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Sample records for dnase ii genes

  1. Biogenesis and proteolytic processing of lysosomal DNase II.

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    Susumu Ohkouchi

    Full Text Available Deoxyribonuclease II (DNase II is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected--a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.

  2. Human T-cell leukemia virus types I and II exhibit different DNase I protection patterns

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    Altman, R.; Harrich, D.; Garcia, J.A.; Gaynor, R.B.

    1988-01-01

    Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are human retroviruses which normally infect T-lymphoid cells. HTLV-I infection is associated with adult T-cell leukemia-lymphoma, and HTLV-II is associated with an indolent form of hairy-cell leukemia. To identify potential transcriptional regulatory elements of these two related human retroviruses, the authors performed DNase I footprinting of both the HTLV-I and HTLV-II long terminal repeats (LTRs) by using extracts prepared from uninfected T cells, HTLV-I and HTLV-II transformed T cells, and HeLa cells. Five regions of the HTLV-I LTR and three regions of the HTLV-II LTR showed protection by DNase I footprinting. All three of the 21-base-pair repeats previously shown to be important in HTLV transcriptional regulation were protected in the HTLV-I LTR, whereas only one of these repeats was protected in the HTLV-II LTR. Several regions exhibited altered protection in extracts prepared from lymphoid cells as compared with HeLa cells, but there were minimal differences in the protection patterns between HTLV-infected and uninfected lymphoid extracts. A number of HTLV-I and HTLV-II LTR fragments which contained regions showing protection in DNase I footprinting were able to function as inducible enhancer elements in transient CAT gene expression assays in the presence of the HTLV-II tat protein. The alterations in the pattern of the cellular proteins which bind to the HTLV-I and HTLV-II LTRs may in part be responsible for differences in the transcriptional regulation of these two related viruses

  3. Impacts of zero valent iron, natural zeolite and Dnase on the fate of antibiotic resistance genes during thermophilic and mesophilic anaerobic digestion of swine manure.

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    Zhang, Junya; Sui, Qianwen; Zhong, Hui; Meng, Xiaoshan; Wang, Ziyue; Wang, Yawei; Wei, Yuansong

    2018-06-01

    This study investigated the fate of antibiotic resistance genes (ARGs) during mesophilic (mAD) and thermophilic digestion (tAD) of swine manure through zero valent iron (ZVI), natural zeolite and Dnase addition. Changes of microbial community, intI1, heavy metal resistance genes (MRGs) and virulence factors (VFs) were followed to clarify the influencing factors to ARGs reduction. Results showed that AD could realize ARGs reduction with tAD superior to mAD, and ZVI and natural zeolite could further enhance the reduction, especially for natural zeolite addition at mAD. The reduction efficiency of the relative abundance of ARGs was increased by 33.3% and 138.5% after ZVI and natural zeolite addition, respectively, but Dnase deteriorated ARGs reduction at mAD. Most of ARGs could be reduced effectively except sulII and tetM. Network analysis and partial redundancy analysis indicated that co-occurrence of MRGs followed by microbial community contributed the most to the variation of ARGs fate among treatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. [rhDNase: scientific background, cloning and production].

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    Shak, S

    1995-07-01

    Despite the hopes raised by the first attempts in gene therapy, direct correction of the defect in CFTR protein associated with cystic fibrosis is still beyond clinical reach. Therefore we have to set upon the consequences of the defect. Respiratory distress and progressive lung destruction in cystic fibrosis can be accounted for by infectious exacerabations and the accumulation of viscous purulent secretions in the airways. For a long time we have known that purulent secretions that accumulate in the airways of patients with cystic fibrosis contain large amounts of DNA, a complex macromolecule that contributes mostly to the viscosity and hinders the mucociliary function. Hence we hypothesized that enzymatic cleaving of DNA molecules by desoxyribonuclease (DNase) should reduce the viscosity of sputum, and slow or prevent the deterioration of pulmonary function. Using the techniques of molecular biology and genetic engineering, we identified the gene of human DNase I, which was cloned in mammalian cells to produce large amounts of a glycosylated protein for therapeutic use. Catalytic amounts of rhDNase greatly reduce the viscosity of purulent cystic fibrosis sputum, transforming it within minutes from a nonflowing viscous gel to a flowing liquid. This effect was associated with a decrease in size of DNA fragments in the sputum. Our studies suggested that inhalation of a rhDNase aerosol might be a simple direct approach to reduce the viscosity of purulent secretions and thereby help patients with cystic fibrosis clear their airways and breathe more easily.

  5. Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni.

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    Brown, Helen L; Reuter, Mark; Hanman, Kate; Betts, Roy P; van Vliet, Arnoud H M

    2015-01-01

    The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.

  6. How Cations Can Assist DNase I in DNA Binding and Hydrolysis

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    Guéroult, Marc; Picot, Daniel; Abi-Ghanem, Joséphine; Hartmann, Brigitte; Baaden, Marc

    2010-01-01

    DNase I requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA. However, the number and the location of DNase I ion-binding sites remain unclear, as well as the role of these counter-ions. Using molecular dynamics simulations, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them strongly bind Ca2+ while the other two sites coordinate Mg2+. These theoretical results are strongly supported by revisiting crystallographic structures that contain bpDNase I. One Ca2+ stabilizes the functional DNase I structure. The presence of Mg2+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Importantly, Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. These results improve our understanding of the essential role of cations in the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions. PMID:21124947

  7. Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

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    Ma, Wenxiu; Ay, Ferhat; Lee, Choli; Gulsoy, Gunhan; Deng, Xinxian; Cook, Savannah; Hesson, Jennifer; Cavanaugh, Christopher; Ware, Carol B; Krumm, Anton; Shendure, Jay; Blau, C Anthony; Disteche, Christine M; Noble, William S; Duan, ZhiJun

    2018-06-01

    The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Antibiofilm Effect of DNase against Single and Mixed Species Biofilm

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    Sharma, Komal

    2018-01-01

    Biofilms are aggregates of microorganisms that coexist in socially coordinated micro-niche in a self-produced polymeric matrix on pre-conditioned surfaces. The biofilm matrix reduces the efficacy of antibiofilm strategies. DNase degrades the extracellular DNA (e-DNA) present in the matrix, rendering the matrix weak and susceptible to antimicrobials. In the current study, the effect of DNase I was evaluated during biofilm formation (pre-treatment), on preformed biofilms (post-treatment) and both (dual treatment). The DNase I pre-treatment was optimized for P. aeruginosa PAO1 (model biofilm organism) at 10 µg/mL and post-treatment at 10 µg/mL with 15 min of contact duration. Inclusion of Mg2+ alongside DNase I post-treatment resulted in 90% reduction in biofilm within only 5 min of contact time (irrespective of age of biofilm). On extension of these findings, DNase I was found to be less effective against mixed species biofilm than individual biofilms. DNase I can be used as potent antibiofilm agent and with further optimization can be effectively used for biofilm prevention and reduction in situ. PMID:29562719

  9. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.

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    Shak, S; Capon, D J; Hellmiss, R; Marsters, S A; Baker, C L

    1990-12-01

    Respiratory distress and progressive lung destruction in cystic fibrosis can be attributed to bacterial persistence and the accumulation of viscous purulent secretions in the airways. More than 30 yr ago it was suggested that the large amounts of DNA in purulent secretions contribute to its viscosity and that bovine pancreatic DNase I could reduce the viscosity. To evaluate the potential clinical utility of recombinant human DNase I (rhDNase) in the treatment of cystic fibrosis, we have cloned, sequenced, and expressed rhDNase. Catalytic amounts of rhDNase greatly reduce the viscosity of purulent cystic fibrosis sputum, transforming it within minutes from a nonflowing viscous gel to a flowing liquid. The reduction in viscosity is associated with a decrease in size of DNA in the sputum. Inhalation of a rhDNase aerosol may be a simple direct approach that will help individuals with cystic fibrosis and other patients with pneumonia or bronchitis to clear their airways of purulent secretions.

  10. Aerosolized recombinant human DNase I for the treatment of cystic fibrosis.

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    Shak, S

    1995-02-01

    Respiratory symptoms, recurrent infectious exacerbations, and progressive lung destruction in cystic fibrosis can be attributed to bacterial persistence and the accumulation of viscous purulent secretions in the airways. Purulent secretions contain high concentrations of extracellular DNA, a viscous material released by leukocytes. To evaluate the potential clinical utility of recombinant human DNase I (rhDNase or Pulmozyme), the human enzyme was cloned, sequenced, and expressed. In in vitro studies, rhDNase has been shown to reduce the viscoelasticity, reduce the adhesiveness, and improve the mucociliary transportability of cystic fibrosis sputum. In short-term phase 1 and phase 2 clinical trials, rhDNase has been shown to be safely tolerated and to improve the FEV1, FVC, and symptoms of dyspnea. A long-term placebo-controlled phase 3 study was performed in 968 adults and children (> or = 5 years) with cystic fibrosis to determine the effect of rhDNase on the risk of respiratory exacerbations requiring parenteral antibiotics and on the FEV1. Compared with placebo-treated patients, patients treated with rhDNase once daily or twice daily experienced a reduced risk of respiratory exacerbations by 28% (p = 0.04) and 37% (p = 0.01), respectively, and had a mean improvement in FEV1 of 5.8% (p < 0.01) and 5.6% (p < 0.01), respectively. Compared with placebo-treated patients, patients treated with rhDNase spent 2.7 fewer days receiving parenteral antibiotics (p = 0.04) and spent 1.3 fewer days in the hospital (p = 0.06) over the 6-month treatment period. Inhalation of rhDNase did not cause anaphylaxis but was associated with upper airway symptoms (ie, voice alteration, hoarseness, pharyngitis) that were generally mild and transient. In conclusion, aerosol administration of rhDNase was safely tolerated, reduced the risk of infectious exacerbations requiring parenteral antibiotics, and improved pulmonary function and patient well-being.

  11. Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

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    Simona Kamenšek

    2015-06-01

    Full Text Available Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8, after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

  12. Mapping 3D genome architecture through in situ DNase Hi-C.

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    Ramani, Vijay; Cusanovich, Darren A; Hause, Ronald J; Ma, Wenxiu; Qiu, Ruolan; Deng, Xinxian; Blau, C Anthony; Disteche, Christine M; Noble, William S; Shendure, Jay; Duan, Zhijun

    2016-11-01

    With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves ∼4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.

  13. Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

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    Sinicropi, D; Baker, D L; Prince, W S; Shiffer, K; Shak, S

    1994-11-01

    A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.

  14. An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

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    Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

    2015-08-15

    A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. The effect of 3'- methyl-N,N-dimethylaminoasobenzene and gamma rays on the serum level of deoxyribonuclease II of the rat and the isolation of deoxyribonuclease II from the human spleen

    International Nuclear Information System (INIS)

    Jansen van Rensburg, E.

    1979-01-01

    The purpose of this investigation was twofold, viz.: 1. To determine the influence of ionising radiation (physical carcinogen) on deoxyribonuclease II in serum. 2. To develop a sensitive method for determining deoxyribonuclease II, viz. a radioimmunoassay (RlA). With this object in view, the influence of a strong physical carcinogen, viz. 60 Co gamma rays, on the activity of DNase II in rat serum was studied. For comparative studies, the influence of a strong chemical carcinogen (3'-methyl-N,N-dimethylaminoasobenzene) on rat serum DNase II was determined. In addition, attempts were made to isolate, purify and characterise DNase II from human spleen. These attempts were made as the first step in the development of a RlA. The in vivo studies of the influence of 3'MeDAB over a long period (40 weeks) showed that the DNase II activity increased in the serum and liver nuclei of rats during the pre-cancer phase. This increase may, together with other parameters, be useful in the early diagnosis of liver cancer. The most important contribution of this investigation is the possible use of a serum DNase II RlA together with a battery RlAs for other indicators (biochemical) for the determination of radiation exposure in man [af

  16. Human β-globin locus control region: Analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice

    International Nuclear Information System (INIS)

    Caterina, J.J.; Ryan, T.M.; Pawlik, K.M.; Townes, T.M.; Brinster, R.L.; Behringer, R.R.; Palmiter, R.D.

    1991-01-01

    The human β-globin locus control region (LCR) is essential for high-level expression of human var-epsilon-, γ-, and β-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human β-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human β-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletion of both 5' and 3' sequences, a 373-base-pair (BP) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 19-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained

  17. Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.

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    Saluz, H P; Feavers, I M; Jiricny, J; Jost, J P

    1988-01-01

    Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand. Images PMID:3413118

  18. Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence.

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    Minh Tran, Tuan; MacIntyre, April; Khokhani, Devanshi; Hawes, Martha; Allen, Caitilyn

    2016-11-01

    Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach

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    Sinclair Andrew H

    2009-09-01

    Full Text Available Abstract Background Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems. Results We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 × 104 cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method. Conclusion This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 105 cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

  20. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

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    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-01-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [ 3 H]Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes

  1. Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis.

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    Amanda L McGuire

    Full Text Available A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.

  2. Chromatin accessibility data sets show bias due to sequence specificity of the DNase I enzyme.

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    Hashem Koohy

    Full Text Available DNase I is an enzyme which cuts duplex DNA at a rate that depends strongly upon its chromatin environment. In combination with high-throughput sequencing (HTS technology, it can be used to infer genome-wide landscapes of open chromatin regions. Using this technology, systematic identification of hundreds of thousands of DNase I hypersensitive sites (DHS per cell type has been possible, and this in turn has helped to precisely delineate genomic regulatory compartments. However, to date there has been relatively little investigation into possible biases affecting this data.We report a significant degree of sequence preference spanning sites cut by DNase I in a number of published data sets. The two major protocols in current use each show a different pattern, but for a given protocol the pattern of sequence specificity seems to be quite consistent. The patterns are substantially different from biases seen in other types of HTS data sets, and in some cases the most constrained position lies outside the sequenced fragment, implying that this constraint must relate to the digestion process rather than events occurring during library preparation or sequencing.DNase I is a sequence-specific enzyme, with a specificity that may depend on experimental conditions. This sequence specificity is not taken into account by existing pipelines for identifying open chromatin regions. Care must be taken when interpreting DNase I results, especially when looking at the precise locations of the reads. Future studies may be able to improve the sensitivity and precision of chromatin state measurement by compensating for sequence bias.

  3. A novel DNase like compound that inhibits virus propagation from Asian green mussel, Perna viridis (Linn.)

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    Iqbal, A.N.M.Z.; Khan, M.S.

    to counteract some viruses. Keywords: Antiviral property, Bivalves, DNase-like bioactivity, Plasmid pBR 322 Virus mediated pathological conditions such as Chickenpox (Varicella zoster virus, VZV), Japanese encephalitis, Chickungunya, Dengue, infectious... is an object of future research endeavor. DNAse like PPC with an antiviral property can be a potential candidate to develop an effective drugs against the DNA viruses like varicella zoster virus and Herpes viruses. Detailed work is underway to further...

  4. DNaseI Protects against Paraquat-Induced Acute Lung Injury and Pulmonary Fibrosis Mediated by Mitochondrial DNA

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    Guo Li

    2015-01-01

    Full Text Available Background. Paraquat (PQ poisoning is a lethal toxicological challenge that served as a disease model of acute lung injury and pulmonary fibrosis, but the mechanism is undetermined and no effective treatment has been discovered. Methods and Findings. We demonstrated that PQ injures mitochondria and leads to mtDNA release. The mtDNA mediated PBMC recruitment and stimulated the alveolar epithelial cell production of TGF-β1 in vitro. The levels of mtDNA in circulation and bronchial alveolar lavage fluid (BALF were elevated in a mouse of PQ-induced lung injury. DNaseI could protect PQ-induced lung injury and significantly improved survival. Acute lung injury markers, such as TNFα, IL-1β, and IL-6, and marker of fibrosis, collagen I, were downregulated in parallel with the elimination of mtDNA by DNaseI. These data indicate a possible mechanism for PQ-induced, mtDNA-mediated lung injury, which may be shared by other causes of lung injury, as suggested by the same protective effect of DNaseI in bleomycin-induced lung injury model. Interestingly, increased mtDNA in the BALF of patients with amyopathic dermatomyositis-interstitial lung disease can be appreciated. Conclusions. DNaseI targeting mtDNA may be a promising approach for the treatment of PQ-induced acute lung injury and pulmonary fibrosis that merits fast tracking through clinical trials.

  5. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses.

    Science.gov (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. Safety and Efficacy of Tissue Plasminogen Activator and DNase for Complicated Pleural Effusions Secondary to Abdominal Pathology.

    Science.gov (United States)

    Majid, Adnan; Ochoa, Sebastian; Chatterji, Sumit; Fernandez-Bussy, Sebastian; Kheir, Fayez; Rivera, Estefania; Cheng, George; Folch, Erik

    2017-03-01

    Exudative pleural effusions may arise secondary to inflammation of intra-abdominal structures. Pleural space loculations can complicate these effusions, preventing adequate chest tube drainage and leading to consideration of surgical intervention. Previous studies have demonstrated that intrapleural administration of tissue plasminogen activator (tPA) combined with human recombinant DNase can improve fluid drainage and reduce surgery for patients with loculated parapneumonic effusions; however, the efficacy of this treatment has not been evaluated for complicated pleural effusions attributed to intra-abdominal inflammation. We assessed the safety and efficacy of tPA/DNase for 17 pleural effusions associated with nonmalignant intra-abdominal pathology that did not drain adequately after placement of one or more chest tubes. Efficacy was measured by comparing post- to pretreatment fluid drainage rates, volumetric assessment of pleural fluid on radiographic images before and after treatment, and clinical improvement, including the need for surgical intervention. Symptomatic relief was assessed using the Borg scale for breathlessness. After a median of two doses of tPA/DNase, 23.5% of patients had chest pain and none had pleural bleeding. The volume of pleural fluid drained increased from a median of 325 ml to 890 ml per 24 hours after therapy (P = 0.018). The area of pleural space opacity on chest radiographs decreased from a median of 42.8-17.8% of the hemithorax (P = 0.001). tPA/DNase reduced the pleural fluid volume on chest computed tomographic imaging from a median of 294.4 ml to 116.1 ml. Borg scores improved from a median of 3 (interquartile range = 1-6) to 0 (interquartile range = 0-2) after therapy (P = 0.001). The median duration of chest tube placement and hospital stay were 4 and 11 days, respectively. Two patients required surgical intervention for lung entrapment. Overall, treatment was considered successful for 88.2% of patients

  7. Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

    International Nuclear Information System (INIS)

    Lindler, L.E.; Macrina, F.L.

    1987-01-01

    We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci

  8. Discovery of non-directional and directional pioneer transcription factors by modeling DNase profile magnitude and shape

    Science.gov (United States)

    Lewis, Sophia; Barkal, Amira A; van Hoff, John Peter; Karun, Vivek; Jaakkola, Tommi; Gifford, David K

    2014-01-01

    Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs. PMID:24441470

  9. Characterization of anticancer, DNase and antifungal activity of pumpkin 2S albumin.

    Science.gov (United States)

    Tomar, Prabhat Pratap Singh; Nikhil, Kumar; Singh, Anamika; Selvakumar, Purushotham; Roy, Partha; Sharma, Ashwani Kumar

    2014-06-13

    The plant 2S albumins exhibit a spectrum of biotechnologically exploitable functions. Among them, pumpkin 2S albumin has been shown to possess RNase and cell-free translational inhibitory activities. The present study investigated the anticancer, DNase and antifungal activities of pumpkin 2S albumin. The protein exhibited a strong anticancer activity toward breast cancer (MCF-7), ovarian teratocarcinoma (PA-1), prostate cancer (PC-3 and DU-145) and hepatocellular carcinoma (HepG2) cell lines. Acridine orange staining and DNA fragmentation studies indicated that cytotoxic effect of pumpkin 2S albumin is mediated through induction of apoptosis. Pumpkin 2S albumin showed DNase activity against both supercoiled and linear DNA and exerted antifungal activity against Fusarium oxysporum. Secondary structure analysis by CD showed that protein is highly stable up to 90°C and retains its alpha helical structure. These results demonstrated that pumpkin 2S albumin is a multifunctional protein with host of potential biotechnology applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. pDHS-SVM: A prediction method for plant DNase I hypersensitive sites based on support vector machine.

    Science.gov (United States)

    Zhang, Shanxin; Zhou, Zhiping; Chen, Xinmeng; Hu, Yong; Yang, Lindong

    2017-08-07

    DNase I hypersensitive sites (DHSs) are accessible chromatin regions hypersensitive to cleavages by DNase I endonucleases. DHSs are indicative of cis-regulatory DNA elements (CREs), all of which play important roles in global gene expression regulation. It is helpful for discovering CREs by recognition of DHSs in genome. To accelerate the investigation, it is an important complement to develop cost-effective computational methods to identify DHSs. However, there is a lack of tools used for identifying DHSs in plant genome. Here we presented pDHS-SVM, a computational predictor to identify plant DHSs. To integrate the global sequence-order information and local DNA properties, reverse complement kmer and dinucleotide-based auto covariance of DNA sequences were applied to construct the feature space. In this work, fifteen physical-chemical properties of dinucleotides were used and Support Vector Machine (SVM) was employed. To further improve the performance of the predictor and extract an optimized subset of nucleotide physical-chemical properties positive for the DHSs, a heuristic nucleotide physical-chemical property selection algorithm was introduced. With the optimized subset of properties, experimental results of Arabidopsis thaliana and rice (Oryza sativa) showed that pDHS-SVM could achieve accuracies up to 87.00%, and 85.79%, respectively. The results indicated the effectiveness of proposed method for predicting DHSs. Furthermore, pDHS-SVM could provide a helpful complement for predicting CREs in plant genome. Our implementation of the novel proposed method pDHS-SVM is freely available as source code, at https://github.com/shanxinzhang/pDHS-SVM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Gene expression profiles in stages II and III colon cancers

    DEFF Research Database (Denmark)

    Thorsteinsson, Morten; Kirkeby, Lene T; Hansen, Raino

    2012-01-01

    PURPOSE: A 128-gene signature has been proposed to predict outcome in patients with stages II and III colorectal cancers. In the present study, we aimed to reproduce and validate the 128-gene signature in external and independent material. METHODS: Gene expression data from the original material...... were retrieved from the Gene Expression Omnibus (GEO) (n¿=¿111) in addition to a Danish data set (n¿=¿37). All patients had stages II and III colon cancers. A Prediction Analysis of Microarray classifier, based on the 128-gene signature and the original training set of stage I (n¿=¿65) and stage IV (n...... correctly predicted as stage IV-like, and the remaining patients were predicted as stage I-like and unclassifiable, respectively. Stage II patients could not be stratified. CONCLUSIONS: The 128-gene signature showed reproducibility in stage III colon cancer, but could not predict recurrence in stage II...

  12. Safety and Efficacy of Intrapleural Tissue Plasminogen Activator and DNase during Extended Use in Complicated Pleural Space Infections

    Directory of Open Access Journals (Sweden)

    Jason R. McClune

    2016-01-01

    Full Text Available The use of intrapleural therapy with tissue plasminogen activator and DNase improves outcomes in patients with complicated pleural space infections. However, little data exists for the use of combination intrapleural therapy after the initial dosing period of six doses. We sought to describe the safety profile and outcomes of intrapleural therapy beyond this standard dosing. A retrospective review of patients receiving intrapleural therapy with tissue plasminogen activator and DNase was performed at two institutions. We identified 101 patients from January 2013 to August 2015 receiving intrapleural therapy for complicated pleural space infection. The extended use of intrapleural tissue plasminogen activator and DNase therapy beyond six doses was utilized in 20% (20/101 of patients. The mean number of doses in those undergoing extended dosing was 9.8 (range of 7–16. Within the population studied there appears to be no statistically significant increased risk of complications, need for surgical referral, or outcome differences when comparing those receiving standard or extended dosing intrapleural therapy. Future prospective study of intrapleural therapy as an alternative option for patients who fail initial pleural drainage and are unable to tolerate/accept a surgical intervention appears a potential area of study.

  13. Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers

    International Nuclear Information System (INIS)

    Boswell, G.S.; Dimitrijevich, S.D.; Gracy, R.W.

    1989-01-01

    A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developed an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([3H]thymidine, [14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations

  14. DNase Sda1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition.

    Directory of Open Access Journals (Sweden)

    Satoshi Uchiyama

    Full Text Available Group A Streptococcus (GAS has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

  15. Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints.

    Science.gov (United States)

    Schwessinger, Ron; Suciu, Maria C; McGowan, Simon J; Telenius, Jelena; Taylor, Stephen; Higgs, Doug R; Hughes, Jim R

    2017-10-01

    In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k -mer-based analysis of DNase footprints to determine any k -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. © 2017 Schwessinger et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Eco RI RFLP in the human IGF II gene

    Energy Technology Data Exchange (ETDEWEB)

    Cocozza, S; Garofalo, S; Robledo, R; Monticelli, A; Conti, A; Chiarotti, L; Frunzio, R; Bruni, C B; Varrone, S

    1988-03-25

    The probe was a 500 bp cDNA containing exons 2-3 and 4 of the human IGF II gene. The clone was isolated by screening a human liver cDNA library with synthetic oligonucleotides. Eco RI digestion of genomic DNA and hybridization with the IGF II probe detects a two allele polymorphism with allelic fragments of 13.5 kb and 10.5 kb. The frequency was studied 38 unrelated Caucasians: Human IGF II gene was localized on the short arm of chromosome 11 (p15) by in situ hybridization. Codominant segregation was observed in 2 Caucasian families (10 individuals).

  17. Cloning and expression analysis of a new anther-specific gene ...

    Indian Academy of Sciences (India)

    sequencing gene differential expression analysis (Chen et al. ... DNase digestion (Takara, Shiga, Japan), so that any remain- ..... ing the early moments of pollen germination (Guyon et al. 2000). The steady-state transcript level of PGPS/D3 ...

  18. Rapid duplication and loss of nbs-encoding genes in eurosids II

    International Nuclear Information System (INIS)

    Si, W.; Gu, L.; Yang, S.; Zhang, X.; Memon, S.

    2015-01-01

    Eurosids basically evolved from the core Eudicots Rosids. The Rosids consist of two large assemblages, Eurosids I (Fabids) and Eurosids II (Malvids), which belong to the largest group of Angiosperms, comprising of >40,000 and ∼ 15,000 species, respectively. Although the evolutionary patterns of the largest class of disease resistance genes consisting of a nucleotide binding site (NBS) and leucine-rich repeats (LRRs) have been studied in many species, systemic research of NBS-encoding genes has not been performed in different orders of Eurosids II. Here, five Eurosids II species, Gossypium raimondii, Theobroma cacao, Carica papaya, Citrus clementina, and Arabidopsis thaliana, distributing in three orders, were used to gain insights into the evolutionary patterns of the NBS-encoding genes. Our data showed that frequent copy number variations of NBS-encoding genes were found among these species. Phylogenetic tree analysis and the numbers of the NBS-encoding genes in the common ancestor of these species showed that species-specific NBS clades, including multi-copy and single copy numbers are dominant among these genes. However, not a single clade was found with only five copies, which come from all of the five species, respectively, suggesting rapid turn-over with birth and death of the NBS-encoding genes among Eurosids II species. In addition, a strong positive correlation was observed between the Toll/interleukin receptor (TIR)) type NBS-encoding genes and species-specific genes, indicating rapid gene loss and duplication. Whereas, non- TIR type NBS-encoding genes in these five species showed two distinct evolutionary patterns. (author)

  19. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    Science.gov (United States)

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  20. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

    Directory of Open Access Journals (Sweden)

    Tuan Minh Tran

    2016-06-01

    Full Text Available Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

  1. Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II.

    Science.gov (United States)

    Joshi, Ricky S; Nikolaou, Christoforos; Roca, Joaquim

    2018-01-03

    Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.

  2. DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

    Science.gov (United States)

    Villarreal, Jessica Varela; Jungfer, Christina; Obst, Ursula; Schwartz, Thomas

    2013-09-01

    Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems. © 2013 Elsevier B.V. All rights reserved.

  3. Overview of BioCreative II gene mention recognition

    NARCIS (Netherlands)

    Smith, L.; Tanabe, L.K.; Johnson, R.; Kuo, C.-J.; Chung, I-F.; Hsu, C.-N.; Lin, Y.-S.; Klinger, R.; Friedrich, C.M.; Ganchev, K.; Torii, M.; Liu, H.; Haddow, B.; Struble, C.A.; Povinelli, R.J.; Vlachos, A.; Baumgartner (jr.), W.A.; Hunter, L.; Carpenter, B.; Tsai, R.T.-H.; Dai, H.-J.; Liu, F.; Chen, Y.; Sun, C.; Katrenko, S.; Adriaans, P.; Blaschke, C.; Torres, R.; Neves, M.; Nakov, P.; Divoli, A.; Maña-López, M.; Mata, J.; Wilbur, W.J.

    2008-01-01

    Nineteen teams presented results for the Gene Mention Task at the BioCreative II Workshop. In this task participants designed systems to identify substrings in sentences corresponding to gene name mentions. A variety of different methods were used and the results varied with a highest achieved F1

  4. Syndromes associated with Homo sapiens pol II regulatory genes.

    Science.gov (United States)

    Bina, M; Demmon, S; Pares-Matos, E I

    2000-01-01

    The molecular basis of human characteristics is an intriguing but an unresolved problem. Human characteristics cover a broad spectrum, from the obvious to the abstract. Obvious characteristics may include morphological features such as height, shape, and facial form. Abstract characteristics may be hidden in processes that are controlled by hormones and the human brain. In this review we examine exaggerated characteristics presented as syndromes. Specifically, we focus on human genes that encode transcription factors to examine morphological, immunological, and hormonal anomalies that result from deletion, insertion, or mutation of genes that regulate transcription by RNA polymerase II (the Pol II genes). A close analysis of abnormal phenotypes can give clues into how sequence variations in regulatory genes and changes in transcriptional control may give rise to characteristics defined as complex traits.

