert, Benjamin; Gadot, Nicolas; Puisieux, Isabelle; Herceg, Zdenko; Delcros, Jean-Guy; Bernet, Agnès; Mehlen, Patrick; Dante, Robert

    2016-01-01

    In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer. PMID:27378792

  3. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. PMID:27184763

  4. Propionate induces the bovine cytosolic phosphoenolpyruvate carboxykinase promoter activity.

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-08-01

    Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical enzyme within the metabolic networks for gluconeogenesis, hepatic energy metabolism, and tricarboxylic acid cycle function, and is controlled by several transcription factors including hepatic nuclear factor 4α (HNF4α). The primary objective of the present study was to determine whether propionate regulates bovine PCK1 transcription. The second objective was to determine the action of cyclic AMP (cAMP), glucocorticoids, and insulin, hormonal cues known to modulate glucose metabolism, on bovine PCK1 transcriptional activity. The proximal promoter of the bovine PCK1 gene was ligated to a Firefly luciferase reporter and transfected into H4IIE hepatoma cells. Cells were exposed to treatments for 23 h and luciferase activity was determined in cell lysates. Activity of the PCK1 promoter was linearly induced by propionate, and maximally increased 7-fold with 2.5 mM propionate, which was not muted by 100 nM insulin. Activity of the PCK1 promoter was increased 1-fold by either 1.0 mM cAMP or 5.0µM dexamethasone, and 2.2-fold by their combination. Induction by cAMP and dexamethasone was repressed 50% by 100 nM insulin. Propionate, cAMP, and dexamethasone acted synergistically to induce the PCK1 promoter activity. Propionate-responsive regions, identified by 5' deletion analysis, were located between -1,238 and -409 bp and between -85 and +221 bp. Deletions of the core sequences of the 2 putative HNF4α sites decreased the responsiveness to propionate by approximately 40%. These data indicate that propionate regulates its own metabolism through transcriptional stimulation of the bovine PCK1 gene. This induction is mediated, in part, by the 2 putative HNF4α binding sites in the bovine PCK1 promoter. PMID:27289145

  5. The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

    Antoshechkin, I; Bogenhagen, D F; Mastrangelo, I A

    1997-01-01

    The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided e...

  6. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

    Rasmussen, Kasper D.; Jia, Guangshuai; Johansen, Jens V.; Pedersen, Marianne T.; Rapin, Nicolas; Bagger, Frederik O.; Porse, Bo T; Bernard, Olivier A; Christensen, Jesper; Helin, Kristian

    2015-01-01

    The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CpG islands and promoters from aberrant DNA methylation. Rasmussen et al. used a novel Tet2-dependent leukemia mouse model to show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements.

  7. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na+-H+ exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of [3H]thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na+-H+ antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity

  8. Synthesis, DNA interaction and antimicrobial activities of three rimantadine analogues

    Liu, Bing-Mi; Zhang, Jun [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Wang, Xin, E-mail: wangxinlnu@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Zhang, Li-Ping; Liu, Yang [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Niu, Hua-Ying [Jinan Dachpharm Development Co., Ltd., Jinan 250100 (China); Liu, Bin, E-mail: liubinzehao@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China)

    2015-03-15

    The interactions of three rimantadine analogues (RAs) with calf thymus deoxyribonucleic acid (ct-DNA) in buffer solution (pH 7.4) were investigated using berberine (BR) as a probe by various methods. Fluorescence studies revealed that the RAs interacted with DNA in vitro and the quenchings were all static. Furthermore, the binding modes of these compounds to DNA were disclosed as groove binding supported by absorption spectroscopy, viscosity measurement and denatured DNA experiment. The antimicrobial activities of the RAs were also evaluated in Staphylococcus aureus and Escherichia coli, and they all exhibited good bacteriostasic effects. The results might provide an important reference for investigation of the molecular mechanism associated with the DNA binding of the RAs. - Highlights: • Three rimantadine analogues were synthesized. • The RAs effectively quenched the intrinsic fluorescence of DNA via a static combination. • These analogues can bind to DNA via groove binding mode. • The antimicrobial activities of three analogues were also evaluated by the disk diffusion method.

  9. Synthesis, DNA interaction and antimicrobial activities of three rimantadine analogues

    The interactions of three rimantadine analogues (RAs) with calf thymus deoxyribonucleic acid (ct-DNA) in buffer solution (pH 7.4) were investigated using berberine (BR) as a probe by various methods. Fluorescence studies revealed that the RAs interacted with DNA in vitro and the quenchings were all static. Furthermore, the binding modes of these compounds to DNA were disclosed as groove binding supported by absorption spectroscopy, viscosity measurement and denatured DNA experiment. The antimicrobial activities of the RAs were also evaluated in Staphylococcus aureus and Escherichia coli, and they all exhibited good bacteriostasic effects. The results might provide an important reference for investigation of the molecular mechanism associated with the DNA binding of the RAs. - Highlights: • Three rimantadine analogues were synthesized. • The RAs effectively quenched the intrinsic fluorescence of DNA via a static combination. • These analogues can bind to DNA via groove binding mode. • The antimicrobial activities of three analogues were also evaluated by the disk diffusion method

  10. Chromatin inactivation precedes de novo dna methylation during the progressive epigenetic silencing of the rassf1a promoter

    Strunnikova Maria; Schagdarsurengin, Undraga; Kehlen, Astrid; Garbe, James C.; Stampfer, Martha R.; Dammann, Reinhard

    2005-02-23

    Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1 A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.