  5. A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

    Science.gov (United States)

    Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

    2012-01-01

    Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

  6. Coexpression landscape in ATTED-II: usage of gene list and gene network for various types of pathways.

    Science.gov (United States)

    Obayashi, Takeshi; Kinoshita, Kengo

    2010-05-01

    Gene coexpression analyses are a powerful method to predict the function of genes and/or to identify genes that are functionally related to query genes. The basic idea of gene coexpression analyses is that genes with similar functions should have similar expression patterns under many different conditions. This approach is now widely used by many experimental researchers, especially in the field of plant biology. In this review, we will summarize recent successful examples obtained by using our gene coexpression database, ATTED-II. Specifically, the examples will describe the identification of new genes, such as the subunits of a complex protein, the enzymes in a metabolic pathway and transporters. In addition, we will discuss the discovery of a new intercellular signaling factor and new regulatory relationships between transcription factors and their target genes. In ATTED-II, we provide two basic views of gene coexpression, a gene list view and a gene network view, which can be used as guide gene approach and narrow-down approach, respectively. In addition, we will discuss the coexpression effectiveness for various types of gene sets.

  7. Regulated gene expression in cultured type II cells of adult human lung.

    Science.gov (United States)

    Ballard, Philip L; Lee, Jae W; Fang, Xiaohui; Chapin, Cheryl; Allen, Lennell; Segal, Mark R; Fischer, Horst; Illek, Beate; Gonzales, Linda W; Kolla, Venkatadri; Matthay, Michael A

    2010-07-01

    Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

  8. Angiotensin-II type 1 receptor gene polymorphism and diabetic microangiopathy

    DEFF Research Database (Denmark)

    Tarnow, L; Cambien, Francois; Rossing, P

    1996-01-01

    with proliferative retinopathy and without diabetic retinopathy was found either: 77 (50%) / 66 (42%) / 13 (8%) vs. 42 (63%) / 22 (33%) / 3 (4%) had AA/AC/CC genotypes, respectively. CONCLUSIONS: The A1166-->C polymorphism in the angiotensin-II type 1 receptor gene does not contribute to the genetic susceptibility...... is present particularly in vascular smooth muscle cells, myocardium and the kidney. A transversion of adenine to cytosine at nucleotide position 1166 in the gene coding for the angiotensin-II type 1 receptor has been associated with hypertension in the non-diabetic population. METHODS: We studied...... the relationship between the A1166-->C polymorphism in the angiotensin-II type 1 receptor gene in patients with insulin dependent diabetes mellitus (IDDM) and diabetic nephropathy (121 men, 77 women, age 41 +/- 10 years, diabetes duration 27 +/- 8 years) and in IDDM patients with normoalbuminuria (116 men, 74...

  9. Regulated gene expression in cultured type II cells of adult human lung

    OpenAIRE

    Ballard, Philip L.; Lee, Jae W.; Fang, Xiaohui; Chapin, Cheryl; Allen, Lennell; Segal, Mark R.; Fischer, Horst; Illek, Beate; Gonzales, Linda W.; Kolla, Venkatadri; Matthay, Michael A.

    2010-01-01

    Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at ∼95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days...

  10. Mesoporous silica nanoparticles supported copper(II) and nickel(II) Schiff base complexes: Synthesis, characterization, antibacterial activity and enzyme immobilization

    Science.gov (United States)

    Tahmasbi, Leila; Sedaghat, Tahereh; Motamedi, Hossein; Kooti, Mohammad

    2018-02-01

    Mesoporous silica nanoparticles (MSNs) were prepared by sol-gel method and functionalized with 3-aminopropyltriethoxysilane. Schiff base grafted mesoporous silica nanoparticle was synthesized by the condensation of 2-hydroxy-3-methoxybenzaldehyde and amine-functionalized MSNs. The latter material was then treated with Cu(II) and Ni(II) salts separately to obtain copper and nickel complexes anchored mesoporous composites. The newly prepared hybrid organic-inorganic nanocomposites have been characterized by several techniques such as FT-IR, LA-XRD, FE-SEM, TEM, EDS, BET and TGA. The results showed all samples have MCM-41 type ordered mesoporous structure and functionalization occurs mainly inside the mesopore channel. The presence of all elements in synthesized nanocomposites and the coordination of Schiff base via imine nitrogen and phenolate oxygen were confirmed. MSNs and all functionalized MSNs have uniform spherical nanoparticles with a mean diameter less than 100 nm. The as-synthesized mesoporous nanocomposites were investigated for antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria, as carrier for gentamicin and also for immobilization of DNase, coagulase and amylase enzymes. MSN-SB-Ni indicated bacteriocidal effect against S.aureus and all compounds were found to be good carrier for gentamicin. Results of enzyme immobilization for DNase and coagulase and α-amylase revealed that supported metal complexes efficiently immobilized enzymes.

  11. Potential novel bZIP-like gene for resistance to Erysiphe necator ...

    African Journals Online (AJOL)

    AJL

    2012-06-19

    Jun 19, 2012 ... In this study, a novel bZIP-like gene was isolated from Chinese wild Vitis ... Results reveal that it was in lower lever in flower than in leaf, stem, tendril and fruit. .... First-strand cDNA was synthesized from 1 µg of DNase treated.

  12. Identification and characterization of the human type II collagen gene (COL2A1).

    OpenAIRE

    Cheah, Kathryn; Stoker, N.G.; Griffin, J.R.; Grosveld, Frank; Solomon, E.

    1985-01-01

    textabstractThe gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis shows that this clone is highly homologous to the chicken alpha 1(II) collagen gene. These data together suggest that CosHcol1 contains the human alpha 1(II) collagen gene COL2A1. The clone appears...

  13. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    Science.gov (United States)

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

  14. Study on the Imprinting Status of Insulin-Like Growth Factor II (IGF-II Gene in Villus during 6–10 Gestational Weeks

    Directory of Open Access Journals (Sweden)

    Jianhong Chen

    2010-01-01

    Full Text Available Objective. To compare the difference of imprinting status of insulin-like growth factor II (IGF-II gene in villus between normal embryo development group and abnormal embryo development group and to investigate the relationship between karyotype and the imprinting status of IGF-II gene. Methods. A total of 85 pregnant women with singleton pregnancy were divided into two groups: one with abnormal embryo development (n=38 and the other with normal embryo development (n=47. Apa I polymorphism of IGF-II gene in chorionic villus was assayed with reverse transcriptase polymerase chain reaction (RT-PCR and restriction fragment length polymorphism (RFLP. The relationship between chromosomal abnormal karyotype and IGF-II gene imprinting status was analyzed by primary cell culture and G-banding chromosomal karyotype analysis. Results. IGF-II imprinting loss rate was higher in the abnormal embryo development group than the normal embryo development group (44.7% versus 31.6%, but without significant difference (P>.05. The percentage of abnormal chromosomes of chorionic villus in the abnormal embryo development group was 42.5%, in which IGF-II imprinting loss rate reached 64.7%. No abnormal karyotypes were found in the normal embryo development group. However, there was significant difference in IGF-II imprinting loss rate between two groups (P>.05. Conclusion. During weeks 6–10 of gestation, abnormal embryonic development is correlated with chromosomal abnormalities. The imprinting status of IGF-II gene played important roles in embryonic development, and imprinting loss might be related to chromosomal abnormalities.

  15. Epigenetic repression of male gametophyte-specific genes in the Arabidopsis sporophyte

    DEFF Research Database (Denmark)

    Hoffmann, Robert D; Palmgren, Michael Broberg

    2013-01-01

    Tissue formation, the identity of cells, and the functions they fulfill, are results of gene regulation. The male gametophyte of plants, pollen, is outstanding in this respect as several hundred genes expressed in pollen are not expressed in the sporophyte. How pollen-specific genes are down......-regulated in the sporophyte has yet to be established. In this study, we have performed a bioinformatics analysis of publicly available genome-wide epigenetics data of several sporophytic tissues. By combining this analysis with DNase I footprinting data, we assessed means by which the repression of pollen-specific genes...

  16. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    Science.gov (United States)

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

    OpenAIRE

    Hoopes, B C; McClure, W R

    1985-01-01

    We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have con...

  18. Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype

    International Nuclear Information System (INIS)

    Kowara, Renata; Karaczyn, Aldona; Cheng, Robert Y.S.; Salnikow, Konstantin; Kasprzak, Kazimierz S.

    2005-01-01

    B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells

  19. MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen

    Directory of Open Access Journals (Sweden)

    Judith A. James

    2012-12-01

    Full Text Available Anthrax Lethal Toxin consists of Protective Antigen (PA and Lethal Factor (LF, and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus, B6 (H-2b, and B6.H2k (H-2k. IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.

  20. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

    Science.gov (United States)

    Hiesel, Rudolf; Brennicke, Axel

    1983-01-01

    The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

  1. Small RNAs were involved in homozygous state-associated silencing of a marker gene (Neomycin phosphotransferase II: nptII) in transgenic tomato plants.

    Science.gov (United States)

    Deng, Lei; Pan, Yu; Chen, Xuqing; Chen, Guoping; Hu, Zongli

    2013-07-01

    Homozygous state-associated co-suppression is not a very common phenomenon. In our experiments, two transgenic plants 3A29 and 1195A were constructed by being transformed with the constructs pBIN-353A and pBIN119A containing nptII gene as a marker respectively. The homozygous progeny from these two independent transgenic lines 3A29 and 1195A, displayed kanamycin-sensitivity and produced a short main root without any lateral roots as untransformed control (wild-type) seedlings when germinated on kanamycin media. For the seedlings derived from putative hemizygous plants, the percentage of the seedlings showing normal growth on kanamycin media was about 50% and lower than the expected percentage (75%). Southern analysis of the genomic DNA confirmed that the homozygous and hemizygous plants derived from the same lines contained the same multiple nptII transgenes, which were located on the same site of chromosome. Northern analysis suggested that the marker nptII gene was expressed in the primary and the hemizygous transformants, but it was silenced in the homozygous transgenic plants. Further Northern analysis indicated that antisense and sense small nptII-derived RNAs were present in the transgenic plants and the blotting signal of nptII-derived small RNA was much higher in the homozygous transgenic plants than that of hemizygous transgenic plants. Additionally, read-through transcripts from the TRAMP gene to the nptII gene were detected. These results suggest that the read-through transcripts may be involved in homozygous state-associated silencing of the nptII transgene in transgenic tomato plants and a certain threshold level of the nptII-derived small RNAs is required for the homozygous state-associated co-suppression of the nptII transgene. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. DNA sequence of the Peromyscus leucopus MHC class II gene Aa (MhcPeleAa)

    Energy Technology Data Exchange (ETDEWEB)

    Crew, M.D.; Bates, L.M. [Univ. of Arkansas for Medical Sciences, Little Rock, AR (United States)

    1996-09-01

    The genus Peromyscus has been extensively studied by populations biologists and ecologists for over eighty years, with P. leucopus (the white-footed mouse) being one of the most intensively investigated species. Polymorphic major histocompatibility complex (MHC) genes have proven useful in population genetic studies and might be helpful in understanding the population dynamics of Peromyscus species which are ubiquitously distributed over North and Central America. Polymorphism of P. leucopus MHC (MhcPele) class II genes was evident by restriction fragment length polymorphism (RFLP) analyses using human and mouse probes and Pele class II loci exhibited degrees of polymorphism similar to H2 class II genes (A-like>E-like). 8 refs., 2 figs.

  3. A comparative gene analysis with rice identified orthologous group II HKT genes and their association with Na(+) concentration in bread wheat.

    Science.gov (United States)

    Ariyarathna, H A Chandima K; Oldach, Klaus H; Francki, Michael G

    2016-01-19

    Although the HKT transporter genes ascertain some of the key determinants of crop salt tolerance mechanisms, the diversity and functional role of group II HKT genes are not clearly understood in bread wheat. The advanced knowledge on rice HKT and whole genome sequence was, therefore, used in comparative gene analysis to identify orthologous wheat group II HKT genes and their role in trait variation under different saline environments. The four group II HKTs in rice identified two orthologous gene families from bread wheat, including the known TaHKT2;1 gene family and a new distinctly different gene family designated as TaHKT2;2. A single copy of TaHKT2;2 was found on each homeologous chromosome arm 7AL, 7BL and 7DL and each gene was expressed in leaf blade, sheath and root tissues under non-stressed and at 200 mM salt stressed conditions. The proteins encoded by genes of the TaHKT2;2 family revealed more than 93% amino acid sequence identity but ≤52% amino acid identity compared to the proteins encoded by TaHKT2;1 family. Specifically, variations in known critical domains predicted functional differences between the two protein families. Similar to orthologous rice genes on chromosome 6L, TaHKT2;1 and TaHKT2;2 genes were located approximately 3 kb apart on wheat chromosomes 7AL, 7BL and 7DL, forming a static syntenic block in the two species. The chromosomal region on 7AL containing TaHKT2;1 7AL-1 co-located with QTL for shoot Na(+) concentration and yield in some saline environments. The differences in copy number, genes sequences and encoded proteins between TaHKT2;2 homeologous genes and other group II HKT gene families within and across species likely reflect functional diversity for ion selectivity and transport in plants. Evidence indicated that neither TaHKT2;2 nor TaHKT2;1 were associated with primary root Na(+) uptake but TaHKT2;1 may be associated with trait variation for Na(+) exclusion and yield in some but not all saline environments.

  4. Understanding Epistatic Interactions between Genes Targeted by Non-coding Regulatory Elements in Complex Diseases

    Directory of Open Access Journals (Sweden)

    Min Kyung Sung

    2014-12-01

    Full Text Available Genome-wide association studies have proven the highly polygenic architecture of complex diseases or traits; therefore, single-locus-based methods are usually unable to detect all involved loci, especially when individual loci exert small effects. Moreover, the majority of associated single-nucleotide polymorphisms resides in non-coding regions, making it difficult to understand their phenotypic contribution. In this work, we studied epistatic interactions associated with three common diseases using Korea Association Resource (KARE data: type 2 diabetes mellitus (DM, hypertension (HT, and coronary artery disease (CAD. We showed that epistatic single-nucleotide polymorphisms (SNPs were enriched in enhancers, as well as in DNase I footprints (the Encyclopedia of DNA Elements [ENCODE] Project Consortium 2012, which suggested that the disruption of the regulatory regions where transcription factors bind may be involved in the disease mechanism. Accordingly, to identify the genes affected by the SNPs, we employed whole-genome multiple-cell-type enhancer data which discovered using DNase I profiles and Cap Analysis Gene Expression (CAGE. Assigned genes were significantly enriched in known disease associated gene sets, which were explored based on the literature, suggesting that this approach is useful for detecting relevant affected genes. In our knowledge-based epistatic network, the three diseases share many associated genes and are also closely related with each other through many epistatic interactions. These findings elucidate the genetic basis of the close relationship between DM, HT, and CAD.

  5. Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints

    OpenAIRE

    Schwessinger, R; Suciu, MC; McGowan, SJ; Telenius, J; Taylor, S; Higgs, DR; Hughes, JR

    2017-01-01

    In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor bin...

  6. Loss of Major DNase I Hypersensitive Sites in Duplicatedglobin Gene Cluster Incompletely Silences HBB Gene Expression

    Czech Academy of Sciences Publication Activity Database

    Reading, N. S.; Shooter, C.; Song, J.; Miller, R.; Agarwal, A.; Láníková, Lucie; Clark, B.; Thein, S.L.; Divoký, V.; Prchal, J.T.

    2016-01-01

    Roč. 37, č. 11 (2016), s. 1153-1156 ISSN 1059-7794 R&D Projects: GA MŠk(CZ) LH15223 Institutional support: RVO:68378050 Keywords : globin genes * regulation * sickle cell disease * HBB duplication Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.601, year: 2016

  7. A Caenorhabditis elegans RNA polymerase II gene, ama-1 IV, and nearby essential genes.

    Science.gov (United States)

    Rogalski, T M; Riddle, D L

    1988-01-01

    The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20 degrees but are arrested as larvae at 25 degrees, and two others are fertile at 20 degrees and sterile at 25 degrees. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25 degrees is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four gamma-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development.

  8. Characterization and evolution of MHC class II B genes in Galápagos marine iguanas (Amblyrhynchus cristatus).

    Science.gov (United States)

    Glaberman, Scott; Moreno, Maria A; Caccone, Adalgisa

    2009-08-01

    Major histocompatibility complex (MHC) class II molecules play a key role in the adaptive immune system of vertebrates. Class II B genes appear to evolve in a very different manner in mammals and birds. Orthology is commonly observed among mammal loci, while genes tend to cluster phylogenetically within bird species. Here we present class II B data from a representative of another major group of amniotes, the squamates (i.e. lizards, snakes, amphisbaenians), with the ultimate goal of placing mammalian and avian MHC evolution into a broader context. In this study, eight class II B cDNA sequences were obtained from the Galápagos marine iguana (Amblyrhynchus cristatus) which were divided into five locus groups, Amcr-DAB1 through -DAB5, based on similarities along most of the coding and noncoding portions of the transcribed gene. All marine iguana sequences were monophyletic with respect to class II genes from other vertebrates indicating that they originated from a common ancestral locus after squamates split from other reptiles. The beta-1 domain, which is involved in antigen binding, exhibited signatures of positive selection as well as interlocus gene conversion in both long and short tracts-a pattern also observed in birds and fish, but not in mammals. On the other hand, the beta-2 domain was divergent between gene groups, which is characteristic of mammals. Based on these results, we preliminarily show that squamate class II B genes have been shaped by a unique blend of evolutionary forces that have been observed in differing degrees in other vertebrates.

  9. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

    International Nuclear Information System (INIS)

    Gilmour, D.S.; Lis, J.T.

    1986-01-01

    By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

  10. SVMRFE based approach for prediction of most discriminatory gene target for type II diabetes

    Directory of Open Access Journals (Sweden)

    Atul Kumar

    2017-06-01

    Full Text Available Type II diabetes is a chronic condition that affects the way our body metabolizes sugar. The body's important source of fuel is now becoming a chronic disease all over the world. It is now very necessary to identify the new potential targets for the drugs which not only control the disease but also can treat it. Support vector machines are the classifier which has a potential to make a classification of the discriminatory genes and non-discriminatory genes. SVMRFE a modification of SVM ranks the genes based on their discriminatory power and eliminate the genes which are not involved in causing the disease. A gene regulatory network has been formed with the top ranked coding genes to identify their role in causing diabetes. To further validate the results pathway study was performed to identify the involvement of the coding genes in type II diabetes. The genes obtained from this study showed a significant involvement in causing the disease, which may be used as a potential drug target.

  11. Interaction between the thyroid hormone receptor and co-factors on the promoter of the gene encoding phospho enol pyruvate carboxykinase

    NARCIS (Netherlands)

    Schmidt, E. D.; van Beeren, M.; Glass, C. K.; Wiersinga, W. M.; Lamers, W. H.

    1993-01-01

    Using transient transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phospho-enol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at

  12. Selective visualization of gene structure with ultraviolet light

    International Nuclear Information System (INIS)

    Wang, Z.; Becker, M.M.

    1988-01-01

    The ability of the ultraviolet (UV) footprinting technique to detect chromatin has been investigated in vitro. Two basic types of chromatin, a phased nucleosome and a phased nucleosome containing a phased H1 protein, have been reconstituted onto a cloned 5S ribosomal RNA gene from sea urchin. The histone-DNA interactions in each complex have been probed with exonuclease III, DNase I, dimethyl sulfate, and UV light. Whereas DNase I and exonuclease III readily detect interactions between histones and DNA, UV light and dimethyl sulfate do not. In contrast to histone-DNA interactions, we demonstrate that intimate sequence-specific contacts between the same sea urchin 5S DNA and the Xenopus laevis transcription factor IIIA (TFIIIA) are readily detected with UV light. Since the sensitivity of UV light for TFIIIA contacts is similar to its sensitivity for other regulatory protein-DNA contacts, these studies demonstrate the feasibility of using UV light to selectively visualize regulatory protein-DNA interactions in vivo with little or no interference from histone-DNA interactions

  13. Characterization of class II alpha genes and DLA-D region allelic associations in the dog.

    Science.gov (United States)

    Sarmiento, U M; Storb, R F

    1988-10-01

    Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

  14. Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

    DEFF Research Database (Denmark)

    Phylactides, M.; Rowntree, R.; Nuthall, H.

    2002-01-01

    hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2,3,10,16,17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhancer/reporter...

  15. Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling.

    Science.gov (United States)

    Liu, Cun-Fei; Zhang, Jia; Shen, Kai; Gao, Ping-Jin; Wang, Hai-Ya; Jin, Xin; Meng, Chao; Fang, Ning-Yuan

    2015-04-01

    Vascular adventitia and adventitia‑derived reactive oxygen species (ROS) contribute to vascular remodeling following vascular injury. A previous ex vivo study in adventitial fibroblasts showed that catalase, one of most important anti‑oxide enzymes, was downregulated by angiotensin II (AngII). The aim of the present study was to investigate whether adventitial gene transfer of catalase affects AngII‑induced vascular remodeling in vivo. Adenoviruses co‑expressing catalase and enhanced green fluorescent protein (eGFP) or expressing eGFP only were applied to the adventitial surface of common carotid arteries of Sprague‑Dawley rats. Alzet minipumps administering AngII (0.75 mg/kg/day) were then implanted subcutaneously for 14 days. Systolic blood pressure and biological parameters of vascular remodeling were measured in each group. Adventitial fibroblasts were cultured and p38 mitogen‑activated protein kinase (MAPK) phosphorylation was measured using western blot analysis. The results showed that adventitial gene transfer of catalase had no effect on AngII‑induced systolic blood pressure elevation. However, catalase adenovirus transfection significantly inhibited AngII‑induced media hypertrophy compared with that of the control virus (Padventitial α‑smooth muscle actin expression. Furthermore, catalase transfection significantly inhibited the AngII‑induced increase in p38MAPK phosphorylation. In conclusion, the results of the present study demonstrated that adventitial gene transfer of catalase significantly attenuated AngII‑induced vascular remodeling in rats via inhibition of adventitial p38MAPK phosphorylation.

  16. ColoLipidGene: signature of lipid metabolism-related genes to predict prognosis in stage-II colon cancer patients

    Science.gov (United States)

    Vargas, Teodoro; Moreno-Rubio, Juan; Herranz, Jesús; Cejas, Paloma; Molina, Susana; González-Vallinas, Margarita; Mendiola, Marta; Burgos, Emilio; Aguayo, Cristina; Custodio, Ana B.; Machado, Isidro; Ramos, David; Gironella, Meritxell; Espinosa-Salinas, Isabel; Ramos, Ricardo; Martín-Hernández, Roberto; Risueño, Alberto; De Las Rivas, Javier; Reglero, Guillermo; Yaya, Ricardo; Fernández-Martos, Carlos; Aparicio, Jorge; Maurel, Joan; Feliu, Jaime; de Molina, Ana Ramírez

    2015-01-01

    Lipid metabolism plays an essential role in carcinogenesis due to the requirements of tumoral cells to sustain increased structural, energetic and biosynthetic precursor demands for cell proliferation. We investigated the association between expression of lipid metabolism-related genes and clinical outcome in intermediate-stage colon cancer patients with the aim of identifying a metabolic profile associated with greater malignancy and increased risk of relapse. Expression profile of 70 lipid metabolism-related genes was determined in 77 patients with stage II colon cancer. Cox regression analyses using c-index methodology was applied to identify a metabolic-related signature associated to prognosis. The metabolic signature was further confirmed in two independent validation sets of 120 patients and additionally, in a group of 264 patients from a public database. The combined analysis of these 4 genes, ABCA1, ACSL1, AGPAT1 and SCD, constitutes a metabolic-signature (ColoLipidGene) able to accurately stratify stage II colon cancer patients with 5-fold higher risk of relapse with strong statistical power in the four independent groups of patients. The identification of a group of 4 genes that predict survival in intermediate-stage colon cancer patients allows delineation of a high-risk group that may benefit from adjuvant therapy, and avoids the toxic and unnecessary chemotherapy in patients classified as low-risk group. PMID:25749516

  17. Lack of evidence that the XqYq pairing tips at meiosis in the mouse show hypersensitivity to DNAse I.

    Science.gov (United States)

    Separovic, E R; Chandley, A C

    1987-01-01

    In situ nick translation procedures have been applied to meiotic metaphase I divisions of the normal and XY, Sxr mouse. Unlike in man, where the pairing tips of the XY bivalent show a special sensitivity to DNAse I nicking, no such sensitivity can be detected for either of these types of mouse. Hypersensitivity in the D-band equivalent region of the X chromosome does, however, exist, this site being early replicating in somatic cells and housing the X inactivation centre (Xce).

  18. Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance.

    Science.gov (United States)

    Ghanem, S

    2011-01-01

    In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.

  19. Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

    Science.gov (United States)

    Bastien, C.; Machlin, S.; Zhang, Y.; Donaldson, K.; Hanson, R. S.

    1989-01-01

    Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome cL, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome cL structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed. PMID:16348074

  20. DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells.

    Science.gov (United States)

    Murphy, Shawn P; Holtz, Renae; Lewandowski, Nicole; Tomasi, Thomas B; Fuji, Hiroshi

    2002-09-15

    MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.

  1. Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting

    Directory of Open Access Journals (Sweden)

    van der Meijde Jolanda

    2007-08-01

    Full Text Available Abstract Background Fasting has dramatic effects on small intestinal transport function. However, little is known on expression of intestinal transport and phase I/II metabolism genes during fasting and the role the fatty acid-activated transcription factor PPARα may play herein. We therefore investigated the effects of fasting on expression of these genes using Affymetrix GeneChip MOE430A arrays and quantitative RT-PCR. Results After 24 hours of fasting, expression levels of 33 of the 253 analyzed transporter and phase I/II metabolism genes were changed. Upregulated genes were involved in transport of energy-yielding molecules in processes such as glycogenolysis (G6pt1 and mitochondrial and peroxisomal oxidation of fatty acids (Cact, Mrs3/4, Fatp2, Cyp4a10, Cyp4b1. Other induced genes were responsible for the inactivation of the neurotransmitter serotonin (Sert, Sult1d1, Dtd, Papst2, formation of eicosanoids (Cyp2j6, Cyp4a10, Cyp4b1, or for secretion of cholesterol (Abca1 and Abcg8. Cyp3a11, typically known because of its drug metabolizing capacity, was also increased. Fasting had no pronounced effect on expression of phase II metabolic enzymes, except for glutathione S-transferases which were down-regulated. Time course studies revealed that some genes were acutely regulated, whereas expression of other genes was only affected after prolonged fasting. Finally, we identified 8 genes that were PPARα-dependently upregulated upon fasting. Conclusion We have characterized the response to fasting on expression of transporters and phase I/II metabolic enzymes in murine small intestine. Differentially expressed genes are involved in a variety of processes, which functionally can be summarized as a increased oxidation of fat and xenobiotics, b increased cholesterol secretion, c increased susceptibility to electrophilic stressors, and d reduced intestinal motility. This knowledge increases our understanding of gut physiology, and may be of relevance

  2. Distinct mutations in yeast TAF(II)25 differentially affect the composition of TFIID and SAGA complexes as well as global gene expression patterns.

    Science.gov (United States)

    Kirschner, Doris B; vom Baur, Elmar; Thibault, Christelle; Sanders, Steven L; Gangloff, Yann-Gaël; Davidson, Irwin; Weil, P Anthony; Tora, Làszlò

    2002-05-01

    The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.

  3. A comparison of 12-gene colon cancer assay gene expression in African American and Caucasian patients with stage II colon cancer.

    Science.gov (United States)

    Govindarajan, Rangaswamy; Posey, James; Chao, Calvin Y; Lu, Ruixiao; Jadhav, Trafina; Javed, Ahmed Y; Javed, Awais; Mahmoud, Fade A; Osarogiagbon, Raymond U; Manne, Upender

    2016-06-18

    African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer. We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student's t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1). Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups. The distribution of

  4. A comparison of 12-gene colon cancer assay gene expression in African American and Caucasian patients with stage II colon cancer

    International Nuclear Information System (INIS)

    Govindarajan, Rangaswamy; Posey, James; Chao, Calvin Y.; Lu, Ruixiao; Jadhav, Trafina; Javed, Ahmed Y.; Javed, Awais; Mahmoud, Fade A.; Osarogiagbon, Raymond University; Manne, Upender

    2016-01-01

    African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer. We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student’s t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1). Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups. The distribution of Recurrence Score

  5. Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes

    Directory of Open Access Journals (Sweden)

    Feuer Gerold

    2004-11-01

    Full Text Available Abstract Background Human T-lymphotropic virus type-1 (HTLV-1 is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export. Results Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing ~33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes. Conclusions We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.

  6. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    Science.gov (United States)

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

  7. Transcriptional up-regulation of antioxidant genes by PPARδ inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Kim, Hyo Jung; Ham, Sun Ah; Paek, Kyung Shin; Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Han, Chang Woo; Seo, Han Geuk

    2011-01-01

    Research highlights: → Activation of PPARδ by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. → Agonist-activated PPARδ suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. → GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. → Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) δ as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPARδ by GW501516, a specific agonist of PPARδ, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPARδ suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPARδ-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  8. Identification of 42 Genes Linked to Stage II Colorectal Cancer Metastatic Relapse

    Directory of Open Access Journals (Sweden)

    Rabeah A. Al-Temaimi

    2016-04-01

    Full Text Available Colorectal cancer (CRC is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT. Resultant genes were classified based on gene ontology (GO, which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings.

  9. Comparative Genomic Analysis of Neutrophilic Iron(II Oxidizer Genomes for Candidate Genes in Extracellular Electron Transfer

    Directory of Open Access Journals (Sweden)

    Shaomei He

    2017-08-01

    Full Text Available Extracellular electron transfer (EET is recognized as a key biochemical process in circumneutral pH Fe(II-oxidizing bacteria (FeOB. In this study, we searched for candidate EET genes in 73 neutrophilic FeOB genomes, among which 43 genomes are complete or close-to-complete and the rest have estimated genome completeness ranging from 5 to 91%. These neutrophilic FeOB span members of the microaerophilic, anaerobic phototrophic, and anaerobic nitrate-reducing FeOB groups. We found that many microaerophilic and several anaerobic FeOB possess homologs of Cyc2, an outer membrane cytochrome c originally identified in Acidithiobacillus ferrooxidans. The “porin-cytochrome c complex” (PCC gene clusters homologous to MtoAB/PioAB are present in eight FeOB, accounting for 19% of complete and close-to-complete genomes examined, whereas PCC genes homologous to OmbB-OmaB-OmcB in Geobacter sulfurreducens are absent. Further, we discovered gene clusters that may potentially encode two novel PCC types. First, a cluster (tentatively named “PCC3” encodes a porin, an extracellular and a periplasmic cytochrome c with remarkably large numbers of heme-binding motifs. Second, a cluster (tentatively named “PCC4” encodes a porin and three periplasmic multiheme cytochromes c. A conserved inner membrane protein (IMP encoded in PCC3 and PCC4 gene clusters might be responsible for translocating electrons across the inner membrane. Other bacteria possessing PCC3 and PCC4 are mostly Proteobacteria isolated from environments with a potential niche for Fe(II oxidation. In addition to cytochrome c, multicopper oxidase (MCO genes potentially involved in Fe(II oxidation were also identified. Notably, candidate EET genes were not found in some FeOB, especially the anaerobic ones, probably suggesting EET genes or Fe(II oxidation mechanisms are different from the searched models. Overall, based on current EET models, the search extends our understanding of bacterial EET and

  10. Gene Expression Profile in the Early Stage of Angiotensin II-induced Cardiac Remodeling: a Time Series Microarray Study in a Mouse Model

    Directory of Open Access Journals (Sweden)

    Meng-Qiu Dang

    2015-01-01

    Full Text Available Background/Aims: Angiotensin II (Ang II plays a critical role in the cardiac remodeling contributing to heart failure. However, the gene expression profiles induced by Ang II in the early stage of cardiac remodeling remain unknown. Methods: Wild-type male mice (C57BL/6 background, 10-weeek-old were infused with Ang II (1500 ng/kg/min for 7 days. Blood pressure was measured. Cardiac function and remodeling were examined by echocardiography, H&E and Masson staining. The time series microarrays were then conducted to detected gene expression profiles. Results: Microarray results identified that 1,489 genes were differentially expressed in the hearts at day 1, 3 and 7 of Ang II injection. These genes were further classified into 26 profiles by hierarchical cluster analysis. Of them, 4 profiles were significant (No. 19, 8, 21 and 22 and contained 904 genes. Gene Ontology showed that these genes mainly participate in metabolic process, oxidation-reduction process, extracellular matrix organization, apoptotic process, immune response, and others. Significant pathways included focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, MAPK and insulin signaling pathways, which were known to play important roles in Ang II-induced cardiac remodeling. Moreover, gene co-expression networks analysis suggested that serine/cysteine peptidase inhibitor, member 1 (Serpine1, also known as PAI-1 localized in the core of the network. Conclusions: Our results indicate that many genes are mainly involved in metabolism, inflammation, cardiac fibrosis and hypertrophy. Serpine1 may play a central role in the development of Ang II-induced cardiac remodeling at the early stage.

  11. Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression

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    Chun-Ki Kim

    2012-01-01

    Full Text Available Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1 and glutamine-cysteine ligase (GCL. Furthermore, KGBE administration suppressed NF-κB-mediated expression of atherogenic inflammatory genes (TNF-α, IL-1β, iNOS, COX-2, ICAM-1, and VCAM-1, without altering serum cholesterol levels, in ApoE-/- mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-κB activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-α-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-κB-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis.

  12. Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

    Science.gov (United States)

    Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

    1992-01-01

    A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

  13. Linkage of the gene that encodes the alpha 1 chain of type V collagen (COL5A1) to type II Ehlers-Danlos syndrome (EDS II).

    Science.gov (United States)

    Loughlin, J; Irven, C; Hardwick, L J; Butcher, S; Walsh, S; Wordsworth, P; Sykes, B

    1995-09-01

    Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective tissue with skin, ligaments and blood vessels being the main sites affected. The commonest variant (EDS II) exhibits an autosomal dominant mode of inheritance and is characterized by joint hypermobility, cigarette paper scars, lax skin and excessive bruising. As yet no gene has been linked to EDS II, nor has linkage been established to a specific region of the genome. However, several candidate genes encoding proteins of the extracellular matrix have been excluded. Using an intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1 gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a single large pedigree.

  14. Identification and characterization of the human type II collagen gene (COL2A1).

    NARCIS (Netherlands)

    K.S.E. Cheah (Kathryn); N.G. Stoker; J.R. Griffin; F.G. Grosveld (Frank); E. Solomon

    1985-01-01

    textabstractThe gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis

  15. Evolution of major histocompatibility complex class I and class II genes in the brown bear

    Directory of Open Access Journals (Sweden)

    Kuduk Katarzyna

    2012-10-01

    Full Text Available Abstract Background Major histocompatibility complex (MHC proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN exceeded the rate of synonymous substitutions (dS at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

  16. Evolution of major histocompatibility complex class I and class II genes in the brown bear.

    Science.gov (United States)

    Kuduk, Katarzyna; Babik, Wiesław; Bojarska, Katarzyna; Sliwińska, Ewa B; Kindberg, Jonas; Taberlet, Pierre; Swenson, Jon E; Radwan, Jacek

    2012-10-02

    Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South-north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

  17. Evolution of major histocompatibility complex class I and class II genes in the brown bear

    Science.gov (United States)

    2012-01-01

    Background Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia. PMID:23031405

  18. Feature selection and classification of MAQC-II breast cancer and multiple myeloma microarray gene expression data.

    Directory of Open Access Journals (Sweden)

    Qingzhong Liu

    Full Text Available Microarray data has a high dimension of variables but available datasets usually have only a small number of samples, thereby making the study of such datasets interesting and challenging. In the task of analyzing microarray data for the purpose of, e.g., predicting gene-disease association, feature selection is very important because it provides a way to handle the high dimensionality by exploiting information redundancy induced by associations among genetic markers. Judicious feature selection in microarray data analysis can result in significant reduction of cost while maintaining or improving the classification or prediction accuracy of learning machines that are employed to sort out the datasets. In this paper, we propose a gene selection method called Recursive Feature Addition (RFA, which combines supervised learning and statistical similarity measures. We compare our method with the following gene selection methods: Support Vector Machine Recursive Feature Elimination (SVMRFE, Leave-One-Out Calculation Sequential Forward Selection (LOOCSFS, Gradient based Leave-one-out Gene Selection (GLGS. To evaluate the performance of these gene selection methods, we employ several popular learning classifiers on the MicroArray Quality Control phase II on predictive modeling (MAQC-II breast cancer dataset and the MAQC-II multiple myeloma dataset. Experimental results show that gene selection is strictly paired with learning classifier. Overall, our approach outperforms other compared methods. The biological functional analysis based on the MAQC-II breast cancer dataset convinced us to apply our method for phenotype prediction. Additionally, learning classifiers also play important roles in the classification of microarray data and our experimental results indicate that the Nearest Mean Scale Classifier (NMSC is a good choice due to its prediction reliability and its stability across the three performance measurements: Testing accuracy, MCC values, and

  19. BanII dimorphic site located in the third intron of the human apolipoprotein AI (APOA1) gene

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, R T; Kresnak, M T; Frossard, P M

    1988-02-11

    A 0.7kb fragment generated by AvaII digestion of pBL13AI, a 0.965kb full-length human apolipoprotein AI cDNA was cloned into the EcoRI site of pBR322. The apoAI cDNA was isolated from a lambdagt10 human fetal liver cDNA library. BanII (GPuGCPyC) (International Biotechnologies, Inc.) identifies two invariant bands at 1122bp and 417bp, and a single two-allele polymorphism with bands at either 274bp or 452bp. The human apolipoprotein AI-CIII-AIV gene complex has been localized on the long arm of chromosome 11 by Southern blot analysis of human-chinese hamster cell hybrids. Co-dominant segregation has been observed in two families (13 individuals). The BanII restriction map was constructed from DNA sequence data of the human apoAI gene. The 452bp fragment is generated by the loss of a BanII dimorphic site in the third intron of the apoAI gene, between the 178bp and the 274bp fragments.

  20. Interaction between alcohol dehydrogenase II gene, alcohol consumption, and risk for breast cancer

    OpenAIRE

    St?rmer, T; Wang-Gohrke, S; Arndt, V; Boeing, H; Kong, X; Kreienberg, R; Brenner, H

    2002-01-01

    MaeIII Restriction Fragment Length Polymorphism in exon 3 of the alcohol dehydrogenase II was assessed in serum from 467 randomly selected German women and 278 women with invasive breast cancer to evaluate the interaction between a polymorphism of the alcohol dehydrogenase II gene, alcohol consumption and risk for breast cancer. In both groups, usual consumption of different alcoholic beverages was asked for using semiquantitative food frequency questionnaires. We used multivariable logistic ...

  1. DNA polymorphism of HLA class II genes in primary biliary cirrhosis

    DEFF Research Database (Denmark)

    Morling, Niels; Dalhoff, K; Fugger, L

    1992-01-01

    We investigated the DNA restriction fragment length polymorphism of the major histocompatibility complex class II genes: HLA-DRB, -DQA, -DQB, DPA, -DPB, the serologically defined HLA-A, B, C, DR antigens, and the primed lymphocyte typing defined HLA-DP antigens in 23 Danish patients with primary...... than 0.05, 'corrected' P greater than 0.05). No DNA fragments specific for DRB1*0301 (DR3) could be identified. The frequencies in PBC of other genetic markers including DRw8, DRB1*08, HLA-DP antigens, DPA, and DPB genes did not differ significantly from those in controls. The associations between PBC...

  2. BcII RFLP for the human vimentin gene

    Energy Technology Data Exchange (ETDEWEB)

    Marcus, E M; Smith, B A; Telenius, H; Ponder, B A.J.; Mathew, C G.P. [Haddow Laboratories, Surrey (England); Landsvater, R M; Buys, C H.C.M. [State Univ. of Groningen (Netherlands); Ferrari, S [Temple University Medical School, Philadelphia, PA (USA)

    1988-09-26

    A 1.1 kb cDNA clone (hp4F1) encoding the human vimentin gene was identified in a human library by screening with 4F1, a hamster vimentin cDNA. BcII (TGATCA) recognizes a two allele polymorphism: bands A1 at 8.1 kb, and A2 at 3.6 kb. The allele frequency was determined in 47 unrelated Caucasian individuals. The RFLP was mapped to chromosome 10pter-10q23 using somatic cell hybrids and to 10p13 by in situ hybridization. Co-dominant segregation was observed in 2 informative families.

  3. Giant panda genomic data provide insight into the birth-and-death process of mammalian major histocompatibility complex class II genes.

    Directory of Open Access Journals (Sweden)

    Qiu-Hong Wan

    Full Text Available To gain an understanding of the genomic structure and evolutionary history of the giant panda major histocompatibility complex (MHC genes, we determined a 636,503-bp nucleotide sequence spanning the MHC class II region. Analysis revealed that the MHC class II region from this rare species contained 26 loci (17 predicted to be expressed, of which 10 are classical class II genes (1 DRA, 2 DRB, 2 DQA, 3 DQB, 1 DYB, 1 DPA, and 2 DPB and 4 are non-classical class II genes (1 DOA, 1 DOB, 1 DMA, and 1 DMB. The presence of DYB, a gene specific to ruminants, prompted a comparison of the giant panda class II sequence with those of humans, cats, dogs, cattle, pigs, and mice. The results indicated that birth and death events within the DQ and DRB-DY regions led to major lineage differences, with absence of these regions in the cat and in humans and mice respectively. The phylogenetic trees constructed using all expressed alpha and beta genes from marsupials and placental mammals showed that: (1 because marsupials carry loci corresponding to DR, DP, DO and DM genes, those subregions most likely developed before the divergence of marsupials and placental mammals, approximately 150 million years ago (MYA; (2 conversely, the DQ and DY regions must have evolved later, but before the radiation of placental mammals (100 MYA. As a result, the typical genomic structure of MHC class II genes for the giant panda is similar to that of the other placental mammals and corresponds to BTNL2 approximately DR1 approximately DQ approximately DR2 approximately DY approximately DO_box approximately DP approximately COL11A2. Over the past 100 million years, there has been birth and death of mammalian DR, DQ, DY, and DP genes, an evolutionary process that has brought about the current species-specific genomic structure of the MHC class II region. Furthermore, facing certain similar pathogens, mammals have adopted intra-subregion (DR and DQ and inter-subregion (between DQ and DP

  4. DNA polymorphism of HLA class II genes in pauciarticular juvenile rheumatoid arthritis

    DEFF Research Database (Denmark)

    Morling, N; Friis, J; Fugger, L

    1991-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) class II genes: HLA-DRB, -DQA, -DQB, DPA, and -DPB in 54 patients with pauciarticular juvenile rheumatoid arthritis (PJRA) and in healthy Danes. The frequencies of DNA fragments a...

  5. Glucose 6P binds and activates HlyIIR to repress Bacillus cereus haemolysin hlyII gene expression.

    Directory of Open Access Journals (Sweden)

    Elisabeth Guillemet

    Full Text Available Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. We have previously shown that B. cereus Haemolysin II (HlyII induces macrophage cell death by apoptosis. In this work, we investigated the regulation of the hlyII gene. We show that HlyIIR, the negative regulator of hlyII expression in B. cereus, is especially active during the early bacterial growth phase. We demonstrate that glucose 6P directly binds to HlyIIR and enhances its activity at a post-transcriptional level. Glucose 6P activates HlyIIR, increasing its capacity to bind to its DNA-box located upstream of the hlyII gene, inhibiting its expression. Thus, hlyII expression is modulated by the availability of glucose. As HlyII induces haemocyte and macrophage death, two cell types that play a role in the sequestration of nutrients upon infection, HlyII may induce host cell death to allow the bacteria to gain access to carbon sources that are essential components for bacterial growth.

  6. IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor

    DEFF Research Database (Denmark)

    Versteyhe, Soetkin; Klaproth, Birgit; Borup, Rehannah

    2013-01-01

    Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor...... for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice......, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts...

  7. Gene expression dose-response changes in microarrays after exposure of human peripheral lung epithelial cells to nickel(II).

    Science.gov (United States)

    Cheng, Robert Y S; Zhao, Ailian; Alvord, W Gregory; Powell, Douglas A; Bare, Robert M; Masuda, Akira; Takahashi, Takashi; Anderson, Lucy M; Kasprzak, Kazimierz S

    2003-08-15

    Occupational exposure to nickel compounds is associated with lung cancer risk; both genotoxic and epigenetic mechanisms have been proposed. For comprehensive examination of the acute effects of nickel(II) acetate on gene expression in cultured human peripheral lung epithelial HPL1D cells, microarray analyses were carried out with cDNA chips (approximately 8000 cDNAs). Cells were exposed for 24 h to nontoxic (50, 100, and 200 microM) or toxic (400, 800, and 1600 microM) nickel(II) concentrations. Cluster analysis was applied to the 868 genes with > or = 2-fold change at any concentration. Two main clusters showed marked up- or down-regulation at the highest, toxic concentrations. The data further subdivided into 10 highly cohesive clusters with high probability, and of these only 2 had the same response trend at low nontoxic as at high concentrations, an observation of clear relevance to the process of high- to low-dose extrapolation in risk assessment. There were 113 genes showing > or = 2-fold change at the three lower nontoxic concentrations, those most relevant to in vivo carcinogenesis. In addition to expected responses of metallothionein, ferritin, and heat-shock proteins, the results revealed for the first time changed expression of some potential cancer-related genes in response to low-dose Ni(II): RhoA, dyskerin, interferon regulatory factor 1, RAD21 homologue, and tumor protein, translationally controlled. Overall, most of the genes impacted by nontoxic concentrations of nickel(II) acetate related to gene transcription, protein synthesis and stability, cytoskeleton, signaling, metabolism, cell membrane, and extracellular matrix.

  8. Molecular analysis of pericentrin gene (PCNT) in a series of 24 Seckel/microcephalic osteodysplastic primordial dwarfism type II (MOPD II) families.

    Science.gov (United States)

    Willems, M; Geneviève, D; Borck, G; Baumann, C; Baujat, G; Bieth, E; Edery, P; Farra, C; Gerard, M; Héron, D; Leheup, B; Le Merrer, M; Lyonnet, S; Martin-Coignard, D; Mathieu, M; Thauvin-Robinet, C; Verloes, A; Colleaux, L; Munnich, A; Cormier-Daire, V

    2010-12-01

    Microcephalic osteodysplastic primordial dwarfism type II (MOPD II, MIM 210720) and Seckel syndrome (SCKL, MIM 210600) belong to the primordial dwarfism group characterised by intrauterine growth retardation, severe proportionate short stature, and pronounced microcephaly. MOPD II is distinct from SCKL by more severe growth retardation, radiological abnormalities, and absent or mild mental retardation. Seckel syndrome is associated with defective ATR dependent DNA damage signalling. In 2008, loss-of-function mutations in the pericentrin gene (PCNT) have been identified in 28 patients, including 3 SCKL and 25 MOPDII cases. This gene encodes a centrosomal protein which plays a key role in the organisation of mitotic spindles. The aim of this study was to analyse PCNT in a large series of SCKL-MOPD II cases to further define the clinical spectrum associated with PCNT mutations. Among 18 consanguineous families (13 SCKL and 5 MOPDII) and 6 isolated cases (3 SCKL and 3 MOPD II), 13 distinct mutations were identified in 5/16 SCKL and 8/8 MOPDII including five stop mutations, five frameshift mutations, two splice site mutations, and one apparent missense mutation affecting the last base of exon 19. Moreover, we demonstrated that this latter mutation leads to an abnormal splicing with a predicted premature termination of translation. The clinical analysis of the 5 SCKL cases with PCNT mutations showed that they all presented minor skeletal changes and clinical features compatible with MOPDII diagnosis. It is therefore concluded that, despite variable severity, MOPDII is a genetically homogeneous condition due to loss-of-function of pericentrin.

  9. Prevalence of qnr, intI, and intII genes in extendedspectrum beta ...

    African Journals Online (AJOL)

    Purpose: To investigate the prevalence of qnr, intI, and intII genes in extended spectrum betalactamase (ESBL)-producing Escherichia coli isolated from clinical samples in Kerman, Iran. Methods: A total of 127 E. coli were collected from clinical samples in Kerman hospitals. The antibiotic susceptibility test was performed ...

  10. Magnified fluorescence detection of silver(I) ion in aqueous solutions by using nano-graphite-DNA hybrid and DNase I.

    Science.gov (United States)

    Wei, Yin; Li, Bianmiao; Wang, Xu; Duan, Yixiang

    2014-08-15

    This paper describes a novel approach utilizing nano-graphite-DNA hybrid and DNase I for the amplified detection of silver(I) ion in aqueous solutions for the first time. Nano-graphite can effectively quench the fluorescence of dye-labeled cytosine-rich single-stranded DNA due to its strong π-π stacking interactions; however, in the presence of Ag(+), C-Ag(+)-C coordination induces the probe to fold into a hairpin structure, which does not adsorb on the surface of nano-graphite and thus retains the dye fluorescence. Meanwhile, the hairpin structure can be cleaved by DNase I, and in such case Ag(+) is delivered from the complex. The released Ag(+) then binds other dye-labeled single-stranded DNA on the nano-graphite surface, and touches off another target recycling, resulting in the successive release of dye-labeled single-stranded DNA from the nano-graphite, which leads to significant amplification of the signal. The present magnification sensing system exhibits high sensitivity toward Ag(+) with a limit of detection of 0.3nM (S/N=3), which is much lower than the standard for Ag(+) in drinking water recommended by the Environmental Protection Agency (EPA). The selectivity of the sensor for Ag(+) against other biologically and environmentally related metal ions is outstanding due to the high specificity of C-Ag(+)-C formation. Moreover, the sensing system is used for the determination of Ag(+) in river water samples with satisfying results. The proposed assay is simple, cost-effective, and might open the door for the development of new assays for other metal ions or biomolecules. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

    Science.gov (United States)

    Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

    2018-06-01

    To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

  12. The roles of MHC class II genes and post-translational modification in celiac disease.

    Science.gov (United States)

    Sollid, Ludvig M

    2017-08-01

    Our increasing understanding of the etiology of celiac disease, previously considered a simple food hypersensitivity disorder caused by an immune response to cereal gluten proteins, challenges established concepts of autoimmunity. HLA is a chief genetic determinant, and certain HLA-DQ allotypes predispose to the disease by presenting posttranslationally modified (deamidated) gluten peptides to CD4 + T cells. The deamidation of gluten peptides is mediated by transglutaminase 2. Strikingly, celiac disease patients generate highly disease-specific autoantibodies to the transglutaminase 2 enzyme. The dual role of transglutaminase 2 in celiac disease is hardly coincidental. This paper reviews the genetic mapping and involvement of MHC class II genes in disease pathogenesis, and discusses the evidence that MHC class II genes, via the involvement of transglutaminase 2, influence the generation of celiac disease-specific autoantibodies.

  13. NASA's GeneLab Phase II: Federated Search and Data Discovery

    Science.gov (United States)

    Berrios, Daniel C.; Costes, Sylvain V.; Tran, Peter B.

    2017-01-01

    GeneLab is currently being developed by NASA to accelerate 'open science' biomedical research in support of the human exploration of space and the improvement of life on earth. Phase I of the four-phase GeneLab Data Systems (GLDS) project emphasized capabilities for submission, curation, search, and retrieval of genomics, transcriptomics and proteomics ('omics') data from biomedical research of space environments. The focus of development of the GLDS for Phase II has been federated data search for and retrieval of these kinds of data across other open-access systems, so that users are able to conduct biological meta-investigations using data from a variety of sources. Such meta-investigations are key to corroborating findings from many kinds of assays and translating them into systems biology knowledge and, eventually, therapeutics.

  14. NASAs GeneLab Phase II: Federated Search and Data Discovery

    Science.gov (United States)

    Berrios, Daniel C.; Costes, Sylvain; Tran, Peter

    2017-01-01

    GeneLab is currently being developed by NASA to accelerate open science biomedical research in support of the human exploration of space and the improvement of life on earth. Phase I of the four-phase GeneLab Data Systems (GLDS) project emphasized capabilities for submission, curation, search, and retrieval of genomics, transcriptomics and proteomics (omics) data from biomedical research of space environments. The focus of development of the GLDS for Phase II has been federated data search for and retrieval of these kinds of data across other open-access systems, so that users are able to conduct biological meta-investigations using data from a variety of sources. Such meta-investigations are key to corroborating findings from many kinds of assays and translating them into systems biology knowledge and, eventually, therapeutics.

  15. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K

    2001-01-01

    but independent of binding to pocket-binding proteins, suggesting a novel mechanism for E2F-mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol. 20, 2014-2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down....... These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5' deletion analysis of the PAI-1 promoter showed that multiple sites...

  16. Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II

    OpenAIRE

    Faust, Daniela M.; Renkawitz-Pohl, Renate; Falkenburg, Dieter; Gasch, Alexander; Bialojan, Siegfried; Young, Richard A.; Bautz, Ekkehard K. F.

    1986-01-01

    Genomic clones of Drosophila melanogaster were isolated from a λ library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of ∼2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)+-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion p...

  17. Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes.

    Science.gov (United States)

    Kumar, Rahul

    2016-01-01

    Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

  18. DNA typing of HLA class II genes in native inhabitants of Chukotka

    Energy Technology Data Exchange (ETDEWEB)

    Krylov, M.Yu.; Erdesz, S.; Alexeeva, L.I. [Institute of Rheumatology, Moscow (Russian Federation)

    1995-06-01

    Polymorphism of HLA class II genes was studied in native Chukotka inhabitants with the use of DNA oligotyping. The characteristics of the distribution of allelic variants of the loci HLA-DRB1, -DQA1, -DQB1, and -DPB1 were revealed; they were similar to those of other Subarctic Mongoloid populations and different from those for comparable populations of other climatic and geographic zones. Our data suggest that the specific features found for the distributions of some alleles of the loci examined are related to the geographic variation in the HLA gene system studied. 20 refs., 4 tabs.

  19. Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

    DEFF Research Database (Denmark)

    Hwang, C S; Mandrup, S; MacDougald, O A

    1996-01-01

    Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob...... gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes...... to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C...

  20. The in vitro transcription of a rainbow trout (Salmo gairdnerii) protamine gene. II. Controlled mutation of the cap site region.

    Science.gov (United States)

    Jankowski, J M; Dixon, G H

    1985-02-01

    A series of plasmids containing new fusion genes in which the trout protamine gene is placed under the control of the complete herpes virus (HSV-1) tk promoter Pvu II-Bgl II fragment (pM8), or a shortened thymidine kinase (tk) promoter in which the region between the TATA box and the cap site is altered by using the Pvu II-Mlu I fragment (pM7), have been constructed. An additional recombinant plasmid was constructed in which the Bgl II-Ava II fragment of the protamine gene containing the entire protamine promoter but missing the protamine coding region was cloned into pBR322 between the Xho II 1666 and Hind III sites (pP5). For in vitro transcription, a HeLa cell lysate system was prepared and the RNA transcription products, after glyoxalation, were electrophoretically analyzed on 5% polyacrylamide gels. In constructing pM8 the DNA sequence between the tk promoter and the cap site was present while in pM7 it was deleted. Similar multiple transcripts were seen in both cases, indicating that the region between the promoter and the cap site has no effect upon transcription in vitro. The multiple transcripts appear to be due to the presence of a cryptic promoter in the complementary strand of the protamine gene. The activity of this cryptic promoter has been confirmed by comparison of the transcription of plasmid pP5, in which the protamine mRNA coding region has been deleted, with a previously described plasmid, pJBRP (Jankowski JM and Dixon GH (1984) Can. J. Biochem. Cell. Biol. 62, 291-300), containing the intact protamine gene.

  1. Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Nana Pei

    Full Text Available Increased expression of angiotensin II type 2 receptor (AT2R induces apoptosis in numerous tumor cell lines, with either Angiotensin II-dependent or Angiotensin II-independent regulation, but its molecular mechanism remains poorly understood. Here, we used PCR Array analysis to determine the gene and microRNA expression profiles in human prostate cancer cell lines transduced with AT2R recombinant adenovirus. Our results demonstrated that AT2R over expression leads to up-regulation of 6 apoptosis-related genes (TRAIL-R2, BAG3, BNIPI, HRK, Gadd45a, TP53BP2, 2 cytokine genes (IL6 and IL8 and 1 microRNA, and down-regulation of 1 apoptosis-related gene TNFSF10 and 2 cytokine genes (BMP6, BMP7 in transduced DU145 cells. HRK was identified as an up-regulated gene in AT2R-transduced PC-3 cells by real-time RT-PCR. Next, we utilized siRNAs to silence the up-regulated genes to further determine their roles on AT2R overexpression mediated apoptosis. The results showed downregulation of Gadd45a reduced the apoptotic effect by ∼30% in DU145 cells, downregulation of HRK reduced AT2R-mediated apoptosis by more than 50% in PC-3 cells, while downregulation of TRAIL-R2 enhanced AT2R-mediated apoptosis more than 4 times in DU145 cells. We also found that the effects on AT2R-mediated apoptosis caused by downregulation of Gadd45a, TRAIL-R2 and HRK were independent in activation of p38 MAPK, p44/42 MAPK and p53. Taken together, our results demonstrated that TRAIL-R2, Gadd45a and HRK may be novel target genes for further study of the mechanism of AT2R-mediated apoptosis in prostate cancer cells.

  2. Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

    Science.gov (United States)

    Seal, S N; Davis, D L; Burch, J B

    1991-05-01

    The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.

  3. Characterization of a DUF820 family protein Alr3200 of the ...

    Indian Academy of Sciences (India)

    The hypothetical protein 'Alr3200' of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterialspecies. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have aDNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a ...

  4. Insulin gene polymorphisms in type 1 diabetes, Addison's disease and the polyglandular autoimmune syndrome type II

    Directory of Open Access Journals (Sweden)

    Hahner Stefanie

    2008-07-01

    Full Text Available Abstract Background Polymorphisms within the insulin gene can influence insulin expression in the pancreas and especially in the thymus, where self-antigens are processed, shaping the T cell repertoire into selftolerance, a process that protects from β-cell autoimmunity. Methods We investigated the role of the -2221Msp(C/T and -23HphI(A/T polymorphisms within the insulin gene in patients with a monoglandular autoimmune endocrine disease [patients with isolated type 1 diabetes (T1D, n = 317, Addison's disease (AD, n = 107 or Hashimoto's thyroiditis (HT, n = 61], those with a polyglandular autoimmune syndrome type II (combination of T1D and/or AD with HT or GD, n = 62 as well as in healthy controls (HC, n = 275. Results T1D patients carried significantly more often the homozygous genotype "CC" -2221Msp(C/T and "AA" -23HphI(A/T polymorphisms than the HC (78.5% vs. 66.2%, p = 0.0027 and 75.4% vs. 52.4%, p = 3.7 × 10-8, respectively. The distribution of insulin gene polymorphisms did not show significant differences between patients with AD, HT, or APS-II and HC. Conclusion We demonstrate that the allele "C" of the -2221Msp(C/T and "A" -23HphI(A/T insulin gene polymorphisms confer susceptibility to T1D but not to isolated AD, HT or as a part of the APS-II.

  5. Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

    Science.gov (United States)

    Monerawela, Chandre; James, Tharappel C; Wolfe, Kenneth H; Bond, Ursula

    2015-03-01

    Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  6. Pvu-II RFLP at the human lipoprotein lipase (LPL) gene locus

    Energy Technology Data Exchange (ETDEWEB)

    Li, S; Oka, K; Galton, D; Stocks, J

    1988-03-25

    Human lipoprotein lipase (LPL) cDNA, 1.6 Kbp, was isolated from human adipose cDNA library and subcloned into Bam HI site of pIB131. Pvu-II identifies a two allele polymorphism with bands at 7.0 Kb, 4.4 Kb and 2.5 Kb. The frequency was studied in 34 caucasians. The gene was assigned to 8p22. Co-dominant inheritance was demonstrated in a 2 generation family.

  7. MHC class II genes in the European badger (Meles meles) : Characterization, patterns of variation, and transcription analysis

    NARCIS (Netherlands)

    Sin, Yung Wa; Dugdale, Hannah L.; Newman, Chris; Macdonald, David W.; Burke, Terry

    The major histocompatibility complex (MHC) comprises many genes, some of which are polymorphic with numerous alleles. Sequence variation among alleles is most pronounced in exon 2 of the class II genes, which encodes the alpha 1 and beta 1 domains that form the antigen-binding site (ABS) for the

  8. De novo dominant mutation of SOX10 gene in a Chinese family with Waardenburg syndrome type II.

    Science.gov (United States)

    Chen, Kaitian; Zong, Ling; Liu, Min; Zhan, Yuan; Wu, Xuan; Zou, Wenting; Jiang, Hongyan

    2014-06-01

    Waardenburg syndrome is a rare genetic disorder, inherited as an autosomal dominant trait. The condition is characterized by sensorineural hearing loss and pigment disturbances of the hair, skin, and iris. The de novo mutation in the SOX10 gene, responsible for Waardenburg syndrome type II, is rarely seen. The present study aimed to identify the genetic causes of Waardenburg syndrome type II in a Chinese family. Clinical and molecular evaluations were conducted in a Chinese family with Waardenburg syndrome type II. A novel SOX10 heterozygous c.259-260delCT mutation was identified. Heterozygosity was not observed in the parents and sister of the proband, indicating that the mutation has arisen de novo. The novel frameshift mutation, located in exon 3 of the SOX10 gene, disrupted normal amino acid coding from Leu87, leading to premature termination at nucleotide 396 (TGA). The high mobility group domain of SOX10 was inferred to be partially impaired. The novel heterozygous c.259-260delCT mutation in the SOX10 gene was considered to be the cause of Waardenburg syndrome in the proband. The clinical and genetic characterization of this family would help elucidate the genetic heterogeneity of SOX10 in Waardenburg syndrome type II. Moreover, the de novo pattern expanded the mutation data of SOX10. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin.

    Science.gov (United States)

    Mohan Kumar, Dipu; Lin, Mingqun; Xiong, Qingming; Webber, Mathew James; Kural, Comert; Rikihisa, Yasuko

    2015-11-03

    Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. Ehrlichia chaffeensis, an

  10. DNA polymorphism of HLA class II genes in pauciarticular juvenile rheumatoid arthritis

    DEFF Research Database (Denmark)

    Morling, N; Friis, J; Fugger, L

    1991-01-01

    associated with the following HLA class II genes were increased in PJRA when compared to normal controls: DRB1*08 (DRw8) (35.2% vs 10.3%, RR = 4.6, p less than 10(-3), DRB3*01/02/03 (DRw52) (76.3% vs 48.1%, RR 3.5, p less than 10(-3)), DQA1*0401 (41.0% vs 7.4%, RR = 7.9, p less than 10(-3)), DQA1*0501 (55...... of DNA fragments associated with the following HLA class II genes were decreased in PJRA although not statistically significantly so after 'correction' of p values: DRB1*04 (14.8% vs 40.2%, RR = 0.27; p less than 10(-3)), DRB1*07 (0% vs 25.9%, RR = 0.04, p less than 10(-3)), DRB4*0101 (DRw53) (25.9% vs...... 53.6%, RR = 0.31, p less than 10(-3)), DQA1*0102 (11.6% vs 36.0%, RR = 0.25, p less than 10(-4)), and DQA1*0201 (2.6% vs 34.2%, RR = 0.05, p less than 10(-2)).(ABSTRACT TRUNCATED AT 250 WORDS)...

  11. An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

    Science.gov (United States)

    Itzhaki, H; Maxson, J M; Woodson, W R

    1994-09-13

    The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

  12. The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

    International Nuclear Information System (INIS)

    Katayama, Seiichi; Ashizawa, Koji; Gohma, Hiroshi; Fukuhara, Tadahiro; Narumi, Kazunori; Tsuzuki, Yasuhiro; Tatemoto, Hideki; Nakada, Tadashi; Nagai, Kenji

    2006-01-01

    The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17α-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ERα-selective agonist), diarylpropionitrile (DPN, an ERβ-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ERα-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ERβ-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

  13. The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

    Science.gov (United States)

    Katayama, Seiichi; Ashizawa, Koji; Gohma, Hiroshi; Fukuhara, Tadahiro; Narumi, Kazunori; Tsuzuki, Yasuhiro; Tatemoto, Hideki; Nakada, Tadashi; Nagai, Kenji

    2006-12-15

    The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17alpha-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ERalpha-selective agonist), diarylpropionitrile (DPN, an ERbeta-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ERalpha-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ERbeta-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

  14. Soluble TGF-β type II receptor gene therapy reduces TGF-β activity in irradiated lung tissue and protects lungs from radiation-induced injury

    International Nuclear Information System (INIS)

    Vujaskovic, Z.; Rabbani, Z.; Zhang, X.; Samulski, T.V.; Li, C.-Y.; Anscher, M.S.

    2003-01-01

    Full text: The objective was to determine whether administration of recombinant human adenoviral vector carrying soluble TGF-β1 type II receptor (TβR-II) gene reduces availability of active TGFβ1 and protects lung from radiation-induced injury. Female Fisher-344 rats were randomized into four groups to receive: 1) Control 2) Adenoviral green fluorescent protein vector (AdGFP) alone 3) Radiation (RT) + Adenoviral vector with TGF-β1 type II receptor gene (AdexTβR-II-Fc) 4) RT alone. Animals were irradiated to right hemithorax using a single dose of 30 Gy. The packaging and production of a recombinant adenovirus carrying the fused human TβR-II-IgG1 Fc gene was achieved by use of the AdEasy system. The treatment vector AdexTbR-II-Fc (1.5*1010 PFU) and control vector AdGFP (1*109 PFU) were injected i.v. 24 hrs after RT. Respiratory rate was measured as an index of pulmonary function weekly for 5 weeks post RT. Structural damage was scored histologically. Immunohistochemistry was performed to identify activated macrophages. ELISA was used to quantify active TGF-β1 in tissue homogenate. Western blot was used to determine TβR-II expression in plasma and lung tissue. Animals receiving treatment vector AdexTbR-II-Fc have elevated plasma levels of soluble TβR-II at 24 and 48 hours after injection. In the RT+AdexTbR-II-Fc group, there was a significant reduction in respiratory rate (p = 0.002) at four weeks after treatment compared to RT alone group. Histology revealed a significant reduction in lung structural damage in animals receiving gene therapy after RT vs RT alone (p=0.0013). There was also a decrease in the number of activated macrophage (p= 0.02) in RT+AdexTbR-II-Fc group vs RT alone. The tissue protein expression of active TGF-β1 was significantly reduced in rats receiving RT+AdexTbR-II-Fc treatment (p<0.05). This study shows the ability of adenovirus mediated soluble TβR-II gene therapy to reduce tissue levels of active TGF-β1 and ameliorate radiation

  15. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes

    Directory of Open Access Journals (Sweden)

    Ruifang Ma

    2017-08-01

    Full Text Available Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10 produced 425.60 μg·g−1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  16. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

    Science.gov (United States)

    Duan, X; Li, X; Xue, Q; Abo-el-Saad, M; Xu, D; Wu, R

    1996-04-01

    We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

  17. TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription.

    Science.gov (United States)

    Gómez-Herreros, Fernando; Zagnoli-Vieira, Guido; Ntai, Ioanna; Martínez-Macías, María Isabel; Anderson, Rhona M; Herrero-Ruíz, Andrés; Caldecott, Keith W

    2017-08-10

    DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.

  18. Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis.

    Science.gov (United States)

    Takemoto, Y; Sakatani, M; Takami, S; Tachibana, T; Higaki, J; Ogihara, T; Miki, T; Katsuya, T; Tsuchiyama, T; Yoshida, A; Yu, H; Tanio, Y; Ueda, E

    1998-06-01

    Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SACE in this disease has been found. A study of the association of angiotensin II receptor gene polymorphisms with sarcoidosis was therefore undertaken. ACE (I/D), angiotensin II type 1 receptor (AGTR1), and angiotensin II type 2 receptor (AGTR2) gene polymorphisms were investigated by polymerase chain reaction (PCR) and SACE levels were measured in three groups of patients: those with sarcoidosis or tuberculosis and normal controls. There was no difference in allele frequency of AGTR1 and AGTR2 polymorphism among the three groups. Neither AGTR1 nor AGTR2 polymorphisms were associated with sarcoidosis. SACE activity was higher in patients with sarcoidosis with the AGTR1 A/C genotype than in others. However, this tendency was not detected in patients with tuberculosis. The AGTR1 allele C is associated with high activity of SACE in patients with sarcoidosis. It is another predisposing factor for high levels of SACE in patients with sarcoidosis and is considered to be an independent factor from the ACE D allele for high levels of SACE in sarcoidosis. This fact could be one of the explanations for the increased SACE activity in sarcoidosis.

  19. Genetic and expression studies of SMN2 gene in Russian patients with spinal muscular atrophy type II and III

    Directory of Open Access Journals (Sweden)

    Schiöth Helgi B

    2011-07-01

    Full Text Available Abstract Background Spinal muscular atrophy (SMA type I, II and III is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (SMN1. SMN2 is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies while most SMA type II patients carry three SMN2 copies and SMA III patients have three or four SMN2 copies. The SMN1 gene produces a full-length transcript (FL-SMN while SMN2 is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMNΔ7 mRNA. Methods In this study we performed quantification of the SMN2 gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the SMN1 gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMNΔ7 mRNA ratio as SMA biomarker. Results Comparison of the SMN2 copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III and presence of four copies of the SMN2 gene. In both asymptomatic cases we found an increased number of SMN2 copies in the healthy carriers and a biallelic SMN1 absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls. Conclusions We suggest that the SMN2 gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMNΔ7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy.

  20. Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

    Science.gov (United States)

    Krawczyk, Z; Wu, C

    1987-08-15

    We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

  1. Ultrasensitive aptamer-based multiplexed electrochemical detection by coupling distinguishable signal tags with catalytic recycling of DNase I.

    Science.gov (United States)

    Tang, Dianping; Tang, Juan; Li, Qunfang; Su, Biling; Chen, Guonan

    2011-10-01

    This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.

  2. Methylation of class II transactivator gene promoter IV is not associated with susceptibility to Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Lincoln Matthew R

    2008-07-01

    Full Text Available Abstract Background Multiple sclerosis (MS is a complex trait in which alleles at or near the class II loci HLA-DRB1 and HLA-DQB1 contribute significantly to genetic risk. The MHC class II transactivator (MHC2TA is the master controller of expression of class II genes, and methylation of the promoter of this gene has been previously been shown to alter its function. In this study we sought to assess whether or not methylation of the MHC2TA promoter pIV could contribute to MS disease aetiology. Methods In DNA from peripheral blood mononuclear cells from a sample of 50 monozygotic disease discordant MS twins the MHC2TA promoter IV was sequenced and analysed by methylation specific PCR. Results No methylation or sequence variation of the MHC2TA promoter pIV was found. Conclusion The results of this study cannot support the notion that methylation of the pIV promoter of MHC2TA contributes to MS disease risk, although tissue and timing specific epigenetic modifications cannot be ruled out.

  3. [Analysis of SOX10 gene mutation in a family affected with Waardenburg syndrome type II].

    Science.gov (United States)

    Zheng, Lei; Yan, Yousheng; Chen, Xue; Zhang, Chuan; Zhang, Qinghua; Feng, Xuan; Hao, Shen

    2018-02-10

    OBJECTIVE To detect potential mutation of SOX10 gene in a pedigree affected with Warrdenburg syndrome type II. METHODS Genomic DNA was extracted from peripheral blood samples of the proband and his family members. Exons and flanking sequences of MITF, PAX3, SOX10, SNAI2, END3 and ENDRB genes were analyzed by chip capturing and high throughput sequencing. Suspected mutations were verified with Sanger sequencing. RESULTS A c.127C>T (p.R43X) mutation of the SOX10 gene was detected in the proband, for which both parents showed a wild-type genotype. CONCLUSION The c.127C>T (p.R43X) mutation of SOX10 gene probably underlies the ocular symptoms and hearing loss of the proband.

  4. A network-based predictive gene-expression signature for adjuvant chemotherapy benefit in stage II colorectal cancer.

    Science.gov (United States)

    Cao, Bangrong; Luo, Liping; Feng, Lin; Ma, Shiqi; Chen, Tingqing; Ren, Yuan; Zha, Xiao; Cheng, Shujun; Zhang, Kaitai; Chen, Changmin

    2017-12-13

    The clinical benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) is controversial. This study aimed to explore novel gene signature to predict outcome benefit of postoperative 5-Fu-based therapy in stage II CRC. Gene-expression profiles of stage II CRCs from two datasets with 5-Fu-based adjuvant chemotherapy (training dataset, n = 212; validation dataset, n = 85) were analyzed to identify the indicator. A systemic approach by integrating gene-expression and protein-protein interaction (PPI) network was implemented to develop the predictive signature. Kaplan-Meier curves and Cox proportional hazards model were used to determine the survival benefit of adjuvant chemotherapy. Experiments with shRNA knock-down were carried out to confirm the signature identified in this study. In the training dataset, we identified 44 PPI sub-modules, by which we separate patients into two clusters (1 and 2) having different chemotherapeutic benefit. A predictor of 11 PPI sub-modules (11-PPI-Mod) was established to discriminate the two sub-groups, with an overall accuracy of 90.1%. This signature was independently validated in an external validation dataset. Kaplan-Meier curves showed an improved outcome for patients who received adjuvant chemotherapy in Cluster 1 sub-group, but even worse survival for those in Cluster 2 sub-group. Similar results were found in both the training and the validation dataset. Multivariate Cox regression revealed an interaction effect between 11-PPI-Mod signature and adjuvant therapy treatment in the training dataset (RFS, p = 0.007; OS, p = 0.006) and the validation dataset (RFS, p = 0.002). From the signature, we found that PTGES gene was up-regulated in CRC cells which were more resistant to 5-Fu. Knock-down of PTGES indicated a growth inhibition and up-regulation of apoptotic markers induced by 5-Fu in CRC cells. Only a small proportion of stage II CRC patients could benefit from adjuvant therapy. The 11-PPI-Mod as

  5. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    Energy Technology Data Exchange (ETDEWEB)

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  6. Novel roles for metallothionein-I + II (MT-I + II) in defense responses, neurogenesis, and tissue restoration after traumatic brain injury: insights from global gene expression profiling in wild-type and MT-I + II knockout mice

    DEFF Research Database (Denmark)

    Penkowa, Milena; Cáceres, Mario; Borup, Rehannah

    2006-01-01

    of the somatosensorial cortex and killed at 0, 1, 4, 8, and 16 days postlesion (dpl) using Affymetrix genechips/oligonucleotide arrays interrogating approximately 10,000 different murine genes (MG_U74Av2). Hierarchical clustering analysis of these genes readily shows an orderly pattern of gene responses at specific...... and opened new avenues that were confirmed by immunohistochemistry. Data in KO, MT-I-overexpressing, and MT-II-injected mice strongly suggest a role of these proteins in postlesional activation of neural stem cells....

  7. [Chromomeric organization of interphase chromosomes in Drosophila melanogaster].

    Science.gov (United States)

    Zhuimulev, I F; Beliaeva, E S; Zykova, T Iu; Semeshin, V F; Demakov, S A; Demakova, O V; Goncharov, F P; Khoroshko, V A; Boldyreva, L V; Kokoza, E B; Pokholkiova, G V

    2013-01-01

    As a result of treatment of bioinformatic data on the genome localization of structural proteins, histone modifications, DNase-hypersensitive regions, replication origins (taken from modENCODE) and their cytological localization to polytene chromosome structures, it is shown here that two types of interphase chromosomes -polytene chromosomes from salivary glands and from mitotically dividing cells cultures - demonstrate identical pictures of interband/band, i. e. the same localization and length on physical map and the same sets of proteins. In the interbands of both chromosome types we find the proteins that control initiation of transcription (RNA-polymerase II, transcription factors), replication (ORC2) as well as proteins modifying nucleosome structure (WDS, NURF) and proteins of insulators (BEAF). The nucleosome density and H1 histone concentration in the interbands are depleted; localization of DNase-hypersensitive regions corresponds strictly to the interbands. So, we conclude that both polytene and cell line interphase chromosomes are arranged according to general principle and polytene chromosomes represent precise model of interphase chromosomes. The interbands play a critical role in the initiation of transcription and replication. The interbands of interphase chromosomes are the sites of 5' parts of genes, while the 3' gene ends are located in the adjacent bands. The constancy of interbands decondensation results in the conclusion that the "interbands" genes are constantly active, i. e. they contain "house-keeping" genes. The large late replicating bands contain genes that do not have direct contact to the adjoining interbands are usually polygenic and contain tissue-specific genes.

  8. The Circadian Rhythm Gene Arntl2 Is a Metastasis Susceptibility Gene for Estrogen Receptor-Negative Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Ngoc-Han Ha

    2016-09-01

    Full Text Available Breast cancer mortality is primarily due to metastasis rather than primary tumors, yet relatively little is understood regarding the etiology of metastatic breast cancer. Previously, using a mouse genetics approach, we demonstrated that inherited germline polymorphisms contribute to metastatic disease, and that these single nucleotide polymorphisms (SNPs could be used to predict outcome in breast cancer patients. In this study, a backcross between a highly metastatic (FVB/NJ and low metastatic (MOLF/EiJ mouse strain identified Arntl2, a gene encoding a circadian rhythm transcription factor, as a metastasis susceptibility gene associated with progression, specifically in estrogen receptor-negative breast cancer patients. Integrated whole genome sequence analysis with DNase hypersensitivity sites reveals SNPs in the predicted promoter of Arntl2. Using CRISPR/Cas9-mediated substitution of the MOLF promoter, we demonstrate that the SNPs regulate Arntl2 transcription and affect metastatic burden. Finally, analysis of SNPs associated with ARNTL2 expression in human breast cancer patients revealed reproducible associations of ARNTL2 expression quantitative trait loci (eQTL SNPs with disease-free survival, consistent with the mouse studies.

  9. Cis-acting elements in the promoter region of the human aldolase C gene.

    Science.gov (United States)

    Buono, P; de Conciliis, L; Olivetta, E; Izzo, P; Salvatore, F

    1993-08-16

    We investigated the cis-acting sequences involved in the expression of the human aldolase C gene by transient transfections into human neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the 5'-flanking DNA direct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease of CAT activity. Gel shift and DNase I footprinting analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Sp1 or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysis shows a half palindromic ERE motif (GGTCA), in elements B and D. Region D binds a transactivating factor which appears also essential to stabilize the initiation complex.

  10. The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin.

    Science.gov (United States)

    Cavaliere, Rosalia; Ball, Jessica L; Turnbull, Lynne; Whitchurch, Cynthia B

    2014-08-01

    Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene

    DEFF Research Database (Denmark)

    Blume, N; Petersen, J S; Andersen, L C

    1992-01-01

    is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency...

  12. Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo

    2012-01-01

    and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining...

  13. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

    International Nuclear Information System (INIS)

    Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

    1988-01-01

    Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

  14. Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene.

    NARCIS (Netherlands)

    Heuvel, L.P.W.J. van den; Koul, K. Op de; Knots, E.; Knoers, N.V.A.M.; Monnens, L.A.H.

    2001-01-01

    BACKGROUND: At present the genetic defect for autosomal recessive and autosomal dominant hypophosphataemic rickets with hypercalciuria (HHRH) is unknown. Type II sodium/phosphate cotransporter (NPT2) gene is a serious candidate for being the causative gene in either or both autosomal recessive and

  15. Zn(II)-dipicolylamine-based metallo-lipids as novel non-viral gene vectors.

    Science.gov (United States)

    Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zhao, Zhi-Gang

    2017-08-01

    In this study, a series of Zn(II)-dipicolylamine (Zn-DPA) based cationic lipids bearing different hydrophobic tails (long chains, α-tocopherol, cholesterol or diosgenin) were synthesized. Structure-activity relationship (SAR) of these lipids was studied in detail by investigating the effects of several structural aspects including the type of hydrophobic tails, the chain length and saturation degree. In addition, several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could condense DNA into nanosized particles with appropriate size and zeta-potentials. MTT-based cell viability assays showed that lipoplexes 5 had low cytotoxicity. The in vitro gene transfection studies showed the hydrophobic tails clearly affected the TE, and hexadecanol-containing lipid 5b gives the best TE, which was 2.2 times higher than bPEI 25k in the presence of 10% serum. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

  16. Sequence Analysis of Inducible Prophage phIS3501 Integrated into the Haemolysin II Gene of Bacillus thuringiensis var israelensis ATCC35646

    Directory of Open Access Journals (Sweden)

    Bouziane Moumen

    2012-01-01

    Full Text Available Diarrheic food poisoning by bacteria of the Bacillus cereus group is mostly due to several toxins encoded in the genomes. One of them, cytotoxin K, was recently identified as responsible for severe necrotic syndromes. Cytotoxin K is similar to a class of proteins encoded by genes usually annotated as haemolysin II (hlyII in the majority of genomes of the B. cereus group. The partially sequenced genome of Bacillus thuringiensis var israelensis ATCC35646 contains several potentially induced prophages, one of them integrated into the hlyII gene. We determined the complete sequence and established the genomic organization of this prophage-designated phIS3501. During induction of excision of this prophage with mitomycin C, intact hlyII gene is formed, thus providing to cells a genetic ability to synthesize the active toxin. Therefore, this prophage, upon its excision, can be implicated in the regulation of synthesis of the active toxin and thus in the virulence of bacterial host. A generality of selection for such systems in bacterial pathogens is indicated by the similarity of this genetic arrangement to that of Staphylococcus aureus  β-haemolysin.

  17. A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II

    International Nuclear Information System (INIS)

    Wang Haoyang; Hou Yanning; Cui Yingxia; Huang Yufeng; Shi Yichao; Xia Xinyi; Lu Hongyong; Wang Yunhua; Li Xiaojun

    2009-01-01

    Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM 125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C→A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II

  18. Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

    International Nuclear Information System (INIS)

    Kedzierska, Barbara; Glinkowska, Monika; Iwanicki, Adam; Obuchowski, Michal; Sojka, Piotr; Thomas, Mark S.; Wegrzyn, Grzegorz

    2003-01-01

    The bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the 'lysis-versus-lysogeny' decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA + or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII

  19. Association analysis of class II cytokine and receptor genes in vitiligo patients.

    Science.gov (United States)

    Traks, Tanel; Karelson, Maire; Reimann, Ene; Rätsep, Ranno; Silm, Helgi; Vasar, Eero; Kõks, Sulev; Kingo, Külli

    2016-05-01

    The loss of melanocytes in vitiligo is mainly attributed to defective autoimmune mechanisms and lately autoinflammatory mediators have become more emphasized. Among these, a number of class II cytokines and their receptors have displayed altered expression patterns in vitiligo. Thus, we selected 30 SNPs from the regions of respective genes to be genotyped in Estonian case-control sample (109 and 328 individuals, respectively). For more precise analyses, patients were divided into subgroups based on vitiligo progression activity, age of onset, sex, occurrence of vitiligo among relatives, extent of depigmented areas, appearance of Köbner's phenomenon, existence of halo nevi, occurrence of spontaneous repigmentation, and amount of thyroid peroxidase antibodies. No associations appeared in whole vitiligo group. In subgroups, several allelic and haplotype associations were found. The strongest involved SNPs rs12301088 (near IL26 gene), that was associated with familial vitiligo and existence of halo nevi, and rs2257167 (IFNAR1 gene), that was associated with female vitiligo. Additionally, haplotypes consisting of rs12301088 and rs12321603 alleles (IL26-IL22 genes), that were associated with familial vitiligo and existence of halo nevi. In conclusion, several genetic associations with vitiligo subphenotypes were revealed and functional explanations to these remain to be determined in respective studies. Copyright © 2016. Published by Elsevier Inc.

  20. Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results.

    Science.gov (United States)

    Naegele, Klaudia; Lautenschlager, Irmeli; Gosert, Rainer; Loginov, Raisa; Bir, Katia; Helanterä, Ilkka; Schaub, Stefan; Khanna, Nina; Hirsch, Hans H

    2018-04-22

    Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by >90% indicating the presence of unprotected CMV genomic DNA. Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons. Copyright © 2018 Elsevier B.V. All

  1. Angiotensin II modulates interleukin-1β-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-κB crosstalk

    International Nuclear Information System (INIS)

    Xu, Shanqin; Zhi, Hui; Hou, Xiuyun; Jiang, Bingbing

    2011-01-01

    Highlights: → We examine how angiotensin II modulates ERK-NF-κB crosstalk and gene expression. → Angiotensin II suppresses IL-1β-induced prolonged ERK and NF-κB activation. → ERK-RSK1 signaling is required for IL-1β-induced prolonged NF-κB activation. → Angiotensin II modulates NF-κB responsive genes via regulating ERK-NF-κB crosstalk. → ERK-NF-κB crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1β-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1β-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1β, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE -/- ) mice. VCAM-1 and iNOS expression were higher in ApoE -/- than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE -/- mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells

  2. Differential expression of secretogranin II and chromogranin A genes in the female rat pituitary through sexual maturation and estrous cycle

    International Nuclear Information System (INIS)

    Anouar, Y.; Duval, J.

    1991-01-01

    Secretogranin II (SgII) is a protein of pituitary secretory granules released by LHRH-stimulated gonadotrope cells. Estrogens and androgens are modulators of SgII release. Experiments were performed to determine the regulation of expression of the SgII gene in the female rat pituitary, during sexual maturation and according to the estrous cycle. Age- and cycle-related changes in SgII mRNA content were estimated through cytoplasmic slot blot; SgII content was determined by western blotting; maturation of the protein was controlled through [35S]sulfate labeling. Variations in chromogranin A (CgA), another protein of secretory granules, were analyzed in the same experimental conditions to assess the specificity of SgII regulation. The pituitary SgII concentration increased between days 7 and 21 (2.2-fold) and then declined to the initial 7-day-old value. Simultaneously, the CgA concentration went through a maximum between days 14 and 21 and then strongly dropped to barely detectable levels in the adult pituitary. The SgII mRNA concentration followed roughly the same pattern as the protein. Moreover, the sulfation level remained constant between days 14 and 60. These results demonstrated a regulatory mechanism operating, during sexual maturation, on the SgII gene and not on the protein processing or on storage/release steps. In the 4-day cycling females, the pituitary SgII mRNA and protein contents were the lowest during estrus. They then increased to their highest values in diestrus II. Moreover, the sulfation level of SgII was significantly higher during estrus than during any other stage. Due to its low content level, variations in pituitary CgA could not be demonstrated during the cycle

  3. Glutaric acidemia type II: gene structure and mutations of the electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) gene.

    Science.gov (United States)

    Goodman, Stephen I; Binard, Robert J; Woontner, Michael R; Frerman, Frank E

    2002-01-01

    Glutaric acidemia type II is a human inborn error of metabolism which can be due to defects in either subunit of electron transfer flavoprotein (ETF) or in ETF:ubiquinone oxidoreductase (ETF:QO), but few disease-causing mutations have been described. The ETF:QO gene is located on 4q33, and contains 13 exons. Primers to amplify these exons are presented, together with mutations identified by molecular analysis of 20 ETF:QO-deficient patients. Twenty-one different disease-causing mutations were identified on 36 of the 40 chromosomes.

  4. The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.

    Directory of Open Access Journals (Sweden)

    Archana D Siddam

    2018-03-01

    Full Text Available Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk inhibitor p27Kip1 by interacting with its 5' UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

  5. Isolation and characterization of major histocompatibility complex class II B genes in cranes.

    Science.gov (United States)

    Kohyama, Tetsuo I; Akiyama, Takuya; Nishida, Chizuko; Takami, Kazutoshi; Onuma, Manabu; Momose, Kunikazu; Masuda, Ryuichi

    2015-11-01

    In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

  6. [Mutational frequencies in usherin(USH2A gene) in 26 Colombian individuals with Usher syndrome type II].

    Science.gov (United States)

    López, Greizy; Gelvez, Nancy Yaneth; Tamayo, Martalucía

    2011-03-01

    Usher syndrome is a disorder characterized by progressive retinitis pigmentosa, prelingual sensory hearing loss and vestibular dysfunction. It is the most frequent cause of deaf-blindness in humans. Three clinical types and twelve genetic subtypes have been characterized. Type II is the most common, and among these cases, nearly 80% have mutations in the USH2A gene. The aim of the study was to establish the mutational frequencies for the short isoform of USH2A gene in Usher syndrome type II. Twenty-six Colombian individuals with Usher syndrome type II were included. SSCP analysis for 20 exons of the short isoform was performed and abnormal patterns were sequenced. Sequencing of exon 13 of the USH2A gene was performed for all the individuals because the most frequent mutation is located in this exon. The most frequent mutation was c.2299delG, identified in the 27% (n=8) of the sample. The second mutation, p.R334W, showed a frequency of 15%. A new variant identified in the 5’UTR region, g.129G>T, was present in 1 individual (4%). Four polymorphisms were identified; one of them is a new deletion in exon 20, first reported in this study. Mutations in the usherin short isoform were identified in 38% of a sample of 26 USH2 cases. Molecular diagnosis was established in 7 of the 26.

  7. Co-ordinate control of synthesis of mitochondrial and non-mitochondrial hemoproteins: a binding site for the HAP1 (CYP1) protein in the UAS region of the yeast catalase T gene (CTT1).

    Science.gov (United States)

    Winkler, H; Adam, G; Mattes, E; Schanz, M; Hartig, A; Ruis, H

    1988-01-01

    Control of expression of the Saccharomyces cerevisiae CTT1 (catalase T) gene by the HAP1 (CYP1) gene, a mediator of heme control of mitochondrial cytochromes, was studied. Expression of a CTT1-lacZ fusion in a hap1 mutant showed that the CTT1 promoter is under HAP1 control. As demonstrated by a gel retardation assay, the HAP1 protein binds to a heme control region of the CTT1 gene. This binding in vitro is stimulated by hemin. The HAP1-binding sequence was localized by using DNA fragments spanning different regions, by DNase I footprinting and by methylation interference of DNA-protein binding. The binding site was compared to the HAP1-binding sequences previously characterized in detail (UAS1CYC1, UASCYC7). There is strikingly little similarity between the three sequences, which have only four of those 23 bp in common which are protected from DNase I digestion. However, the pattern of major and minor groove contacts in the complex is quite similar in all three cases. The results obtained show that there is true co-ordinate control of expression of mitochondrial cytochromes and at least some extra-mitochondrial hemoproteins. Heme acts as a metabolic signal in this coordination, which is mediated by the HAP1 protein. Images PMID:2844525

  8. Familial neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin II gene

    NARCIS (Netherlands)

    de Fost, M.; van Trotsenburg, A. S. P.; van Santen, H. M.; Endert, E.; van den Elzen, C.; Kamsteeg, E. J.; Swaab, D. F.; Fliers, E.

    2011-01-01

    Familial neurohypophyseal (central) diabetes insipidus (DI) is caused by mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The majority of cases is inherited in an autosomal dominant way. In this study, we present the clinical features of a mother and her son with autosomal

  9. Familial neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin II gene

    NARCIS (Netherlands)

    Fost, M. de; Trotsenburg, A.S. van; Santen, H.M. van; Endert, E.; Elzen, C. van den; Kamsteeg, E.J.; Swaab, D.F.; Fliers, E.A.

    2011-01-01

    BACKGROUND: Familial neurohypophyseal (central) diabetes insipidus (DI) is caused by mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The majority of cases is inherited in an autosomal dominant way. In this study, we present the clinical features of a mother and her son with

  10. ColoFinder: a prognostic 9-gene signature improves prognosis for 871 stage II and III colorectal cancer patients

    Directory of Open Access Journals (Sweden)

    Mingguang Shi

    2016-03-01

    Full Text Available Colorectal cancer (CRC is a heterogeneous disease with a high mortality rate and is still lacking an effective treatment. Our goal is to develop a robust prognosis model for predicting the prognosis in CRC patients. In this study, 871 stage II and III CRC samples were collected from six gene expression profilings. ColoFinder was developed using a 9-gene signature based Random Survival Forest (RSF prognosis model. The 9-gene signature recurrence score was derived with a 5-fold cross validation to test the association with relapse-free survival, and the value of AUC was gained with 0.87 in GSE39582(95% CI [0.83–0.91]. The low-risk group had a significantly better relapse-free survival (HR, 14.8; 95% CI [8.17–26.8]; P < 0.001 than the high-risk group. We also found that the 9-gene signature recurrence score contributed more information about recurrence than standard clinical and pathological variables in univariate and multivariate Cox analyses when applied to GSE17536(p = 0.03 and p = 0.01 respectively. Furthermore, ColoFinder improved the predictive ability and better stratified the risk subgroups when applied to CRC gene expression datasets GSE14333, GSE17537, GSE12945and GSE24551. In summary, ColoFinder significantly improves the risk assessment in stage II and III CRC patients. The 9-gene prognostic classifier informs patient prognosis and treatment response.

  11. MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen

    OpenAIRE

    Garman, Lori; Dumas, Eric K.; Kurella, Sridevi; Hunt, Jonathan J.; Crowe, Sherry R.; Nguyen, Melissa L.; Cox, Philip M.; James, Judith A.; Farris, A. Darise

    2012-01-01

    Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class I...

  12. Majewski osteodysplastic primordial dwarfism type II (MOPD II) syndrome previously diagnosed as Seckel syndrome: report of a novel mutation of the PCNT gene.

    Science.gov (United States)

    Piane, Maria; Della Monica, Matteo; Piatelli, Gianluca; Lulli, Patrizia; Lonardo, Fortunato; Chessa, Luciana; Scarano, Gioacchino

    2009-11-01

    We report on a 3-year-old boy with prenatal onset of proportionate dwarfism, postnatal severe microcephaly, high forehead with receded hairline, sparse scalp hair, beaked nose, mild retrognathia and hypotonia diagnosed at birth as Seckel syndrome. At age 3 years, he became paralyzed due to a cerebrovascular malformation. Based on the clinical and radiological features showing evidence of skeletal dysplasia, the diagnosis was revised to Majewski osteodysplastic primordial dwarfism type II (MOPD II) syndrome. Western blot analysis of the patient's lymphoblastoid cell line lysate showed the absence of the protein pericentrin. Subsequent molecular analysis identified a novel homozygous single base insertion (c.1527_1528insA) in exon 10 of the PCNT gene, which leads to a frameshift (Treo510fs) and to premature protein truncation. PCNT mutations must be considered diagnostic of MOPD II syndrome. A possible role of pericentrin in the development of cerebral vessels is suggested. Copyright 2009 Wiley-Liss, Inc.

  13. Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

    Science.gov (United States)

    Bastian, Thomas W; Rice, Stephen A

    2009-01-01

    Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

  14. Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

    Science.gov (United States)

    Cao, Yanli; Zheng, Fanglin; Wang, Lei; Zhao, Guolei; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

    2017-07-01

    Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. © 2017 John Wiley & Sons Ltd.

  15. Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis

    OpenAIRE

    Takemoto, Y.; Sakatani, M.; Takami, S.; Tachibana, T.; Higaki, J.; Ogihara, T.; Miki, T.; Katsuya, T.; Tsuchiyama, T.; Yoshida, A.; Yu, H.; Tanio, Y.; Ueda, E.

    1998-01-01

    BACKGROUND—Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SA...

  16. Identification and Construction of Combinatory Cancer Hallmark-Based Gene Signature Sets to Predict Recurrence and Chemotherapy Benefit in Stage II Colorectal Cancer.

    Science.gov (United States)

    Gao, Shanwu; Tibiche, Chabane; Zou, Jinfeng; Zaman, Naif; Trifiro, Mark; O'Connor-McCourt, Maureen; Wang, Edwin

    2016-01-01

    Decisions regarding adjuvant therapy in patients with stage II colorectal cancer (CRC) have been among the most challenging and controversial in oncology over the past 20 years. To develop robust combinatory cancer hallmark-based gene signature sets (CSS sets) that more accurately predict prognosis and identify a subset of patients with stage II CRC who could gain survival benefits from adjuvant chemotherapy. Thirteen retrospective studies of patients with stage II CRC who had clinical follow-up and adjuvant chemotherapy were analyzed. Respective totals of 162 and 843 patients from 2 and 11 independent cohorts were used as the discovery and validation cohorts, respectively. A total of 1005 patients with stage II CRC were included in the 13 cohorts. Among them, 84 of 416 patients in 3 independent cohorts received fluorouracil-based adjuvant chemotherapy. Identification of CSS sets to predict relapse-free survival and identify a subset of patients with stage II CRC who could gain substantial survival benefits from fluorouracil-based adjuvant chemotherapy. Eight cancer hallmark-based gene signatures (30 genes each) were identified and used to construct CSS sets for determining prognosis. The CSS sets were validated in 11 independent cohorts of 767 patients with stage II CRC who did not receive adjuvant chemotherapy. The CSS sets accurately stratified patients into low-, intermediate-, and high-risk groups. Five-year relapse-free survival rates were 94%, 78%, and 45%, respectively, representing 60%, 28%, and 12% of patients with stage II disease. The 416 patients with CSS set-defined high-risk stage II CRC who received fluorouracil-based adjuvant chemotherapy showed a substantial gain in survival benefits from the treatment (ie, recurrence reduced by 30%-40% in 5 years). The CSS sets substantially outperformed other prognostic predictors of stage 2 CRC. They are more accurate and robust for prognostic predictions and facilitate the identification of patients with stage

  17. ins-7 Gene expression is partially regulated by the DAF-16/IIS signaling pathway in Caenorhabditis elegans under celecoxib intervention.

    Directory of Open Access Journals (Sweden)

    Shanqing Zheng

    Full Text Available DAF-16 target genes are employed as reporters of the insulin/IGF-1 like signal pathway (IIS, and this is notably true when Caenorhabditis elegans (C. elegans is used to study the action of anti-aging compounds on IIS activity. However, some of these genes may not be specific to DAF-16, even if their expression levels are altered when DAF-16 is activated. Celecoxib was reported to extend the lifespan of C. elegans through activation of DAF-16. Our results confirmed the function of celecoxib on aging; however, we found that the expression of ins-7, a DAF-16 target gene, was abnormally regulated by celecoxib. ins-7 plays an important role in regulating aging, and its expression is suppressed in C. elegans when DAF-16 is activated. However, we found that celecoxib upregulated the expression of ins-7 in contrast to its role in DAF-16 activation. Our subsequent analysis indicated that the expression level of ins-7 in C. elegans was negatively regulated by DAF-16 activity. Additionally, its expression was also positively regulated by DAF-16-independent mechanisms, at least following external pharmacological intervention. Our study suggests that ins-7 is not a specific target gene of DAF-16, and should not be chosen as a reporter for IIS activity. This conclusion is important in the study of INSs on aging in C. elegans, especially under the circumstance of drug intervention.

  18. ins-7 Gene expression is partially regulated by the DAF-16/IIS signaling pathway in Caenorhabditis elegans under celecoxib intervention.

    Science.gov (United States)

    Zheng, Shanqing; Liao, Sentai; Zou, Yuxiao; Qu, Zhi; Liu, Fan

    2014-01-01

    DAF-16 target genes are employed as reporters of the insulin/IGF-1 like signal pathway (IIS), and this is notably true when Caenorhabditis elegans (C. elegans) is used to study the action of anti-aging compounds on IIS activity. However, some of these genes may not be specific to DAF-16, even if their expression levels are altered when DAF-16 is activated. Celecoxib was reported to extend the lifespan of C. elegans through activation of DAF-16. Our results confirmed the function of celecoxib on aging; however, we found that the expression of ins-7, a DAF-16 target gene, was abnormally regulated by celecoxib. ins-7 plays an important role in regulating aging, and its expression is suppressed in C. elegans when DAF-16 is activated. However, we found that celecoxib upregulated the expression of ins-7 in contrast to its role in DAF-16 activation. Our subsequent analysis indicated that the expression level of ins-7 in C. elegans was negatively regulated by DAF-16 activity. Additionally, its expression was also positively regulated by DAF-16-independent mechanisms, at least following external pharmacological intervention. Our study suggests that ins-7 is not a specific target gene of DAF-16, and should not be chosen as a reporter for IIS activity. This conclusion is important in the study of INSs on aging in C. elegans, especially under the circumstance of drug intervention.

  19. Two novel mutations of CLCN7 gene in Chinese families with autosomal dominant osteopetrosis (type II).

    Science.gov (United States)

    Zheng, Hui; Shao, Chong; Zheng, Yan; He, Jin-Wei; Fu, Wen-Zhen; Wang, Chun; Zhang, Zhen-Lin

    2016-07-01

    Autosomal dominant osteopetrosis type II (ADO-II) is a heritable bone disorder characterized by osteosclerosis, predominantly involving the spine (vertebral end-plate thickening, or rugger-jersey spine), the pelvis ("bone-within-bone" structures) and the skull base. Chloride channel 7 (CLCN7) has been reported to be the causative gene. In this study, we aimed to identify the pathogenic mutation in four Chinese families with ADO-II. All 25 exons of the CLCN7 gene, including the exon-intron boundaries, were amplified and sequenced directly in four probands from the Chinese families with ADO-II. The mutation site was then identified in other family members and 250 healthy controls. In family 1, a known missense mutation c.296A>G in exon 4 of CLCN7 was identified in the proband, resulting in a tyrosine (UAU) to cysteine (UGU) substitution at p.99 (Y99C); the mutation was also identified in his affected father. In family 2, a novel missense mutation c.865G>C in exon 10 was identified in the proband, resulting in a valine (GUC) to leucine (CUC) substitution at p.289 (V289L); the mutation was also identified in her healthy mother and sister. In family 3, a novel missense mutation c.1625C>T in exon 17 of CLCN7 was identified in the proband, resulting in an alanine (GCG) to valine (GUG) substitution at p.542 (A542V); the mutation was also identified in her father. In family 4, a hot spot, R767W (c.2299C>T, CGG>TGG), in exon 24 was found in the proband which once again proved the susceptibility of the site or the similar genetic background in different races. Moreover, two novel mutations, V289L and A542V, occurred at a highly conserved position, found by a comparison of the protein sequences from eight vertebrates, and were predicted to have a pathogenic effect by PolyPhen-2 software, which showed "probably damaging" with a score of approximately 1. These mutation sites were not identified in 250 healthy controls. Our present findings suggest that the novel missense

  20. Cloning and molecular analysis of L-asparaginase II gene (ansB

    Directory of Open Access Journals (Sweden)

    ZEINAT K. MOHAMED

    2015-12-01

    Full Text Available The deamination of L-asparagine to L-aspartic acid and ammonia is catalyzed by L-asparaginases (L-asparagine amino hydrolase. The enzyme L-asparaginase is widely distributed in nature from different living organisms, starting from bacteria till mammals and plants. It has been recently thought to be a therapeutic agent in treatment of various lymphoblastic leukemia diseases. There have been many attempts to isolate microorganisms that produce L-asparaginase. L-ASNase producing bacteria, Escherichia coli MG27, was previously isolated from the River Nile and identified. In this study, ansB gene, encoding L-ASNase II from E. coli MG27, was amplified by PCR, cloned and characterized by DNA sequencing. The DNA sequence was then analyzed using bioinformatics analysis and translated into amino acid sequence. Identification of highly conserved amino acid sequence motifs was conducted by comparison against the InterPro database. Analysis revealed that the protein sequence had a catalytic domain of L-asparaginase type II (IPR004550 that belong to asparaginase/glutaminase family (IPR006034 and has asparaginase/glutaminase conserved site (IPR020827. According to results predicted using PSIpred tool, ansB consists of eight α-helices and 13 β-strands.

  1. Identification and Characterization of Novel Immunomodulatory Bursal-derived Pentapeptide-II (BPP-II)*

    Science.gov (United States)

    Feng, Xiu-Li; Liu, Qing-Tao; Cao, Rui-Bing; Zhou, Bin; Ma, Zhi-Yong; Deng, Wen-Lei; Wei, Jian-Chao; Qiu, Ya-Feng; Wang, Fang-Quan; Gu, Jin-Yan; Wang, Feng-Juan; Zheng, Qi-Sheng; Ishag, Hassan; Chen, Pu-Yan

    2012-01-01

    The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement. PMID:22184121

  2. Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

    Science.gov (United States)

    Feng, Xiu-Li; Liu, Qing-Tao; Cao, Rui-Bing; Zhou, Bin; Ma, Zhi-Yong; Deng, Wen-Lei; Wei, Jian-Chao; Qiu, Ya-Feng; Wang, Fang-Quan; Gu, Jin-Yan; Wang, Feng-Juan; Zheng, Qi-Sheng; Ishag, Hassan; Chen, Pu-Yan

    2012-02-03

    The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

  3. Agrobacterium tumefaciens-MEDIATED IN-PLANTA TRANSFORMATION OF INDONESIAN MAIZE USING pIG121Hm-Cs PLASMID CONTAINING nptII AND hpt GENES

    Directory of Open Access Journals (Sweden)

    Edy Listanto

    2017-05-01

    Full Text Available Maize (Zea mays L. productivity in Indonesia is challenged to be increased using genetic engineering. Recent advances in Agrobacterium tumefaciens-mediated in-planta transforma-tion makes it possible to transform maize with low cost, and simple method. This study aimed to confirm pIG121Hm-Cs plasmid in A. tumefaciens, and to estimate the efficiency level of  A. tumefaciens-mediated in-planta transformation of Indonesian maize by using pIG121Hm-Cs plasmid containing nptII and hpt genes. A series of studies were conducted including confirmation of gene construct of pIG121Hm-Cs plasmid in A. tumefaciens, transformation of four maize lines through A. tumefaciens-mediated in-planta technique, acclimatization of transformant plants and molecular analysis of selected plants using polymerase chain reaction (PCR. The pIG121Hm-Cs plasmid was confirmed via PCR analysis using specific primers of nptII and hpt genes and resulted 700 bp and 500 bp for fragments of nptII and hpt, respectively. After selection, acclimatization and molecular analysis steps, the efficiency levels of transformation of four maize lines were low, ranging from 3.8% to 12.8%. The level of transformation efficiency of ST-27 line was the highest accounting for 12.8% of 45 planted embryos on selection medium based on PCR analysis using specific primer for nptII gene. Overall, A. tumefaciens-mediated in planta transformation on maize floral pistil in this study proved to be successful and rapid. Therefore, this enhanced transformation method will be beneficial for Indonesian maize genetic engineering.

  4. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Directory of Open Access Journals (Sweden)

    Anil Raj

    Full Text Available Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  5. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Science.gov (United States)

    Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

    2015-01-01

    Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  6. Construction of expression vector containing glnA gene and detection of NPT II activity in the transgenic rice calli using 32P-labelled compound

    International Nuclear Information System (INIS)

    Su Jin; Zhang Xueqin; Yan Qiusheng; Chen Zhangliang; You Chongbiao

    1993-08-01

    The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilense Sp7 by PCR technique. the amplified 1.4 kb DNA fragment was cloned at the EcoRV site of Bluescript-SK. Both sequencing and restriction digestion data showed that the 1.4 kb DNA fragment flanked with BamHI site at each end was really the glnA gene of A. brasilense Sp7. The glnA gene was ligated with Bg1 II site of pCo24. As a result, an expression vector pGSC35 with CaMV35S promoter was obtained. Using colony in situ hybridization with α- 32 P-dATP labelled probes to screen the positive clones, another glnA gene expression vector pAGNB92 with rice actin 1 promoter was constructed after three rounds of ligation and transformation. Protoplasts isolated from rice cell suspension line cv. T986 were transformed with glnA expression vectors pGSC35 and pAGNB92 containing neomycin phosphotransferase II (NPTII) gene by using PEG fusion and electroporation. Transformed microcalli were selected on media containing G418 disulfate salt. NPT II activity was detected in 37% of G418 resistant calli by using dot blot hybridization with γ- 32 P-ATP and kanamycin as substrate

  7. HLA non-class II genes may confer type I diabetes susceptibility in a Mapuche (Amerindian) affected family.

    Science.gov (United States)

    Pérez-Bravo, Francisco; Martinez-Laso, Jorge; Martin-Villa, Jose M; Moscoso, Juan; Moreno, Almudena; Serrano-Vela, Juan I; Zamora, Jorge; Asenjo, Silvia; Gleisner, Andrea; Arnaiz-Villena, Antonio

    2006-01-01

    A rare case of type I diabetes is studied in an Amerindian (Mapuche) family from Chile, analyzing glutamic acid decarboxylase, islet-cell autoantibodies and human leukocyte antigen (HLA) genes. The affected sib is the only one that has one specific HLA haplotype combination that differs from the other sibs only in the HLA class I genes. It is concluded that HLA diabetes susceptibility factors may be placed outside the class II region or even that susceptibility factors do not exist in the HLA region in this Amerindian family.

  8. The properties of the single chicken MHC classical class II alpha chain (B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules

    DEFF Research Database (Denmark)

    Salomonsen, Jan; Marston, Denise; Avila, David

    2003-01-01

    for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most...... the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both......In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis...

  9. Identification of two novel critical mutations in PCNT gene resulting in microcephalic osteodysplastic primordial dwarfism type II associated with multiple intracranial aneurysms.

    Science.gov (United States)

    Li, Fei-Feng; Wang, Xu-Dong; Zhu, Min-Wei; Lou, Zhi-Hong; Zhang, Qiong; Zhu, Chun-Yu; Feng, Hong-Lin; Lin, Zhi-Guo; Liu, Shu-Lin

    2015-12-01

    Microcephalic osteodysplastic primordial dwarfism type II (MOPD II) is a highly detrimental human autosomal inherited recessive disorder. The hallmark characteristics of this disease are intrauterine and postnatal growth restrictions, with some patients also having cerebrovascular problems such as cerebral aneurysms. The genomic basis behind most clinical features of MOPD II remains largely unclear. The aim of this work was to identify the genetic defects in a Chinese family with MOPD II associated with multiple intracranial aneurysms. The patient had typical MOPD II syndrome, with subarachnoid hemorrhage and multiple intracranial aneurysms. We identified three novel mutations in the PCNT gene, including one single base alteration (9842A>C in exon 45) and two deletions (Del-C in exon 30 and Del-16 in exon 41). The deletions were co-segregated with the affected individual in the family and were not present in the control population. Computer modeling demonstrated that the deletions may cause drastic changes on the secondary and tertiary structures, affecting the hydrophilicity and hydrophobicity of the mutant proteins. In conclusion, we identified two novel mutations in the PCNT gene associated with MOPD II and intracranial aneurysms, and the mutations were expected to alter the stability and functioning of the protein by computer modeling.

  10. Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

    Science.gov (United States)

    Dergai, Oleksandr; Cousin, Pascal; Gouge, Jerome; Satia, Karishma; Praz, Viviane; Kuhlman, Tracy; Lhôte, Philippe; Vannini, Alessandro; Hernandez, Nouria

    2018-05-01

    RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters. © 2018 Dergai et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Examination of chromosome 7p22 candidate genes RBaK, PMS2 and GNA12 in familial hyperaldosteronism type II.

    Science.gov (United States)

    Jeske, Y W A; So, A; Kelemen, L; Sukor, N; Willys, C; Bulmer, B; Gordon, R D; Duffy, D; Stowasser, M

    2008-04-01

    1. There are two types of familial hyperaldosteronism (FH): FH-I and FH-II. FH-I is caused by a hybrid CYP11B1/CYP11B2 gene mutation. The genetic cause of FH-II, which is more common, is unknown. Adrenal hyperplasia and adenomas are features. We previously reported linkage of FH-II to a approximately 5 Mb region on chromosome 7p22. We subsequently reported finding no causative mutations in the retinoblastoma-associated Kruppel-associated box gene (RBaK), a candidate at 7p22 involved in tumorigenesis and cell cycle control. 2. In the current study we investigated RBaK regulatory regions and two other candidate genes: postmeiotic segregation increased 2 (PMS2, involved in DNA mismatch repair and tumour predisposition) and guanine nucleotide-binding protein alpha-12 (GNA12, a transforming oncogene). 3. The GNA12 and PMS2 genes were examined in two affected (A1, A2) and two unaffected (U1, U2) subjects from a large 7p22-linked FH-II family (family 1). No mutations were found. 4. The RBaK and PMS2 distal promoters were sequenced to -2150 bp from the transcription start site for RBaK and-2800 bp for PMS2. Five unreported single nucleotide polymorphisms (SNPs) were found in subjects A1, A2 but not in U1 or U2; A(-2031 bp)T, T(-2030 bp)G, G(-834 bp)C, C(-821 bp)G in RBaK and A(-876 bp)G in PMS2. Additional affected and unaffected subjects from family 1 and from two other 7p22-linked FH-II families and 58 unrelated normotensive control subjects were genotyped for these SNPs. 5. The five novel SNPs were found to be present in a significant proportion of normotensive controls. The four RBaK promoter SNPs were found to be in linkage disequilibrium in the normal population. The RBaK promoter (-)2031T/2030G/834C/821T allele was found to be in linkage disequilibrium with the causative mutation in FH-II family 1, but not in families 2 and 3. The PMS2 promoter (-)876G allele was also found to be linked to affected phenotypes in family 1. 6. The RBaK and PMS2 promoter SNPs alter the

  12. Phylogenetic analysis of the core histone doublet and DNA topo II genes of Marseilleviridae: evidence of proto-eukaryotic provenance.

    Science.gov (United States)

    Erives, Albert J

    2017-11-28

    While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like "doublets" in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of

  13. Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

    Science.gov (United States)

    Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

    2012-03-01

    The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices

    Directory of Open Access Journals (Sweden)

    Sanders Ian R

    2006-03-01

    Full Text Available Abstract Background The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota, which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. Results In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. Conclusion On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence

  15. Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

    Directory of Open Access Journals (Sweden)

    Jungtae Na

    Full Text Available Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5 as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

  16. Construction and characterization of Gal-chitosan graft methoxy poly (ethylene glycol) (Gal-CS-mPEG) nanoparticles as efficient gene carrier

    Science.gov (United States)

    Jin, Jiting; Fu, Wandong; Liao, Miaofei; Han, Baoqin; Chang, Jing; Yang, Yan

    2017-10-01

    In the present study, galactosylated chitosan (Gal-CS) was conjugated with methoxy poly(ethylene glycol) (mPEG) as a hydrophilic group. The structure of Gal-CS-mPEG polymer was characterized and the nanoparticles (NPs) were prepared using ironic gelation method. The study was designed to investigate the characteristics and functions of Gal-CS-mPEG NPs. The morphology of Gal-CS-mPEG NPs was observed by SEM and it was a compact and spherical shape. The size of the NPs was approximately 200 nm in diameter under the ideal process parameters. The interaction between Gal-CS-mPEG NPs and pDNA, and the protection of pDNA against DNase I and serum degradation by Gal-CS-mPEG NPs were evaluated. Agarose gel electrophoresis results showed that Gal-CS-mPEG NPs had strong interaction with pDNA at the weight ratio of 12:1, 4:1 and 2:1 and could protect pDNA from DNase I and serum degradation. Gal-CS-mPEG NPs exhibited high loading efficiency and sustainable in vitro release. The blood compatibility studies demonstrated that Gal-CS-mPEG NPs had superior compatibility with erythrocytes in terms of aggregation degree and hemolysis level. Gal-CS-mPEG NPs showed no cytotoxicity on L929 cells, which is a normal mouse connective tissue fibroblast, but showed inhibitory effects on the proliferation of Bel-7402 cells, which is a liver cancer cell line. In conclusion, Gal-CS-mPEG NP is a bio-safe and efficient gene carrier with potential application in gene delivery.

  17. HETEROGENEITY OF POLYCLONAL IMMUNOGLOBULINS NUCLEASE ACTIVITY IN RHEUMATOID AND REACTIVE ARTHRITIS

    Directory of Open Access Journals (Sweden)

    M. V. Volkova

    2017-01-01

    Full Text Available Catalytic properties of immunoglobulins are widely studied within recent years. It was found that nuclease activity of immunoglobulins is increased in systemic autoimmune diseases. Given some pathogenetic features of rheumatoid arthritis and reactive arthritis, it is appropriate to clarify the nature of nuclease activity in these diseases. Determination of DNAse activity of immunoglobulins with different DNA substrates, and search for specific substrates for distinct clinical entities could serve these purposes. The aim of present work is to determine DNase activity of the polyclonal class G immunoglobulins in rheumatoid and reactive arthritis using various methods.Different methods are used to evaluate nuclease activity. In this paper we present newly developed and modified techniques for determination of DNAse activity of polyclonal IgGs. Particular attention was paid to the electrophoretic method of DNase activity assessment. Polyclonal IgG isolated from blood serum of patients with rheumatoid arthritis and reactive arthritis were used for assays. In this study, we demonstrated the presence of an inhomogeneous DNase activity of immunoglobulins in relation to different substrates.Along with calf thymus DNA, we used bacterial plasmid DNA and PCR products based on bacterial gene sequences. Levels of DNase activity by rivanol clot method with calf thymus DNA as substrate proved to be higher in patients with rheumatoid arthritis than the control values (p < 0.01. DNase abzyme activity in patients with rheumatoid arthritis was elevated, as compared to the patients with reactive arthritis (p < 0.01.When examining ability of the IgG to hydrolyze procaryotic DNA (bacterial plasmid DNA and PCR products, based on bacterial genes, we obtained heterogeneous results. Different Ig samples showed varying degrees of DNA hydrolysis. Abzyme hydrolysis of DNA substrates longer than 700 bp was more pronounced, as compared to short DNA substrates (100 base pairs

  18. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    Science.gov (United States)

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

  19. Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

    Science.gov (United States)

    Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

    1988-01-01

    Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region. Images PMID:2847031

  20. Sequence Analysis of the Capsid Gene during a Genotype II.4 Dominated Norovirus Season in One University Hospital

    DEFF Research Database (Denmark)

    Holzknecht, Barbara Juliane; Franck, Kristina Træholt; Nielsen, Rikke Thoft

    2015-01-01

    Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible...

  1. Effects of aerobic exercise training on ACE and ADRB2 gene expression, plasma angiotensin II level, and flow-mediated dilation: a study on obese postmenopausal women with prehypertension.

    Science.gov (United States)

    Azadpour, Noushin; Tartibian, Bakhtyar; Koşar, Şükran Nazan

    2017-03-01

    The purpose of this study is to determine the effect of 10 weeks of moderate-intensity aerobic exercise training (MIET) on blood pressure (BP), angiotensin-converting enzyme (ACE) and β2-adrenergic receptor (ADRB2) gene expression in leukocytes, plasma angiotensin II (Ang II), and flow-mediated dilation (FMD) in obese postmenopausal women (PMW) with prehypertension. Twenty-four obese prehypertensive PMW (aged 50-70 y; body mass index ≥30 kg/m) randomly assigned to control (n = 12) and exercise (n = 12) groups. Exercise group performed MIET (25-40 min/d, 3 d/wk at 50%-70% of heart rate reserve) for 10 weeks. Control group maintained their normal daily physical activity level. Body composition, VO2max, BP, ACE and ADRB2 gene expression, plasma Ang II, and FMD were measured before and after the training program. After MIET, systolic and diastolic BPs decreased by 4.6% and 2.4%, respectively (P ACE gene expressions (P ACE (r values range 0.68-0.86) (P ACE and ADRB2 gene expression, decreases Ang II plasma levels, and improves endothelial function in obese PMW, and these alterations are associated with reduction in BP.

  2. Imaging gene expression in gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

    1997-12-31

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

  3. Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus).

    Science.gov (United States)

    Lau, Quintin; Jobbins, Sarah E; Belov, Katherine; Higgins, Damien P

    2013-01-01

    Major histocompatibility complex (MHC) class II molecules have an integral role in the adaptive immune response, as they bind and present antigenic peptides to T helper lymphocytes. In this study of koalas, species-specific primers were designed to amplify exon 2 of the MHC class II DA and DB genes, which contain much of the peptide-binding regions of the α and β chains. A total of two DA α1 domain variants and eight DA β1 (DAB), three DB α1 and five DB β1 variants were amplified from 20 koalas from two free-living populations from South East Queensland and the Port Macquarie region in northern New South Wales. We detected greater variation in the β1 than in the α1 domains as well as evidence of positive selection in DAB. The present study provides a springboard to future investigation of the role of MHC in disease susceptibility in koalas.

  4. Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene.

    Science.gov (United States)

    Le Dréan, Y; Lazennec, G; Kern, L; Saligaut, D; Pakdel, F; Valotaire, Y

    1995-08-01

    We previously reported that the expression of the rainbow trout estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5' flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized an estrogen-responsive element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This element confers estrogen responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0.2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting element involved in the regulation of the gene by estrogen.

  5. Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation.

    Science.gov (United States)

    Zhang, Z X; Kumar, V; Rivera, R T; Pasion, S G; Chisholm, J; Biswas, D K

    1989-10-01

    Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH(1)2C1). We examined six methylatable cytosine residues (5, -CCGG- and 1, -GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that -CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH(1)2C1 cells. Furthermore, the inhibition of PRL gene expression in GH(1)2C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

    Science.gov (United States)

    Sasikumar, Ponnusamy; Paul, Eldho; Gomathi, Sivasamy; Abhishek, Albert; Sasikumar, Sundaresan; Selvam, Govindan Sadasivam

    2016-10-01

    The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real-time PCR experiment shows 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E-0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA - mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA - mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Angiotensin II type 1 receptor (A1166C gene polymorphism and essential hypertension in Egyptian population

    Directory of Open Access Journals (Sweden)

    Marium M. Shamaa

    2016-09-01

    Full Text Available The pathogenesis of essential hypertension (EH is affected by genetic and environmental factors. Mutations in hypertension-related genes can affect blood pressure (BP via alteration of salt and water reabsorption by the nephron. The genes of the renin-angiotensin system (RAS have been extensively studied because of the well documented role of this system in the control of BP. It has been previously shown that Angiotensin II type 1 receptor (ATR1 gene polymorphism could be associated with increased risk of EH. So, in the current study, we evaluated the frequency of ATR1 (A1166C polymorphism in relation to EH in a group of Egyptian population. The study population included 83 hypertensive patients and 60 age and sex matched healthy control subjects. Restriction fragment length polymorphism – Polymerase chain reaction (RFLP – PCR was used for the analysis of A1166C polymorphism of ATR1 genes in peripheral blood samples of all patients and controls. The results revealed that there was a positive risk of developing EH when having the T allele whether in homozygous or heterozygous state. From this work, it was concluded that there was an association between ATR1 (A1166C gene polymorphism and the risk of developing EH.

  8. Evolution of Cis-Regulatory Elements and Regulatory Networks in Duplicated Genes of Arabidopsis.

    Science.gov (United States)

    Arsovski, Andrej A; Pradinuk, Julian; Guo, Xu Qiu; Wang, Sishuo; Adams, Keith L

    2015-12-01

    Plant genomes contain large numbers of duplicated genes that contribute to the evolution of new functions. Following duplication, genes can exhibit divergence in their coding sequence and their expression patterns. Changes in the cis-regulatory element landscape can result in changes in gene expression patterns. High-throughput methods developed recently can identify potential cis-regulatory elements on a genome-wide scale. Here, we use a recent comprehensive data set of DNase I sequencing-identified cis-regulatory binding sites (footprints) at single-base-pair resolution to compare binding sites and network connectivity in duplicated gene pairs in Arabidopsis (Arabidopsis thaliana). We found that duplicated gene pairs vary greatly in their cis-regulatory element architecture, resulting in changes in regulatory network connectivity. Whole-genome duplicates (WGDs) have approximately twice as many footprints in their promoters left by potential regulatory proteins than do tandem duplicates (TDs). The WGDs have a greater average number of footprint differences between paralogs than TDs. The footprints, in turn, result in more regulatory network connections between WGDs and other genes, forming denser, more complex regulatory networks than shown by TDs. When comparing regulatory connections between duplicates, WGDs had more pairs in which the two genes are either partially or fully diverged in their network connections, but fewer genes with no network connections than the TDs. There is evidence of younger TDs and WGDs having fewer unique connections compared with older duplicates. This study provides insights into cis-regulatory element evolution and network divergence in duplicated genes. © 2015 American Society of Plant Biologists. All Rights Reserved.

  9. Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2008-10-01

    Full Text Available Abstract Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS, which spans the promoter and untranslated regions of the ghrelin gene (GHRL. Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2. Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis, as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA genes, including 5' capping, polyadenylation, extensive splicing and short open reading

  10. Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription1

    Science.gov (United States)

    Ido, Ayaka; Iwata, Shinya; Iwata, Yuka; Igarashi, Hisako; Hamada, Takahiro; Sonobe, Seiji; Sugiura, Masahiro; Yukawa, Yasushi

    2016-01-01

    In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin). PMID:26662274

  11. DNA methylation induced changes in chromatin conformation of the promoter of the vitellogenin II gene of Japanese quail during aging.

    Science.gov (United States)

    Gupta, Sanjay; Pathak, Rashmi U; Kanungo, Madhu S

    2006-08-01

    One approach to the understanding of the molecular basis of aging in higher organisms may be to use genes whose timing and rate of expression during the life span run parallel with specific functions that can be monitored. The genes for egg proteins, such as vitellogenin (VTG), which is expressed in the liver, and ovalbumin, lysozyme etc. that are expressed in the oviduct of birds, meet these requirements. Egg laying function is dependent on the production of these proteins, which, in turn, depends on the expression of their genes. In this communication we present the age-related studies on the VTG II gene of the bird, Japanese quail. The gene is expressed only in the liver and its expression is considerably lower in old birds that do not lay eggs. Comparison of the promoter region of the gene carrying the two important cis-acting elements, estrogen responsive element (ERE) and progesterone responsive element (PRE), shows it to be 100% homologous to the corresponding region of the chicken VTG II gene. Methylation of DNA and conformation of chromatin of this region were studied, as they are known to be important for regulation of expression of genes. Our studies show that in the liver of adult female quails which lay eggs, a -CCGG- sequence located in this region is hypomethylated, and the chromatin encompassing this region of the gene is relaxed. In the old, the -CCGG- sequence is hypermethylated and the chromatin is compact. This is correlated with a decrease in the expression of the gene and decrease in egg production. Further, electrophoretic mobility shift assay (EMSA) shows that the levels/affinity of specific trans-acting factors that bind to ERE and PRE present in the region, are not different in adult and old birds. Hence the methylation status of the -CCGG- sequence that is located in-between the ERE and the PRE may be crucial for the conformation of chromatin and availability of these two important cis-acting elements for the binding of the trans

  12. Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of Usher syndrome type II.

    NARCIS (Netherlands)

    Weston, M.D.; Luijendijk, M.W.J.; Humphrey, K.D.; Moller, C.G.; Kimberling, W.J.

    2004-01-01

    Usher syndrome type II (USH2) is a genetically heterogeneous autosomal recessive disorder with at least three genetic subtypes (USH2A, USH2B, and USH2C) and is classified phenotypically as congenital hearing loss and progressive retinitis pigmentosa. The VLGR1 (MASS1) gene in the 5q14.3-q21.1 USH2C

  13. Analysis of the cytochrome c oxidase subunit II (COX2) gene in giant panda, Ailuropoda melanoleuca.

    Science.gov (United States)

    Ling, S S; Zhu, Y; Lan, D; Li, D S; Pang, H Z; Wang, Y; Li, D Y; Wei, R P; Zhang, H M; Wang, C D; Hu, Y D

    2017-01-23

    The giant panda, Ailuropoda melanoleuca (Ursidae), has a unique bamboo-based diet; however, this low-energy intake has been sufficient to maintain the metabolic processes of this species since the fourth ice age. As mitochondria are the main sites for energy metabolism in animals, the protein-coding genes involved in mitochondrial respiratory chains, particularly cytochrome c oxidase subunit II (COX2), which is the rate-limiting enzyme in electron transfer, could play an important role in giant panda metabolism. Therefore, the present study aimed to isolate, sequence, and analyze the COX2 DNA from individuals kept at the Giant Panda Protection and Research Center, China, and compare these sequences with those of the other Ursidae family members. Multiple sequence alignment showed that the COX2 gene had three point mutations that defined three haplotypes, with 60% of the sequences corresponding to haplotype I. The neutrality tests revealed that the COX2 gene was conserved throughout evolution, and the maximum likelihood phylogenetic analysis, using homologous sequences from other Ursidae species, showed clustering of the COX2 sequences of giant pandas, suggesting that this gene evolved differently in them.

  14. A transcription factor active on the epidermal growth factor receptor gene

    International Nuclear Information System (INIS)

    Kageyama, R.; Merlino, G.T.; Pastan, I.

    1988-01-01

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO 4 /polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  15. The Effect of Soy Isoflavone on the Proliferation and Differentiation of Adipose-Derived Mesenchymal Stem Cells into Chondrocytes and Expression of Collagen II and Aggrecan Genes

    Directory of Open Access Journals (Sweden)

    Fatemeh Bamdadpasand Shekarsarayi

    2017-03-01

    Full Text Available Background and Objectives: Due to the lack of blood vessels in cartilage tissue, its damage is not repairable. This study was conducted to investigate the effect of soy isoflavone on proliferation and differentiation of adipose-derived mesenchymal stem cells into chondrocytes and expression of collagen II and aggrecan genes. Methods: In this experimental study, human subcutaneous fat was obtained during liposuction and incubated with collagenase enzyme (type 1 for the breakdown of collagen, and collagenase was deactivated by DMEM medium, and was cultured in the cell sediment after centrifugation, the cells were isolated after the third passage, were placed in chondrogenic medium for differentiate into the cartilage, and were divided into three groups, including control, treatment with TGF-β1, and treatment with soy isoflavones tablets. The tablets were dissolved in distilled water, sterilized by passing through a 0.2 um filter and were added to the culture medium. After 48 hours, cell viability was determined by MTT assay, and after 14 days, collagen II and aggrecan gene expressions were assessed by real-time PCR technique. Data were statistically analyzed by one-way ANOVA and Tukey's post-hoc test using SPSS 20 and p<0.05. Results: The results of MTT assay showed a significant increase in viability in the TGF-β1 group compared to the control and soy isoflavone groups (p<0.05. The RT-PCR indicated a significant increase in the expression of collagen II and aggrecan genes in isoflavones and TGF-β1 groups compared to the control group, and also, the mean CT associated with collagen II gene had a significant increase in isoflavone and TGF-β1groups compared to the control group (p<0.05. Conclusion: Soy in culture medium increases the expression of collagen II and aggrecan genes and cell proliferation, but this increase is not high compared to the TGF-β1 group.

  16. Validation of the 12-gene colon cancer recurrence score as a predictor of recurrence risk in stage II and III rectal cancer patients.

    Science.gov (United States)

    Reimers, Marlies S; Kuppen, Peter J K; Lee, Mark; Lopatin, Margarita; Tezcan, Haluk; Putter, Hein; Clark-Langone, Kim; Liefers, Gerrit Jan; Shak, Steve; van de Velde, Cornelis J H

    2014-11-01

    The 12-gene Recurrence Score assay is a validated predictor of recurrence risk in stage II and III colon cancer patients. We conducted a prospectively designed study to validate this assay for prediction of recurrence risk in stage II and III rectal cancer patients from the Dutch Total Mesorectal Excision (TME) trial. RNA was extracted from fixed paraffin-embedded primary rectal tumor tissue from stage II and III patients randomized to TME surgery alone, without (neo)adjuvant treatment. Recurrence Score was assessed by quantitative real time-polymerase chain reaction using previously validated colon cancer genes and algorithm. Data were analysed by Cox proportional hazards regression, adjusting for stage and resection margin status. All statistical tests were two-sided. Recurrence Score predicted risk of recurrence (hazard ratio [HR] = 1.57, 95% confidence interval [CI] = 1.11 to 2.21, P = .01), risk of distant recurrence (HR = 1.50, 95% CI = 1.04 to 2.17, P = .03), and rectal cancer-specific survival (HR = 1.64, 95% CI = 1.15 to 2.34, P = .007). The effect of Recurrence Score was most prominent in stage II patients and attenuated with more advanced stage (P(interaction) ≤ .007 for each endpoint). In stage II, five-year cumulative incidence of recurrence ranged from 11.1% in the predefined low Recurrence Score group (48.5% of patients) to 43.3% in the high Recurrence Score group (23.1% of patients). The 12-gene Recurrence Score is a predictor of recurrence risk and cancer-specific survival in rectal cancer patients treated with surgery alone, suggesting a similar underlying biology in colon and rectal cancers. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Ups and Downs of Poised RNA Polymerase II in B-Cells.

    Directory of Open Access Journals (Sweden)

    Phuong Dao

    2016-04-01

    Full Text Available Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG, which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression

  18. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations.

    Directory of Open Access Journals (Sweden)

    Lee A Hadwiger

    2015-06-01

    Full Text Available Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid nonhost resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph and the susceptibility of pea to F. solani f sp. pisi (Fspi has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to the chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction.

  19. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations

    Science.gov (United States)

    Hadwiger, Lee A.

    2015-01-01

    Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid “nonhost” resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph) and the susceptibility of pea to F. solani f sp. pisi (Fspi) has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR) genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction. PMID:26124762

  20. Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations.

    Science.gov (United States)

    Hadwiger, Lee A

    2015-01-01

    Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid "nonhost" resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph) and the susceptibility of pea to F. solani f sp. pisi (Fspi) has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR) genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction.

  1. A young root-specific gene (ArMY2) from horseradish encoding a MYR II myrosinase with kinetic preference for the root-specific glucosinolate gluconasturtiin.

    Science.gov (United States)

    Loebers, Andreas; Müller-Uri, Frieder; Kreis, Wolfgang

    2014-03-01

    The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Progranulin gene variability influences the risk for bipolar I disorder, but not bipolar II disorder.

    Science.gov (United States)

    Galimberti, Daniela; Prunas, Cecilia; Paoli, Riccardo A; Dell'Osso, Bernardo; Fenoglio, Chiara; Villa, Chiara; Palazzo, Carlotta; Cigliobianco, Michela; Camuri, Giulia; Serpente, Maria; Scarpini, Elio; Altamura, A Carlo

    2014-11-01

    Recent data have shown that genetic variability in the progranulin (GRN) gene may contribute to the susceptibility to developing bipolar disorder (BD). However, in regard to patients with BD, no information is available on the role of genetic variability and plasma progranulin levels in different types of this disorder. In this study, we performed an association analysis of GRN in an Italian population consisting of 134 patients with BD and 232 controls to evaluate progranulin plasma levels. The presence of the polymorphic variant of the rs5848 single nucleotide polymorphism is protective for the development of bipolar I disorder (BD-I) (odds ratio = 0.55, 95% confidence interval: 0.33-0.93; p = 0.024) but not bipolar II disorder (BD-II) (p > 0.05). In addition, plasma progranulin levels are significantly decreased in BD [mean ± standard deviation (SD) 112 ± 35 versus 183 ± 93 ng/mL in controls; p < 0.001]. Regarding the influence of GRN variability on BD susceptibility, the predisposing genetic background differs between BD-I and BD-II, possibly implying that pathogenic mechanisms differ between the two subtypes of BD. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Developmental status of bioassays in genetic toxicology: a report of Phase II of the US Environmental Protection Agency Gene-Tox program

    Energy Technology Data Exchange (ETDEWEB)

    Brusick, D; Auletta, A

    1985-01-01

    The Gene-Tox Program was structured around two phases of genetic test data evaluation. The first phase consisted of 36 Work Group reports, each evaluating the results and performance of a specific bioassay. The second phase consisted of a plan to summarize the information provided by the Work Groups. The Gene-Tox Coordinating Committee was to be responsible for Phase II, and several subgroups were assigned specific goals in implementing this analysis. This report deals with Goal I which is to identify the developmental status of the individual bioassays reviewed by the Gene-Tox Work Groups in the first phase of the Program. 5 references, 6 tables.

  4. Identifying gene coexpression networks underlying the dynamic regulation of wood-forming tissues in Populus under diverse environmental conditions.

    Science.gov (United States)

    Zinkgraf, Matthew; Liu, Lijun; Groover, Andrew; Filkov, Vladimir

    2017-06-01

    Trees modify wood formation through integration of environmental and developmental signals in complex but poorly defined transcriptional networks, allowing trees to produce woody tissues appropriate to diverse environmental conditions. In order to identify relationships among genes expressed during wood formation, we integrated data from new and publically available datasets in Populus. These datasets were generated from woody tissue and include transcriptome profiling, transcription factor binding, DNA accessibility and genome-wide association mapping experiments. Coexpression modules were calculated, each of which contains genes showing similar expression patterns across experimental conditions, genotypes and treatments. Conserved gene coexpression modules (four modules totaling 8398 genes) were identified that were highly preserved across diverse environmental conditions and genetic backgrounds. Functional annotations as well as correlations with specific experimental treatments associated individual conserved modules with distinct biological processes underlying wood formation, such as cell-wall biosynthesis, meristem development and epigenetic pathways. Module genes were also enriched for DNase I hypersensitivity footprints and binding from four transcription factors associated with wood formation. The conserved modules are excellent candidates for modeling core developmental pathways common to wood formation in diverse environments and genotypes, and serve as testbeds for hypothesis generation and testing for future studies. No claim to original US government works. New Phytologist © 2017 New Phytologist Trust.

  5. Characterization of insulin-like-growth factor II (IGF II mRNA positive hepatic altered foci and IGF II expression in hepatocellular carcinoma during diethylnitrosamine-induced hepatocarcinogenesis in rats

    Directory of Open Access Journals (Sweden)

    Mukherjee Biswajit

    2005-08-01

    Full Text Available Abstract Background Insulin-like-growth factor II (IGF II has been implicated in the pathogenesis of neoplasm of different tissues, including liver of rats and men. This growth factor is believed to exert its effect during cellular proliferation. During the process of development of hepatocellular carcinoma (HCC, different hepatic altered foci appear. They are believed to be the putative precursors of HCC in rats and in men. Thus, to study the role of the gene in a defined model of hepatocarcinogenesis was the target to elucidate its role in various cancer phenotypes during the entire development stage of cancer, right from earlier preneoplastic lesions to HCC Methods Antisense in situ hybridization technique was used here to characterize the type(s of foci in which IGF II mRNA had expressed during the development of hepatocarcinogenesis-induced by diethylnitrosamine and promoted by phenobarbital in rats. Various focal lesions have been categorized depending on the stages and sizes along with IGF II expression patterns in them. Immunohistochemical detection for proliferating cell nuclear antigen (PCNA was made to detect the role of the gene in preneoplastic and neoplastic cellular proliferation. Results IGF II expression was located in the glycogen-storage acidophilic cell foci maximally followed by mixed cell lesions and the least in basophilic lesions. The expression of IGF II was found to be predominant in the HCC. The expression of gene was also located at the peripheral cells of spongiosis hepatis which are believed to be the precursor of ito cell carcinoma. It was noted that there is a direct correlation between IGF II expression and Immunohistochemical detection for PCNA. Conclusion It may be concluded that IGF II gene expression plays an important role during the development of neoplasia and the gene expresses in the sequence of events leading from glycogen-rich-acidophilic lesions to glycogen poor basophilic lesions to HCC with an

  6. Positive selection on MHC class II DRB and DQB genes in the bank vole (Myodes glareolus).

    Science.gov (United States)

    Scherman, Kristin; Råberg, Lars; Westerdahl, Helena

    2014-05-01

    The major histocompatibility complex (MHC) class IIB genes show considerable sequence similarity between loci. The MHC class II DQB and DRB genes are known to exhibit a high level of polymorphism, most likely maintained by parasite-mediated selection. Studies of the MHC in wild rodents have focused on DRB, whilst DQB has been given much less attention. Here, we characterised DQB genes in Swedish bank voles Myodes glareolus, using full-length transcripts. We then designed primers that specifically amplify exon 2 from DRB (202 bp) and DQB (205 bp) and investigated molecular signatures of natural selection on DRB and DQB alleles. The presence of two separate gene clusters was confirmed using BLASTN and phylogenetic analysis, where our seven transcripts clustered according to either DQB or DRB homologues. These gene clusters were again confirmed on exon 2 data from 454-amplicon sequencing. Our DRB primers amplify a similar number of alleles per individual as previously published DRB primers, though our reads are longer. Traditional d N/d S analyses of DRB sequences in the bank vole have not found a conclusive signal of positive selection. Using a more advanced substitution model (the Kumar method) we found positive selection in the peptide binding region (PBR) of both DRB and DQB genes. Maximum likelihood models of codon substitutions detected positively selected sites located in the PBR of both DQB and DRB. Interestingly, these analyses detected at least twice as many positively selected sites in DQB than DRB, suggesting that DQB has been under stronger positive selection than DRB over evolutionary time.

  7. Interfacial stress affects rat alveolar type II cell signaling and gene expression.

    Science.gov (United States)

    Hobi, Nina; Ravasio, Andrea; Haller, Thomas

    2012-07-01

    Previous work from our group (Ravasio A, Hobi N, Bertocchi C, Jesacher A, Dietl P, Haller T. Am J Physiol Cell Physiol 300: C1456-C1465, 2011.) showed that contact of alveolar epithelial type II cells with an air-liquid interface (I(AL)) leads to a paradoxical situation. It is a potential threat that can cause cell injury, but also a Ca(2+)-dependent stimulus for surfactant secretion. Both events can be explained by the impact of interfacial tensile forces on cellular structures. Here, the strength of this mechanical stimulus became also apparent in microarray studies by a rapid and significant change on the transcriptional level. Cells challenged with an I(AL) in two different ways showed activation/inactivation of cellular pathways involved in stress response and defense, and a detailed Pubmatrix search identified genes associated with several lung diseases and injuries. Altogether, they suggest a close relationship of interfacial stress sensation with current models in alveolar micromechanics. Further similarities between I(AL) and cell stretch were found with respect to the underlying signaling events. The source of Ca(2+) was extracellular, and the transmembrane Ca(2+) entry pathway suggests the involvement of a mechanosensitive channel. We conclude that alveolar type II cells, due to their location and morphology, are specific sensors of the I(AL), but largely protected from interfacial stress by surfactant release.

  8. TAF(II)250: a transcription toolbox.

    Science.gov (United States)

    Wassarman, D A; Sauer, F

    2001-08-01

    Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

  9. Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

    Directory of Open Access Journals (Sweden)

    Keith T Ballingall

    Full Text Available Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries. We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201 differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901, which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T

  10. Methylation of WNT target genes AXIN2 and DKK1 as robust biomarkers for recurrence prediction in stage II colon cancer

    NARCIS (Netherlands)

    Kandimalla, R.; Linnekamp, J. F.; van Hooff, S.; Castells, A.; Llor, X.; Andreu, M.; Jover, R.; Goel, A.; Medema, J. P.

    2017-01-01

    Stage II colon cancer (CC) still remains a clinical challenge with patient stratification for adjuvant therapy (AT) largely relying on clinical parameters. Prognostic biomarkers are urgently needed for better stratification. Previously, we have shown that WNT target genes AXIN2, DKK1, APCDD1, ASCL2

  11. Helping Students Understand Gene Regulation with Online Tools: A Review of MEME and Melina II, Motif Discovery Tools for Active Learning in Biology

    Directory of Open Access Journals (Sweden)

    David Treves

    2012-08-01

    Full Text Available Review of: MEME and Melina II, which are two free and easy-to-use online motif discovery tools that can be employed to actively engage students in learning about gene regulatory elements.

  12. MspI and PvuII polymorphisms in the Na,K-ATPase. beta. subunit gene ATP1B1

    Energy Technology Data Exchange (ETDEWEB)

    Shull, M.M.; Pugh, D.G.; Lane, L.K.; Lingrel, J.B. (Univ. of Cincinnati College of Medicine, OH (USA))

    1990-02-25

    ATP1B HH1.2 is a 1.2 kb HindIII fragment from the 3{prime} portion of the human Na,K-ATPase {beta} subunit gene, ATP1B1. MspI identifies a two allele polymorphism (M1: 6.7 kb, M2: 5.3 kb). PvuII also detects a two-allele polymorphism (P1: 5.1 kb, P2: 4.7 kb). ATP1B1 has been assigned to chromosome 1q by somatic cell hybrid analysis. Codominant segregation of the MspI RFLP was observed in one two-generation family (5 individuals). Codominant segregation of the PvuII RFLP was observed in a two-generation (8 individuals) and a three-generation (12 individuals) family.

  13. [Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

    Science.gov (United States)

    Petrova, L P; Prilipov, A G; Katsy, E I

    2017-01-01

    It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

  14. Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

    Science.gov (United States)

    Parsa, A T; Chi, J H; Hurley, P T; Jeyapalan, S A; Bruce, J N

    2001-09-01

    Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. Induction of MHC Class I in rat and human glioma cells after HSV TK

  15. IIS--Integrated Interactome System: a web-based platform for the annotation, analysis and visualization of protein-metabolite-gene-drug interactions by integrating a variety of data sources and tools.

    Science.gov (United States)

    Carazzolle, Marcelo Falsarella; de Carvalho, Lucas Miguel; Slepicka, Hugo Henrique; Vidal, Ramon Oliveira; Pereira, Gonçalo Amarante Guimarães; Kobarg, Jörg; Meirelles, Gabriela Vaz

    2014-01-01

    High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two

  16. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  17. Evaluation of environmental and nutritional factors and sua gene on in vitro biofilm formation of Streptococcus uberis isolates.

    Science.gov (United States)

    Moliva, Melina Vanesa; Cerioli, Florencia; Reinoso, Elina Beatriz

    2017-06-01

    The pathogenesis of Streptococcus uberis is attributed to a combination of extracellular factors and properties such as adherence and biofilm formation. The aim of this work was to evaluate the influence of different factors, additives and bovine milk compounds on S. uberis biofilm formation, as the presence of the sua gene by PCR. Additionally, extracellular DNA and the effect of DNaseI were evaluated in the biofilms yielded. Optimal biofilm development was observed when the pH was adjusted to 7.0 and 37 °C. Additives as glucose and lactose reduced biofilm formation as bovine milk compounds tested. PCR assay showed that not all the isolates yielded sua gene. Extrachromosomal ADN was found in cell-free supernatants, suggesting that DNA released spontaneously to the medium. The results contribute to a better understanding of the factors involved in biofilm production of this important pathogen associated with mastitis in order to promote the design of new therapeutic approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

    NARCIS (Netherlands)

    Dubern, J.F.; Coppoolse, E.R.; Stiekema, W.J.; Bloemberg, G.V.

    2008-01-01

    Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene

  19. [Molecular pathogenesis of Waardenburg syndrome type II resulting from SOX10 gene mutation].

    Science.gov (United States)

    Zhang, Hua; Chen, Hongsheng; Feng, Yong; Qian, Minfei; Li, Jiping; Liu, Jun; Zhang, Chun

    2016-08-01

    To explore the molecular mechanism of Waardenburg syndrome type II (WS2) resulting from SOX10 gene mutation E248fs through in vitro experiment. 293T cells were transiently transfected with wild type (WT) SOX10 and mutant type (MT) E248fs plasmids. The regulatory effect of WT/MT SOX10 on the transcriptional activity of MITF gene and influence of E248fs on WT SOX10 function were determined with a luciferase activity assay. The DNA binding capacity of the WT/MT SOX10 with the promoter of the MITF gene was determined with a biotinylated double-stranded oligonucleotide probe containing the SOX10 binding sequence cattgtc to precipitate MITF and E248fs, respectively. The stability of SOX10 and E248fs were also analyzed. As a loss-of-function mutation, the E248fs mutant failed to transactivate the MITF promoter as compared with the WT SOX10 (P<0.01), which also showed a dominant-negative effect on WT SOX10. The WT SOX10 and E248fs mutant were also able to bind specifically to the cattgtc motif in the MITF promoter, whereas E248fs had degraded faster than WT SOX10. Despite the fact that the E248fs has a dominant-negative effect on SOX10, its reduced stability may down-regulate the transcription of MITF and decrease the synthesis of melanin, which may result in haploinsufficiency of SOX10 protein and cause the milder WS2 phenotype.

  20. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

    Science.gov (United States)

    Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

    2009-01-01

    The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

  1. Inference of RNA polymerase II transcription dynamics from chromatin immunoprecipitation time course data.

    Directory of Open Access Journals (Sweden)

    Ciira wa Maina

    2014-05-01

    Full Text Available Gene transcription mediated by RNA polymerase II (pol-II is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2. The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα and FOXA1 binding in their proximal promoter regions.

  2. [Therapeutic effect of a novel recombinant vaccine encoding chicken collagen type II procollagen gene on collagen-induced arthritis in rat].

    Science.gov (United States)

    Song, Xin-qiang; Luo, Yuan; Wang, Dan; Liu, Shu-guang; Liu, Jin-feng; Yuan, Fang; Xue, Hong; Liu, Nan; Liang, Fei; Sun, Yu-ying; Xi, Yong-zhi

    2006-08-08

    To investigate the therapeutic effect of gene vaccine encoding chicken collagen type II (CC II) on collagen-induced arthritis (CIA) comprehensively. Three groups (CIA) were given a single intravenous injection of plasmid pcDNA-CCOL2A1 (20 microg/kg, 200 microg/kg, 400 microg/kg) respectively and one group (CIA) was injected 200 microg/kg pcDNA3.1 as a control. The effect of gene vaccine (pcDNA-CCOL2A1) was evaluated according to the arthritis score, radiological and histological examinations. The severity of arthritis of CIA rats which were administered 200 microg/kg pcDNA-CCOL2A1 was significantly reduced from the fifth day. According to the radiological and histological examinations, the articular cartilage as well as subchondral bone trabeculae are similar to those of the normal groups, so the bone and articular cartilage structure were protected after treatment with 200 microg/kg pcDNA-CCOL2A1 with a little synovial hyperplasia. The therapeutic effect of 200 microg/kg pcDNA-CCOL2A1 group has significant difference in comparison with that of the pcDNA3.1 group (P 0.05). The new gene vaccine pcDNA-CCOL2A1 has significant therapeutic effect on CIA rats, and the treatment may therefore be an effective strategy for RA patient clinically.

  3. Studies of variability in the PTEN gene among Danish caucasian patients with Type II diabetes mellitus

    DEFF Research Database (Denmark)

    Hansen, L; Jensen, J N; Ekstrøm, C T

    2001-01-01

    Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a novel member in the expanding network of proteins regulating the intracellular effects of insulin. By dephosphorylation of phosphatidyl-inositol-(3, 4, 5)-trisphosphate (PIP3) the PTEN protein...... regulates the insulin-dependent phosphoinositide 3-kinase (PI3K) signalling cassette and accordingly might function as a regulator of insulin sensitivity in skeletal muscle and adipose tissue. In this study we tested PTEN as a candidate gene for insulin resistance and late-onset Type II (non...

  4. Association of SNP variants of MHC Class II DRB gene with thermo-physiological traits in tropical goats.

    Science.gov (United States)

    Yakubu, Abdulmojeed; Salako, Adebowale E; De Donato, Marcos; Peters, Sunday O; Takeet, Michael I; Wheto, Mathew; Okpeku, Moses; Imumorin, Ikhide G

    2017-02-01

    Host defense in vertebrates depend on many secreted regulatory proteins such as major histocompatibility complex (MHC) class II which provide important regulatory and effector functions of T cells. Gene polymorphism in the second exon of Capra-DRB gene in three major Nigerian goat breeds [West African Dwarf (WAD), Red Sokoto (RS), and Sahel (SH)] was analyzed by restriction fragment length polymorphisms (RFLP). Four restriction enzymes, BsaHI, AluI, HaeIII, and SacII, were utilized. The association between the polymorphic sites and some heat tolerance traits were also investigated in a total of 70 WAD, 90 RS, and 50 SH goats. Fourteen different types of alleles identified in the Nigerian goats, four of which were found in the peptide coding region (A57G, Q89R, G104D, and T112I), indicate a high degree of polymorphism at the DRB locus in this species. An obvious excess (P  0.05), except AluI in RS goats and HaeIII in WAD goats (P goat populations, ranged from 0.16 to 0.50. Genotypes AA (BsaHI), GG, GC and CC (AluI) and GG, GA, AA (HaeIII) appeared better in terms of heat tolerance. The heat-tolerant ability of SH and RS goats to the hot and humid tropical environment of Nigeria seemed better than that of the WAD goats. Sex effect (P tropics.

  5. 9-cis-retinoic acid represses estrogen-induced expression of the very low density apolipoprotein II gene.

    Science.gov (United States)

    Schippers, I J; Kloppenburg, M; Snippe, L; Ab, G

    1994-11-01

    The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concentrated on a potential RXR recognition site, which deviates at only one position from a perfect direct A/GGGTCA repeat spaced by one nucleotide (DR-1) and was earlier identified as a common HNF-4/COUP-TF recognition site. However, band shift analysis revealed that this imperfect DR-1 motif does not interact with RXR alpha-homodimers. In accordance with this observation we found that this regulatory element does not mediate transactivation through RXR alpha in the presence of 9-cis-RA. However, our experiments revealed another, unexpected, effect of 9-cis-RA. Instead of stimulating, 9-cis-RA attenuated estrogen-induced expression of transfected estrogen-responsive VLDL-CAT reporter plasmids. This repression appeared to take place through the main estrogen response element (ERE) of the gene. Importantly, 9-cis-RA also strongly repressed the estrogen-induced expression of the endogenous apoVLDLII gene in cultured chicken hepatoma cells.

  6. The NSL Complex Regulates Housekeeping Genes in Drosophila

    Science.gov (United States)

    Raja, Sunil Jayaramaiah; Holz, Herbert; Luscombe, Nicholas M.; Manke, Thomas; Akhtar, Asifa

    2012-01-01

    MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription. PMID:22723752

  7. Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

    Science.gov (United States)

    Lubys, A; Lubienè, J; Kulakauskas, S; Stankevicius, K; Timinskas, A; Janulaitis, A

    1996-07-15

    The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

  8. The Class II trehalose 6-phosphate synthase gene PvTPS9 modulates trehalose metabolism in Phaseolus vulgaris nodules.

    Directory of Open Access Journals (Sweden)

    Aarón Barraza

    2016-11-01

    Full Text Available Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS of common bean (Phaseolus vulgaris, was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1% of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

  9. Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

    Science.gov (United States)

    Ikemoto, Tadahiro; Park, Min Kyun

    2003-10-16

    To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

  10. Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

    Science.gov (United States)

    Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

    2003-07-01

    We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

  11. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron...

  12. The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca(2+) and Mg(2+) dependent-DNase activity and antifungal action on Moniliophthora perniciosa.

    Science.gov (United States)

    Pereira Menezes, Sara; de Andrade Silva, Edson Mario; Matos Lima, Eline; Oliveira de Sousa, Aurizângela; Silva Andrade, Bruno; Santos Lima Lemos, Livia; Peres Gramacho, Karina; da Silva Gesteira, Abelmon; Pirovani, Carlos Priminho; Micheli, Fabienne

    2014-06-11

    The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b. TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae. To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.

  13. The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    Science.gov (United States)

    2014-01-01

    Background The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b. Results TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae. Conclusion To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively. PMID:24920373

  14. Molecular cloning, characterization and localization of chicken type II procollagen gene.

    Science.gov (United States)

    Xi, Caixia; Liu, Nan; Liang, Fei; Guo, Siqi; Sun, Yuying; Yang, Fengtang; Xi, Yongzhi

    2006-01-17

    Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.

  15. RNA polymerase II collision interrupts convergent transcription

    DEFF Research Database (Denmark)

    Hobson, David J; Wei, Wu; Steinmetz, Lars M

    2012-01-01

    Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical...

  16. Comparative Serum Challenges Show Divergent Patterns of Gene Expression and Open Chromatin in Human and Chimpanzee.

    Science.gov (United States)

    Pizzollo, Jason; Nielsen, William J; Shibata, Yoichiro; Safi, Alexias; Crawford, Gregory E; Wray, Gregory A; Babbitt, Courtney C

    2018-03-01

    Humans experience higher rates of age-associated diseases than our closest living evolutionary relatives, chimpanzees. Environmental factors can explain many of these increases in disease risk, but species-specific genetic changes can also play a role. Alleles that confer increased disease susceptibility later in life can persist in a population in the absence of selective pressure if those changes confer positive adaptation early in life. One age-associated disease that disproportionately affects humans compared with chimpanzees is epithelial cancer. Here, we explored genetic differences between humans and chimpanzees in a well-defined experimental assay that mimics gene expression changes that happen during cancer progression: A fibroblast serum challenge. We used this assay with fibroblasts isolated from humans and chimpanzees to explore species-specific differences in gene expression and chromatin state with RNA-Seq and DNase-Seq. Our data reveal that human fibroblasts increase expression of genes associated with wound healing and cancer pathways; in contrast, chimpanzee gene expression changes are not concentrated around particular functional categories. Chromatin accessibility dramatically increases in human fibroblasts, yet decreases in chimpanzee cells during the serum response. Many regions of opening and closing chromatin are in close proximity to genes encoding transcription factors or genes involved in wound healing processes, further supporting the link between changes in activity of regulatory elements and changes in gene expression. Together, these expression and open chromatin data show that humans and chimpanzees have dramatically different responses to the same physiological stressor, and how a core physiological process can evolve quickly over relatively short evolutionary time scales.

  17. Genes for carbon metabolism and the ToxA virulence factor in Pseudomonas aeruginosa are regulated through molecular interactions of PtxR and PtxS.

    Directory of Open Access Journals (Sweden)

    Abdelali Daddaoua

    Full Text Available Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the P(gad and P(kgu for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of Pseudomonas aeruginosa. We show using DNaseI-footprint analysis that in P. aeruginosa PtxR binds to the -35 region of the P(toxA promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the P(kgu and P(gad promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. In vitro, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of P(kgu and P(gad promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the P(toxA promoter, or via the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator.

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

    KAUST Repository

    Alam, Tanvir

    2014-10-02

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

  19. Acinetobacter baumannii clonal lineages I and II harboring different carbapenem-hydrolyzing-β-lactamase genes are widespread among hospitalized burn patients in Tehran.

    Science.gov (United States)

    Mahdian, Somayeh; Sadeghifard, Nourkhoda; Pakzad, Iraj; Ghanbari, Fatemeh; Soroush, Setareh; Azimi, Lila; Rastegar-Lari, Abdolaziz; Giannouli, Maria; Taherikalani, Morovat

    2015-01-01

    The aim of this study was to analyze antimicrobial resistance patterns and their encoding genes and genotypic diversity of Acinetobacter baumannii isolated from burn patients in Tehran, Iran. The presence of extended-spectrum beta-lactamase- and blaOXA-encoding genes among 37 multidrug resistant (MDR) A. baumannii strains isolated from patients hospitalized in a teaching hospital in Tehran was evaluated. Susceptibility to 7 antibiotics was tested by disk agar diffusion and to polymyxin B and colistin was tested by E-test, according to CLSI guidelines. All isolates were then analyzed by PCR for the presence of blaIMP, blaVIM, blaSIMblaOXA-23, blaOXA-24, and blaOXA-58-like carbapenemase genes, and blaOXA-51-like, blaTEM, blaSHV, blaPER, blaVEB, and blaGIM genes. Genotyping of A. baumannii strains was performed by repetitive sequence-based (REP)-PCR and cluster analysis of REP-PCR profiles. A. baumannii isolates were assigned to international clones by multiplex PCR sequence group analysis. Twenty-five A. baumannii isolates were classified as MDR, and 12 were classified as extensively drug resistant. All isolates were susceptible to colistin and polymyxin B. Eighty-one percent of the isolates was resistant to imipenem or meropenem and harbored at least one or both of the blaOXA-23-like or blaOXA-24-like carbapenemase genes. Co-existence of different resistance genes was found among carbapenem-resistant isolates. Multiplex PCR sequence group analysis most commonly assigned A. baumannii isolates to international clones I (18/37; 48.6%) and II (18/37; 48.6%). An alarming increase in resistance to carbapenems and the spread of blaOXA-23-like and/or blaOXA-24-like carbapenemase genes was observed among A. baumannii strains belonging to clonal lineages I and II, isolated from burn patients in Tehran. Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  20. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Verónica Loera-Castañeda

    2014-01-01

    Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  1. Efficient transformation and regeneration of transgenic cassava using the neomycin phosphotransferase gene as aminoglycoside resistance marker gene.

    Science.gov (United States)

    Niklaus, Michael; Gruissem, Wilhelm; Vanderschuren, Hervé

    2011-01-01

    Cassava is one of the most important crops in the tropics. Its industrial use for starch and biofuel production is also increasing its importance for agricultural production in tropical countries. In the last decade cassava biotechnology has emerged as a valuable alternative to the breeding constraints of this highly heterozygous crop for improved trait development of cassava germplasm. Cassava transformation remains difficult and time-consuming because of limitations in selecting transgenic tissues and regeneration of transgenic plantlets. We have recently reported an efficient and robust cassava transformation protocol using the hygromycin phosphotransferase II (hptII) gene as selection marker and the aminoglycoside hygromycin at optimal concentrations to maximize the regeneration of transgenic plantlets. In the present work, we expanded the transformation protocol to the use of the neomycin phosphotransferase II (nptII) gene as selection marker. Several aminoglycosides compatible with the use of nptII were tested and optimal concentrations for cassava transformation were determined. Given its efficiency equivalent to hptII as selection marker with the described protocol, the use of nptII opens new possibilities to engineer transgenic cassava lines with multiple T-DNA insertions and to produce transgenic cassava with a resistance marker gene that is already deregulated in several commercial transgenic crops.

  2. Association of kynurenine aminotransferase II gene C401T polymorphism with immune response in patients with meningitis

    Directory of Open Access Journals (Sweden)

    Leib Stephen L

    2011-04-01

    Full Text Available Abstract Background The kynurenine (KYN pathway has been shown to be altered in several diseases which compromise the central nervous system (CNS including infectious diseases such as bacterial meningitis (BM. The aim of this study was to assess single nucleotide polymorphisms (SNPs in four genes of KYN pathway in patients with meningitis and their correlation with markers of immune response in BM. Methods One hundred and one individuals were enrolled in this study to investigate SNPs in the following genes: indoleamine-2,3-dioxygenase (IDO1 gene, kynureninase (KYNU gene, kynurenine aminotransferase I (CCBL1 gene, and kynurenine aminotransferase II (AADAT gene. SNP analyses were performed by primer-introduced restriction analysis-PCR (PIRA-PCR followed by RFLP. Cytokines were measured using multiplex bead assay while immunoglobulins (IG by immunodiffusion plates and NF-kappaB and c-Jun by dot blot assay. Results The variant allele of SNP AADAT+401C/T showed prevalent frequency in patients with BM. A significant decrease (p AADAT+401C/T. Furthermore, a significant (p p KYNU+715G/A was found with low frequency in the groups, and the SNPs in IDO1+434T/G, KYNU+693G/A, CCBL1+164T/C, and AADAT+650C/T had no frequency in this population. Conclusions This study is the first report of an association of SNP AADAT+401C/T with the host immune response to BM, suggesting that this SNP may affect the host ability in recruitment of leukocytes to the infection site. This finding may contribute to identifying potential targets for pharmacological intervention as adjuvant therapy for BM.

  3. Synthesis and characterization of a lamellar hydroxyapatite/DNA nanohybrid

    Energy Technology Data Exchange (ETDEWEB)

    Zuo Guifu; Wan Yizao; Meng Xianguang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Zhao Qing [School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072 (China); Ren Kaijing [Department of Joint Surgery, Tianjin Hospital, Tianjin 300211 (China); Jia Shiru [Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin University of Science and Technology, 29, 13th Street, TEDA, Tianjin 300457 (China); Wang Jiehua, E-mail: gfzuo@tju.edu.cn [School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072 (China)

    2011-04-15

    Research highlights: {yields} A lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared as a novel gene delivering vector. {yields} Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I. {yields} The protected DNA in the HAp/DNA nanohybrid could be recovered readily under acid conditions. - Abstract: Two-dimensional layered materials exhibit desired functionalities when being used as gene delivery materials. In this study, a novel gene delivering vector, lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared. The structure of HAp/DNA nanohybrid was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy analysis revealed that ion-exchange occurred during the process. Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I and the protected DNA could be recovered readily under acid conditions. Furthermore, the integrity of released DNA was confirmed by UV-vis spectra.

  4. Synthesis and characterization of a lamellar hydroxyapatite/DNA nanohybrid

    International Nuclear Information System (INIS)

    Zuo Guifu; Wan Yizao; Meng Xianguang; Zhao Qing; Ren Kaijing; Jia Shiru; Wang Jiehua

    2011-01-01

    Research highlights: → A lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared as a novel gene delivering vector. → Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I. → The protected DNA in the HAp/DNA nanohybrid could be recovered readily under acid conditions. - Abstract: Two-dimensional layered materials exhibit desired functionalities when being used as gene delivery materials. In this study, a novel gene delivering vector, lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared. The structure of HAp/DNA nanohybrid was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy analysis revealed that ion-exchange occurred during the process. Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I and the protected DNA could be recovered readily under acid conditions. Furthermore, the integrity of released DNA was confirmed by UV-vis spectra.

  5. Casein genes of Bos taurus. II. Isolation and characterization of the β-casein gene

    International Nuclear Information System (INIS)

    Gorodetskii, S.I.; Tkach, T.M.; Kapelinskaya, T.V.

    1988-01-01

    The expression of the casein genes in the cells of the mammary gland is regulated by peptide and steroid hormones. In order to study the controlling mechanisms we have isolated and characterized the β-casein gene. The gene is 8.6 kb long and exceeds by a factor of 7.8 the length of the corresponding mRNA which is encoded by nine exons. The genomic clones incorporate in addition 8.5 kb and 4.5 kb of the 5'- and 3'-flanking regions. We have determined the sequence of the 5- and 3-terminals of the gene and have performed a comparative analysis of the corresponding regions of the rat β-casein gene. Furthermore we have identified the conversed sequences identical or homologous to the potential sections of binding to the nuclear factor CTF/NF-1 by glucocorticoid and progesterone receptors. The regulatory region of the bovine casein gene contains two variants of the TATA signal, flanking the duplication section in the promoter region

  6. Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

    DEFF Research Database (Denmark)

    Hammer, Karin; Jensen, Kaj Frank; Poulsen, Peter

    1987-01-01

    Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of c......II or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation....... In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing...

  7. Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: The class II cytokine receptors and their ligands in mammals and fish

    Directory of Open Access Journals (Sweden)

    Mogensen Knud

    2003-07-01

    Full Text Available Abstract Background The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26 and their receptors (HCRII. Despite the report of a near complete pufferfish (Takifugu rubripes genome sequence, these genes remain undescribed in fish. Results We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF. Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. Conclusion We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish.

  8. Sequence-based model of gap gene regulatory network.

    Science.gov (United States)

    Kozlov, Konstantin; Gursky, Vitaly; Kulakovskiy, Ivan; Samsonova, Maria

    2014-01-01

    The detailed analysis of transcriptional regulation is crucially important for understanding biological processes. The gap gene network in Drosophila attracts large interest among researches studying mechanisms of transcriptional regulation. It implements the most upstream regulatory layer of the segmentation gene network. The knowledge of molecular mechanisms involved in gap gene regulation is far less complete than that of genetics of the system. Mathematical modeling goes beyond insights gained by genetics and molecular approaches. It allows us to reconstruct wild-type gene expression patterns in silico, infer underlying regulatory mechanism and prove its sufficiency. We developed a new model that provides a dynamical description of gap gene regulatory systems, using detailed DNA-based information, as well as spatial transcription factor concentration data at varying time points. We showed that this model correctly reproduces gap gene expression patterns in wild type embryos and is able to predict gap expression patterns in Kr mutants and four reporter constructs. We used four-fold cross validation test and fitting to random dataset to validate the model and proof its sufficiency in data description. The identifiability analysis showed that most model parameters are well identifiable. We reconstructed the gap gene network topology and studied the impact of individual transcription factor binding sites on the model output. We measured this impact by calculating the site regulatory weight as a normalized difference between the residual sum of squares error for the set of all annotated sites and for the set with the site of interest excluded. The reconstructed topology of the gap gene network is in agreement with previous modeling results and data from literature. We showed that 1) the regulatory weights of transcription factor binding sites show very weak correlation with their PWM score; 2) sites with low regulatory weight are important for the model output; 3

  9. Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes).

    Science.gov (United States)

    Strandh, Maria; Lannefors, Mimi; Bonadonna, Francesco; Westerdahl, Helena

    2011-10-01

    The great polymorphism observed in the major histocompatibility complex (MHC) genes is thought to be maintained by pathogen-mediated selection possibly combined with MHC-disassortative mating, guided by MHC-determined olfactory cues. Here, we partly characterize the MHC class I and II B of the blue petrel, Halobaena caerulea (Procellariiformes), a bird with significant olfactory abilities that lives under presumably low pathogen burdens in Subantarctica. Blue petrels are long-lived, monogamous birds which suggest the necessity of an accurate mate choice process. The species is ancestral to songbirds (Passeriformes; many MHC loci), although not to gamefowls (Galliformes; few MHC loci). Considering the phylogenetic relationships and the low subantarctic pathogen burden, we expected few rather than many MHC loci in the blue petrel. However, when we analysed partial MHC class I and class II B cDNA and gDNA sequences we found evidence for as many as at least eight MHC class I loci and at least two class II B loci. These class I and II B sequences showed classical MHC characteristics, e.g. high nucleotide diversity, especially in putative peptide-binding regions where signatures of positive selection was detected. Trans-species polymorphism was found between MHC class II B sequences of the blue petrel and those of thin-billed prion, Pachyptila belcheri, two species that diverged ∼25 MYA. The observed MHC allele richness in the blue petrel may well serve as a basis for mate choice, especially since olfactory discrimination of MHC types may be possible in this species.

  10. Insulin-like growth factor II: complexity of biosynthesis and receptor binding

    DEFF Research Database (Denmark)

    Gammeltoft, S; Christiansen, Jan; Nielsen, F C

    1991-01-01

    Insulin-like growth factor II (IGF-II) belongs to the insulin family of peptides and acts as a growth factor in many fetal tissues and tumors. The gene expression of IGF-II is initiated at three different promoters which gives rise to multiple transcripts. In a human rhabdomyosarcoma cell line......, Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of IGF-II is very...

  11. Striking hematological abnormalities in patients with microcephalic osteodysplastic primordial dwarfism type II (MOPD II): a potential role of pericentrin in hematopoiesis.

    Science.gov (United States)

    Unal, Sule; Alanay, Yasemin; Cetin, Mualla; Boduroglu, Koray; Utine, Eda; Cormier-Daire, Valerie; Huber, Celine; Ozsurekci, Yasemin; Kilic, Esra; Simsek Kiper, Ozlem Pelin; Gumruk, Fatma

    2014-02-01

    Microcephalic osteodysplastic primordial dwarfism type II (MOPD II) is a rare primordial dwarfism that is similar to Seckel syndrome. Seckel syndrome is known to be associated with various hematological abnormalities; however, hematological findings in MOPD II patients have not been previously reported. The present study aimed to describe the hematological findings in a series of eight patients with MOPD II from a single center. The study included eight patients with MOPD II that were analyzed via molecular testing, and physical and laboratory examinations. Molecular testing showed that seven of the eight patients had pericentrin (PCNT) gene mutations. Hematological evaluation showed that 7 (87.5%) patients had thrombocytosis, 6 (75%) had leukocytosis, 5 (62.5%) had both leukocytosis and thrombocytosis, and 2 (25%) had anemia. We report leukocytosis and thrombocytosis as a common hematologic abnormality in patients with MOPD II. The present findings may improve our understanding of the potential function of the PCNT gene in hematopoietic cell proliferation and differentiation. © 2013 Wiley Periodicals, Inc.

  12. Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney

    International Nuclear Information System (INIS)

    Werner, H.; Shen-Orr, Z.; Stannard, B.; Burguera, B.; Roberts, C.T. Jr.; LeRoith, D.

    1990-01-01

    Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I-receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I-peptide mRNA. Similarly, IGF-II-receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I- and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II-receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study

  13. Autosomal dominant familial neurohypophyseal diabetes insipidus caused by a mutation in the arginine-vasopressin II gene in four generations of a Korean family

    Directory of Open Access Journals (Sweden)

    Myo-Jing Kim

    2014-12-01

    Full Text Available Autosomal dominant neurohypophyseal diabetes insipidus is a rare form of central diabetes insipidus that is caused by mutations in the vasopressin-neurophysin II (AVP-NPII gene. It is characterized by persistent polydipsia and polyuria induced by deficient or absent secretion of arginine vasopressin (AVP. Here we report a case of familial neurohypophyseal diabetes insipidus in four generations of a Korean family, caused by heterozygous missense mutation in exon 2 of the AVP-NPII gene (c.286G>T. This is the first report of such a case in Korea.

  14. Isolation of developing secondary xylem specific cellulose synthase ...

    Indian Academy of Sciences (India)

    Total RNA was treated with RNase-free DNase I (Fermentas, USA) ... Twenty-five μL of qRT- PCR reaction included 11.5 μL milli-q water, 200 ng of cDNA, ..... grey shading, class specific region (CSR I and II), plant conserved region (CRP) and.

  15. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

    Science.gov (United States)

    Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

    2014-01-13

    Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The

  16. Screening method for Staphylococcus aureus identification in subclinical bovine mastitis from dairy farms

    Directory of Open Access Journals (Sweden)

    Natapol Pumipuntu

    2017-07-01

    Full Text Available Background: Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area. Aim: Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA], and deoxyribonuclease (DNase test, for S. aureus identification in milk samples. Materials and Methods: A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV, and negative predictive value (NPV among both tests were analyzed and compared to the tube coagulase test (TCT, as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus. Results: A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT. Conclusion: MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and

  17. Amphiphilic graft copolymer based on poly(styrene-co-maleic anhydride) with low molecular weight polyethylenimine for efficient gene delivery

    Science.gov (United States)

    Duan, Xiaopin; Xiao, Jisheng; Yin, Qi; Zhang, Zhiwen; Mao, Shirui; Li, Yaping

    2012-01-01

    Background and methods A new amphiphilic comb-shaped copolymer (SP) was synthesized by conjugating poly(styrene-co-maleic anhydride) with low molecular weight polyethyleneimine for gene delivery. Fourier transform infrared spectrum, 1H nuclear magnetic resonance, and gel permeation chromatography were used to characterize the graft copolymer. Results The buffering capability of SP was similar to that of polyethyleneimine within the endosomal pH range. The copolymer could condense DNA effectively to form complexes with a positive charge (13–30 mV) and a small particle size (130–200 nm) at N/P ratios between 5 and 20, and protect DNA from degradation by DNase I. In addition, SP showed much lower cytotoxicity than polyethyleneimine 25,000. Importantly, the gene transfection activity and cellular uptake of SP-DNA complexes were all markedly higher than that of complexes of polyethyleneimine 25,000 and DNA in MCF-7 and MCF-7/ADR cell lines. Conclusion This work highlights the promise of SP as a safe and efficient synthetic vector for DNA delivery. PMID:23028224

  18. Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

    Science.gov (United States)

    Tajaddod, Mansoureh; Tanzer, Andrea; Licht, Konstantin; Wolfinger, Michael T; Badelt, Stefan; Huber, Florian; Pusch, Oliver; Schopoff, Sandy; Janisiw, Michael; Hofacker, Ivo; Jantsch, Michael F

    2016-10-25

    Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

  19. H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein

    DEFF Research Database (Denmark)

    Runge, Steffen; Nielsen, Finn Cilius; Nielsen, Jacob

    2000-01-01

    H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due to their recip......H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due...

  20. Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter.

    Science.gov (United States)

    Raghothama, K G; Liu, D; Nelson, D E; Hasegawa, P M; Bressan, R A

    1993-12-01

    Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5' deletions of the osmotin promoter cloned into a promoter-less GUSNOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from -108 to -248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to -1052) of enhancer-like sequence and then a sequence upstream of -1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the -248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.

  1. Toxigenic genes, spoilage potential, and antimicrobial resistance of Bacillus cereus group strains from ice cream.

    Science.gov (United States)

    Arslan, Seza; Eyi, Ayla; Küçüksarı, Rümeysa

    2014-02-01

    Bacillus spp. can be recovered from almost every environment. It is also found readily in foods, where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature, and extracellular enzymes. In this study, 29 Bacillus cereus group strains from ice cream were examined for the presence of following virulence genes hblC, nheA, cytK and ces genes, and tested for a range of the extracellular enzymes, and antimicrobial susceptibility. The strains were found to produce extracellular enzymes: proteolytic and lipolytic activity, gelatin hydrolysis and lecithinase production (100%), DNase production (93.1%) and amylase activity (93.1%). Of 29 strains examined, 24 (82.8%) showed hemolytic activity on blood agar. Beta-lactamase enzyme was only produced by 20.7% of B. cereus group. Among 29 B. cereus group from ice cream, nheA was the most common virulence gene detected in 44.8% of the strains, followed by hblC gene with 17.2%. Four (13.8%) of the 29 strains were positive for both hblC gene and nheA gene. Contrarily, cytK and ces genes were not detected in any of the strains. Antimicrobial susceptibility of ice cream isolates was tested to 14 different antimicrobial agents using the disc diffusion method. We detected resistance to penicillin and ampicillin with the same rate of 89.7%. Thirty-one percent of the strains were multiresistant to three or more antibiotics. This study emphasizes that the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin genes, producing extracellular enzymes which may cause spoilage and acquiring antibiotic resistance might hold crucial importance in the food safety and quality. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Microcephalic osteodysplastic primordial dwarfism type II (MOPD II) with multiple vascular complications misdiagnosed as Dubowitz syndrome.

    Science.gov (United States)

    Dieks, Jana-Katharina; Baumer, Alessandra; Wilichowski, Ekkehard; Rauch, Anita; Sigler, Matthias

    2014-09-01

    To date, the genetic basis of Dubowitz syndrome (short stature, microcephaly, facial abnormalities, eczema) is unknown and vascular complications are not known to be associated with this syndrome. In microcephalic osteodysplastic primordial dwarfism type II (MOPD II; disproportionate short statue, microcephaly, facial abnormalities), however, cerebral aneurysms and other vascular abnormalities are frequent complications. MOPD II is a genetic disorder caused by mutations in the pericentrin (PCNT) gene (21q22). We report on a patient who came to our attention as a 22-year-old with subarachnoid bleeding due to a ruptured cranial aneurysm. Until then, the patient was thought and published to have Dubowitz syndrome; previously, he was treated with coronary bypass surgery for extensive coronary angiopathy. Consecutive genetic testing revealed MOPD II. After clinical stabilization, the patient was discharged to a specialized rehabilitation center where he died due to re-rupture of a cranial aneurysm. In patients with short stature-especially when clinical features are accompanied by vascular complications-MOPD II should be considered as a differential diagnosis leading to consecutive genetic testing. After detection of mutations in the PCNT gene, a full vascular status including cerebral imaging and cardiac evaluation needs to be determined in order to analyze vascular abnormalities and initiate prophylactic treatment.

  3. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    International Nuclear Information System (INIS)

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-01-01

    Highlights: → The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. → The core promoter was located in the 5F-1. → Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. → These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  4. Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

    Directory of Open Access Journals (Sweden)

    Yoichiro Shibata

    2012-06-01

    Full Text Available Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

  5. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

    Science.gov (United States)

    Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2014-02-23

    Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the

  6. Scavenger receptor AI/II truncation, lung function and COPD

    DEFF Research Database (Denmark)

    Thomsen, M; Nordestgaard, B G; Tybjaerg-Hansen, A

    2011-01-01

    The scavenger receptor A-I/II (SRA-I/II) on alveolar macrophages is involved in recognition and clearance of modified lipids and inhaled particulates. A rare variant of the SRA-I/II gene, Arg293X, truncates the distal collagen-like domain, which is essential for ligand recognition. We tested whet...

  7. PGMA-Based Cationic Nanoparticles with Polyhydric Iodine Units for Advanced Gene Vectors.

    Science.gov (United States)

    Sun, Yue; Hu, Hao; Yu, Bingran; Xu, Fu-Jian

    2016-11-16

    It is crucial for successful gene delivery to develop safe, effective, and multifunctional polycations. Iodine-based small molecules are widely used as contrast agents for CT imaging. Herein, a series of star-like poly(glycidyl methacrylate) (PGMA)-based cationic vectors (II-PGEA/II) with abundant flanking polyhydric iodine units are prepared for multifunctional gene delivery systems. The proposed II-PGEA/II star vector is composed of one iohexol intermediate (II) core and five ethanolamine (EA) and II-difunctionalized PGMA arms. The amphipathic II-PGEA/II vectors readily self-assemble into well-defined cationic nanoparticles, where massive hydroxyl groups can establish a hydration shell to stabilize the nanoparticles. The II introduction improves cell viabilities of polycations. Moreover, by controlling the suitable amount of introduced II units, the resultant II-PGEA/II nanoparticles can produce fairly good transfection performances in different cell lines. Particularly, the II-PGEA/II nanoparticles induce much better in vitro CT imaging abilities in tumor cells than iohexol (one commonly used commercial CT contrast agent). The present design of amphipathic PGMA-based nanoparticles with CT contrast agents would provide useful information for the development of new multifunctional gene delivery systems.

  8. Biochemical mechanisms involved in the endotoxin-induced type II cell hyperplasia in F344 rat lung

    International Nuclear Information System (INIS)

    Tesfaigzi, J.; Johnson, N.F.; Lechner, J.F.

    1994-01-01

    Proliferative lesions and pulmonary epithelial neoplasms induced in the rat by plutonium inhalation have been shown to be of type II cell origin. Defining the gene changes responsible for the development of the type II proliferative lesions would help to elucidate the genetic events involved in the expansion of initiated type II cells into fully transformed tumor cells. One problem in identifying these gene alterations is dissociating changes in gene expression linked to cell replication or repair from those involved in tumor initiation and progression. The long-term goals of these investigations are to first develop and characterize a model of transient type II cell hyperplasia. Second, changes in gene expression associated with remodeling epithelium will be compared to gene changes exhibited by the 239 Pu-induced hyperplastic lesions

  9. Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

    Science.gov (United States)

    Lu, D; Yang, H; Raizada, M K

    1996-12-01

    Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

  10. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  11. Genetic association analysis of 13 nuclear-encoded mitochondrial candidate genes with type II diabetes mellitus: The DAMAGE study

    DEFF Research Database (Denmark)

    Reiling, Erwin; van Vliet-Ostaptchouk, Jana V; van 't Riet, Esther

    2009-01-01

    ). After a meta-analysis, only one SNP in SIRT4 (rs2522138) remained significant (P=0.01). Extending the second stage with samples from the Danish Steno Study (n=1220 participants) resulted in a common odds ratio (OR) of 0.92 (0.85-1.00), P=0.06. Moreover, in a large meta-analysis of three genome......Mitochondria play an important role in many processes, like glucose metabolism, fatty acid oxidation and ATP synthesis. In this study, we aimed to identify association of common polymorphisms in nuclear-encoded genes involved in mitochondrial protein synthesis and biogenesis with type II diabetes...

  12. The tetracycline resistance determinant Tet 39 and the sulphonamide resistance gene sulII are common among resistant Acinetobacter spp. isolated from integrated fish farms in Thailand

    DEFF Research Database (Denmark)

    Agersø, Yvonne; Petersen, Andreas

    2007-01-01

    Objectives: To determine the genetic basis for tetracycline and sulphonamide resistance and the prevalence of class I and II integrons in oxytetracycline-resistant Acinetobacter spp. from integrated fish farms in Thailand. Methods: A total of 222 isolates were screened for tetracycline resistance...... and Southern blots with sulII and tet(39) probes were performed on selected isolates. Results: The recently identified tetracycline resistance gene tet(39) was demonstrated in 75% (166/222) of oxytetracycline-resistant Acinetobacter spp. from integrated fish farms in Thailand. Isolates that were also...

  13. Use of meta-analysis to combine candidate gene association studies: application to study the relationship between the ESR PvuII polymorphism and sow litter size

    Directory of Open Access Journals (Sweden)

    Alfonso Leopoldo

    2005-07-01

    Full Text Available Abstract This article investigates the application of meta-analysis on livestock candidate gene effects. The PvuII polymorphism of the ESR gene is used as an example. The association among ESR PvuII alleles with the number of piglets born alive and total born in the first (NBA1, TNB1 and later parities (NBA, TNB is reviewed by conducting a meta-analysis of 15 published studies including 9329 sows. Under a fixed effects model, litter size values were significantly lower in the "AA" genotype groups when compared with "AB" and "BB" homozygotes. Under the random effects model, the results were similar although differences between "AA" and "AB" genotype groups were not clearly significant for NBA and TNB. Nevertheless, the most noticeable result was the high and significant heterogeneity estimated among studies. This heterogeneity could be assigned to error sampling, genotype by environment interaction, linkage or epistasis, as referred to in the literature, but also to the hypothesis of population admixture/stratification. It is concluded that meta-analysis can be considered as a helpful analytical tool to synthesise and discuss livestock candidate gene effects. The main difficulty found was the insufficient information on the standard errors of the estimated genotype effects in several publications. Consequently, the convenience of publishing the standard errors or the concrete P-values instead of the test significance level should be recommended to guarantee the quality of candidate gene effect meta-analyses.

  14. Enhanced B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation contributes to ABCC1-mediated chemoresistance and glutathione-mediated survival in acquired topoisomerase II poison-resistant cancer cells.

    Science.gov (United States)

    Chen, Huang-Hui; Chang, Hsin-Huei; Chang, Jang-Yang; Tang, Ya-Chu; Cheng, Yung-Chi; Lin, Li-Mei; Cheng, Shu-Ying; Huang, Chih-Hsiang; Sun, Man-Wu; Chen, Chiung-Tong; Kuo, Ching-Chuan

    2017-12-01

    Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Evaluation of HP0605 and HP0971 genes of efflux pumps in Helicobacter pylori resistance to Metronidazole

    Directory of Open Access Journals (Sweden)

    Mohammad hasan Shirazi

    2009-12-01

    Full Text Available Background: The presence of antibiotic resistance has been reported in H.pylori and it is a major cause of treatment failure. Five families of multidrug efflux pumps are defined in bacteria and resistance-nodulation-division (RND pumps are found mainly in gram negative bacteria. TolC is one of RND pump components and play a critical role in drug resistance. It hasn’t been established that RND family has a role in drug resistance in H.pylori. In this study, we assessed the role of two efflux genes in resistant to metronidazole in H.pylori by evaluation of overexpression TolC genes by RT-PCR method. Methods: In five metronidazole resistant strains of H.pylori, total RNA was extracted. RNA treated with DNase and RNA reverse transcribed to cDNA. Aliquots of the cDNA solution were assayed by RT- PCR for HP0605 and HP0971 genes. The levels of mRNA expression were evaluated by densitometry analysis. Results: All five strains displayed overexpression for HP0605 basis of increased concentration of metronidazole. Three strains showed transcripts for HP0971. One of these had transcripts for HP0971 only in Metronidazole concentration equaled to 16 µg/ml but two strains overexpressed adapt to increase concentration of metronidazole. Conclusion: According to current study, HP0605 and HP0971 genes overexpressed due to increase metronidazole. So, increasing of Metronidazole affects in H.pylori΄s efflux system in transcription level.

  16. Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Kush Shrivastava

    2015-10-01

    Full Text Available Aim: To study the major histocompatibility complex (MHC Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and nucleotide sequencing techniques. Materials and Methods: DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR software. Results: TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE, however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison. Conclusion: The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI, both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

  17. Imaging gene expression in gene therapy

    International Nuclear Information System (INIS)

    Wiebe, Leonard I.

    1997-01-01

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

  18. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    Science.gov (United States)

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  19. [Identification of novel compound heterozygous mutations of USH2A gene in a family with Usher syndrome type II].

    Science.gov (United States)

    Jiang, Haiou; Ge, Chuanqin; Wang, Yiwang; Tang, Genyun; Quan, Qingli

    2015-06-01

    To identify potential mutations in a Chinese family with Usher syndrome type II. Genomic DNA was obtained from two affected and four unaffected members of the family and subjected to amplification of the entire coding sequence and splicing sites of USH2A gene. Mutation detection was conducted by direct sequencing of the PCR products. A total of 100 normal unrelated individuals were used as controls. The patients were identified to be a compound heterozygote for two mutations: c.8272G>T (p.E2758X) in exon 42 from his mother and c.12376-12378ACT>TAA(p.T4126X) in exon 63 of the USH2A gene from his father. Both mutations were not found in either of the two unaffected family members or 100 unrelated controls, and had completely co-segregated with the disease phenotype in the family. Neither mutation has been reported in the HGMD database. The novel compound heterozygous mutations c.8272G>T and c.12376-12378ACT>TAA within the USH2A gene may be responsible for the disease. This result may provide new clues for molecular diagnosis of this disease.

  20. Lack of association between urotensin-II (UTS2 gene polymorphisms (Thr21Met and Ser89Asn and migraine

    Directory of Open Access Journals (Sweden)

    Betül Ozan

    2017-08-01

    Full Text Available Migraine is a common neurovascular brain disorder with heterogeneous clinical presentation, including recurrent headache attacks. The pathophysiology of migraine is complex, and a number of genomic regions have been associated with the development of migraine. In this study, we analyzed the allele and genotype frequencies of the urotensin-II gene (UTS2 polymorphisms, Thr21Met and Ser89Asn, among Turkish patients with migraine. A total of 146 patients with migraine (14 with aura [MA group] and 132 without aura [MO group] were genotyped for Thr21Met and Ser89Asn polymorphisms and compared with 154 age- and sex-matched healthy controls. The UTS2 gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. No significant differences were observed in allele and genotype frequencies for Thr21Met and Ser89Asn polymorphisms between the patients with migraine and control group. Similarly, we did not observe significant differences in allele and genotype frequencies between MA and MO and control group. Moreover, the haplotype analysis showed no association between UTS2 gene haplotypes (MN, MS, TN, and TS and migraine. In summary, Thr21Met and Ser89Asn polymorphisms of the UTS2 gene are not risk factors for migraine in our sample of Turkish migraine patients.

  1. Gene mapping of the Usher syndromes.

    Science.gov (United States)

    Kimberling, W; Smith, R J

    1992-10-01

    USH is an autosomal recessive group of diseases characterized by auditory impairment and visual loss owing to RP. Two common types of USH are known, types I and II. USH type I is characterized by a congenital severe to profound hearing impairment, absent vestibular function, and a progressive pigmentary retinopathy. Persons with type I do not find hearing aids useful, have delayed motor development, and experience progressive night blindness and peripheral visual loss, which usually begins in their second decade. USH type II is characterized by a congenital moderate to severe hearing loss with a down-sloping audiogram, normal vestibular function, and a progressive pigmentary retinopathy. Persons with USH2 find hearing aids beneficial, have normal psychomotor development, and experience progressive night blindness and peripheral visual loss, which usually begins in their third decade. Vestibular dysfunction is the best distinguishing hallmark to differentiate USH type I from type II. One USH type II gene (called USH2) has been assigned to chromosome 1q. One USH type I gene has been tentatively assigned to chromosome 14q. There are other USH genes that have not yet been localized.

  2. Experiment list: SRX1098180 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ensitivity source_name=Cultured K562 cells || transduced gene=dCas9-KRAB || grna target=globin HS2 enhancer ...is=Leukemia Chronic Myelogenous 106502015,0.0,52.4,0 GSM1824418: dCas9 KRAB rep2; Homo sapiens; DNase-Hypers

  3. Fine-scale mapping of the 4q24 locus identifies two independent loci associated with breast cancer risk

    DEFF Research Database (Denmark)

    Guo, Xingyi; Long, Jirong; Zeng, Chenjie

    2015-01-01

    or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor-binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150...

  4. Fowlpoxvirus recombinants coding for the CIITA gene increase the expression of endogenous MHC-II and Fowlpox Gag/Pro and Env SIV transgenes.

    Science.gov (United States)

    Bissa, Massimiliano; Forlani, Greta; Zanotto, Carlo; Tosi, Giovanna; De Giuli Morghen, Carlo; Accolla, Roberto S; Radaelli, Antonia

    2018-01-01

    A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8+ T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4+ T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can enhance the levels of the gag/pro and env gene products only when infection is performed by FP single recombinants. Also, CIITA expression is higher when carried by FP single recombinants than when combined with FPgp or FPenv constructs and can

  5. Association between single nucleotide polymorphism of apoVLDL-II ...

    African Journals Online (AJOL)

    ONOS

    2010-07-05

    Jul 5, 2010 ... Very low density apolipoprotein-II (apoVLDL-II) is a major polypeptide component of avian VLDL. The function of apoVLDL-II is the transport of neutral lipids (triacylglycerol) in the form of VLDL in the plasma. The apoVLDLII gene is dormant in embryos, chicks and roosters but can be activated by estrogen.

  6. The Evaluation of IL6 and ESR1 Gene Polymorphisms in Primary Dysmenorrhea.

    Science.gov (United States)

    Ozsoy, Asker Zeki; Karakus, Nevin; Yigit, Serbulent; Cakmak, Bulent; Nacar, Mehmet Can; Yılmaz Dogru, Hatice

    2016-01-01

    Primary dysmenorrhea is the most common gynecological complaint with painful menstrual cramps in pelvis without any pathology. It affects about half of menstruating women, and it causes significant disruption in quality of life. We investigated the association between IL6 gene promoter and ESR1 gene XbaI and PvuII polymorphisms and primary dysmenorrhea. In this case-control study, 152 unrelated young women with primary dysmenorrhea and 150 unrelated healthy age-matched controls participated. Genomic DNA was isolated and IL6 and ESR1 gene polymorphisms were genotyped using PCR-based RFLP assay. The distribution of genotype and allele frequencies of IL6 gene promoter and ESR1 gene XbaI polymorphisms were not statistically different between patients and controls (p > 0.05). However, the genotype and allele frequencies of ESR1 gene PvuII polymorphism showed statistically significant differences between primary dysmenorrhea patients and controls (p = 0.009 and p = 0.021, respectively). Statistically significant associations were also observed between age and married status of primary dysmenorrhea patients and ESR1 gene PvuII polymorphism (p = 0.044 and p = 0.023, respectively). In combined genotype analyses, AG at ESR1 XbaI and TC at ESR1 PvuII loci encoded a p-value of 0.027. Thus, individuals who are heterozygote at both loci have a lower risk of developing primary dysmenorrhea. Our study suggests no strong association between IL6 gene promoter and ESR1 gene XbaI polymorphisms and primary dysmenorrhea in Turkish women. However, ESR1 gene PvuII polymorphism showed statistically significant differences between primary dysmenorrhea patients and controls. The potential association between ESR1 gene PvuII polymorphism and age and married status of dysmenorrhea patients deserves further consideration.

  7. Immunogenetics of rheumatoid arthritis and primary Sjögren's syndrome: DNA polymorphism of HLA class II genes

    DEFF Research Database (Denmark)

    Morling, Niels; Andersen, V; Fugger, L

    1992-01-01

    . The frequencies of DNA fragments associated with the following HLA class II genes were increased in RA when compared to normal controls: DRB1*04 (DR4) (relative risk, RR = 7.4, P less than 10(-3), DRB4*0101 (DRw53) (RR = 9.6, P less than 10(-3), DQA1*0301 (RR = 9.6, P less than 10(-3), DQB1*0301 (DQw7) (RR = 2.......8, P less than 0.05, 'corrected' P greater than 0.05), and DQB1*0302 (DQw8) (RR = 4.5, P less than 10(-2). Negative associations were found between RA and DRB1*1501 (DR2/DRw15) (RR = 0.2, P less than 10(-2) and DQB1*0602 (DQw6) (RR = 0.2, P less than 10(-2), 'corrected' P greater than 0.......05). The frequencies in RA of other HLA class II associated DNA fragments including DPA and DPB and the antigens DPw1-w6 defined by primed lymphocyte stimulation, did not differ significantly from those in controls. In primary SS, the frequency of HLA-B8 was significantly increased (RR = 9.0, P less than 10...

  8. Association of variants in genes related to the immune response and obesity with BPH in CLUE II.

    Science.gov (United States)

    Lopez, D S; Peskoe, S B; Tsilidis, K K; Hoffman-Bolton, J; Helzlsouer, K J; Isaacs, W B; Smith, M W; Platz, E A

    2014-12-01

    Chronic inflammation and obesity may contribute to the genesis or progression of BPH and BPH-associated lower urinary tract symptoms (LUTS). The influence of variants in genes related to these states on BPH has not been studied extensively. Thus, we evaluated the association of 17 single-nucleotide polymorphisms (SNPs) in immune response genes (IL1B, IL6, IL8, IL10, TNF, CRP, TLR4 and RNASEL) and genes involved in obesity, including insulin regulation (LEP, ADIPOQ, PPARG and TCF7L2), with BPH. BPH cases (N = 568) and age-frequency matched controls (N=568) were selected from among adult male CLUE II cohort participants who responded in 2000 to a mailed questionnaire. BPH was defined as BPH surgery, use of BPH medications or symptomatic BPH (American Urological Association Symptom Index Score ⩾ 15). Controls were men who had not had BPH surgery, did not use BPH medications and whose symptom score was ⩽ 7. Age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression. None of the candidate SNPs was statistically significantly associated with BPH. However, we could not rule out possible weak associations for CRP rs1205 (1082C>T), ADIPOQ rs1501299 (276C>A), PPARG rs1801282 (-49C>G) and TCF7L2 rs7903146 (47833T>C). After summing risk alleles, men with ⩾ 4 had an increased BPH risk compared with those with ⩽ 1 (OR, 1.78; 95% CI, 1.10-2.89; P(trend) = 0.006). SNPs in genes related to immune response and obesity, especially in combination, may be associated with BPH.

  9. Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

    Directory of Open Access Journals (Sweden)

    Reza Saberianfar

    Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

  10. Biologic determinants of tumor recurrence in stage II colon cancer: validation study of the 12-gene recurrence score in cancer and leukemia group B (CALGB) 9581.

    Science.gov (United States)

    Venook, Alan P; Niedzwiecki, Donna; Lopatin, Margarita; Ye, Xing; Lee, Mark; Friedman, Paula N; Frankel, Wendy; Clark-Langone, Kim; Millward, Carl; Shak, Steven; Goldberg, Richard M; Mahmoud, Najjia N; Warren, Robert S; Schilsky, Richard L; Bertagnolli, Monica M

    2013-05-10

    A greater understanding of the biology of tumor recurrence should improve adjuvant treatment decision making. We conducted a validation study of the 12-gene recurrence score (RS), a quantitative assay integrating stromal response and cell cycle gene expression, in tumor specimens from patients enrolled onto Cancer and Leukemia Group B (CALGB) 9581. CALGB 9581 randomly assigned 1,713 patients with stage II colon cancer to treatment with edrecolomab or observation and found no survival difference. The analysis reported here included all patients with available tissue and recurrence (n = 162) and a random (approximately 1:3) selection of nonrecurring patients. RS was assessed in 690 formalin-fixed paraffin-embedded tumor samples with quantitative reverse transcriptase polymerase chain reaction by using prespecified genes and a previously validated algorithm. Association of RS and recurrence was analyzed by weighted Cox proportional hazards regression. Continuous RS was significantly associated with risk of recurrence (P = .013) as was mismatch repair (MMR) gene deficiency (P = .044). In multivariate analyses, RS was the strongest predictor of recurrence (P = .004), independent of T stage, MMR, number of nodes examined, grade, and lymphovascular invasion. In T3 MMR-intact (MMR-I) patients, prespecified low and high RS groups had average 5-year recurrence risks of 13% (95% CI, 10% to 16%) and 21% (95% CI, 16% to 26%), respectively. The 12-gene RS predicts recurrence in stage II colon cancer in CALGB 9581. This is consistent with the importance of stromal response and cell cycle gene expression in colon tumor recurrence. RS appears to be most discerning for patients with T3 MMR-I tumors, although markers such as grade and lymphovascular invasion did not add value in this subset of patients.

  11. Type II cytokeratin gene expression is indicative of early cell differentiation in the chick embryo

    International Nuclear Information System (INIS)

    Charlebois, T.S.

    1988-01-01

    Embryonic development in vertebrates appears to involve a series of inductive tissue interactions that lead to regional specializations, which eventually become elaborated in the basic body plan of the embryo. The inductive interactions leading to early regionalization of the embryo are often particularly difficult to evaluate because of the absence of available morphological or biochemical evidence that such events have occurred. In the 36 hour chick embryo, the regional subdivision of the early ectoderm is evidence by a marked lens-forming bias in the head ectoderm, which is absent in the presumptive dorsal epidermis of the trunk region. As a strategy for isolating genes whose differential expression might reflect this regional subdivision, a cDNA library from 36 hour embryos was prepared and screened for differential hybridization to [ 32 P]cDNA probes synthesized using template RNA isolated from 36 hour head ectoderm and trunk ectoderm. A cDNA clone (T4) was isolated which hybridizes to transcripts present at much higher levels in trunk ectoderm than in head ectoderm. Partial nucleotide and deduced amino acid sequences of this clone indicate that it represents a gene encoding a type II cytokeratin. The distribution of transcripts complementary to the T4 probe was evaluated in early embryos using RNA gel blot analysis and in situ hybridization to tissue sections

  12. A Novel Frameshift Mutation of the USH2A Gene in a Korean Patient with Usher Syndrome Type II.

    Science.gov (United States)

    Boo, Sung Hyun; Song, Min-Jung; Kim, Hee-Jin; Cho, Yang-Sun; Chu, Hosuk; Ko, Moon-Hee; Chung, Won-Ho; Kim, Jong-Won; Hong, Sung Hwa

    2013-03-01

    Usher syndrome type II (USH2) is the most common form of Usher syndrome, characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa. It has been shown that mutations in the USH2A gene are responsible for USH2. The authors herein describe a 34-year-old Korean woman with the typical clinical manifestation of USH2; she had bilateral hearing disturbance and progressive visual deterioration, without vestibular dysfunction. Molecular genetic study of the USH2A gene revealed a novel frameshift mutation (c.2310delA; Glu771LysfsX17). She was heterozygous for this mutation, and no other mutation was found in USH2A, suggesting the possibility of an intronic or large genomic rearrangement mutation. To the best of our knowledge, this is the first report of a genetically confirmed case of USH2 in Korea. More investigations are needed to delineate genotype-phenotype correlations and ethnicity-specific genetic background of Usher syndrome.

  13. Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60.

    Science.gov (United States)

    Aoyagi, N; Wassarman, D A

    2001-10-01

    In vitro, the TAF(II)60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF(II)60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF(II)60 by analyzing four independent Drosophila melanogaster TAF(II)60 mutants. Loss-of-function mutations in Drosophila TAF(II)60 result in lethality, indicating that TAF(II)60 provides a nonredundant function in vivo. Molecular analysis of TAF(II)60 alleles revealed that essential TAF(II)60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF(II)60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF(II)60 from a transgene rescued the lethality of TAF(II)60 mutants and exposed requirements for TAF(II)60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF(II)60 mutant flies implicate TAF(II)60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF(II)60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF(II)60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF(II) proteins.

  14. Loss of Sfpq Causes Long-Gene Transcriptopathy in the Brain

    Directory of Open Access Journals (Sweden)

    Akihide Takeuchi

    2018-05-01

    Full Text Available Summary: Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases. : It has been a long-standing question how mammalian neuronal cells achieve full gene length transcription of extra-long genes. Takeuchi et al. show that RNA-binding protein Sfpq sustains long-gene transcription through Pol II-CTD activation. Loss of Sfpq caused long-gene transcriptopathy, which could be the cause of neurodegenerative and psychiatric disorders. Keywords: RNA-binding protein, transcriptional regulation, RNA polymerase II, cyclin-dependent kinase 9, RBP/transcript-dependent elongation, long-gene transcriptotherapy, neuronal development, neurological and psychiatric diseases, long-gene diseases, long genopathies

  15. Individual members of the light-harvesting complex II chlorophyll a/b-binding protein gene family in pea (Pisum sativum) show differential responses to ultraviolet-B radiation

    International Nuclear Information System (INIS)

    Mackerness, A.H.S.; Liu, L.; Thomas, B.; Thompson, W.F.; Jordan, B.R.; White, M.J.

    1998-01-01

    In the present work, UV-B-repressible and UV-B-inducible genes were identified in the pea, Pisum sativum L., by rapid amplification of 3′ cDNA ends through use of the polymerase chain reaction. Of the UV-B-repressible clones, psUVRub and psUVDeh represent genes encoding Rubisco activase and dehydrin, respectively. A third clone, psUVZinc, did not correspond closely in overall nucleotide sequence to any gene registered in GenBank; however, a short deduced peptide shared similarity with the photosystem-II reaction center X protein of the chlorophyll a+c-containing alga, Odontella sinensis. The UV-B-inducible clones, psUVGluc, psUVAux and psUVRib, were related to genes encoding β-1, 3-glucanase, auxin-repressed protein, and a 40S ribosomal protein, respectively. The modulation of these pea genes indicates how UV-B, through its actions as a physical stressor, affects several important physiological processes in plants. (author)

  16. Genetic variation of the major histocompatibility complex (MHC class II B gene in the threatened Hume's pheasant, Syrmaticus humiae.

    Directory of Open Access Journals (Sweden)

    Weicai Chen

    Full Text Available Major histocompatibility complex (MHC genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae, which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

  17. Heterozygous deletion at the SOX10 gene locus in two patients from a Chinese family with Waardenburg syndrome type II.

    Science.gov (United States)

    Wenzhi, He; Ruijin, Wen; Jieliang, Li; Xiaoyan, Ma; Haibo, Liu; Xiaoman, Wang; Jiajia, Xian; Shaoying, Li; Shuanglin, Li; Qing, Li

    2015-10-01

    Waardenburg syndrome (WS) is a rare disease characterized by sensorineural deafness and pigment disturbance. To date, almost 100 mutations have been reported, but few reports on cases with SOX10 gene deletion. The inheritance pattern of SOX10 gene deletion is still unclear. Our objective was to identify the genetic causes of Waardenburg syndrome type II in a two-generation Chinese family. Clinical evaluations were conducted in both of the patients. Microarray analysis and multiplex ligation-dependent probe amplification (MLPA) were performed to identify disease-related copy number variants (CNVs). DNA sequencing of the SOX10, MITF and SNAI2 genes was performed to identify the pathogenic mutation responsible for WS2. A 280kb heterozygous deletion at the 22q13.1 chromosome region (including SOX10) was detected in both of the patients. No mutation was found in the patients, unaffected family members and 30 unrelated healthy controls. This report is the first to describe SOX10 heterozygous deletions in Chinese WS2 patients. Our result conform the thesis that heterozygous deletions at SOX10 is an important pathogenicity for WS, and present as autosomal dominant inheritance. Nevertheless, heterozygous deletion of the SOX10 gene would be worth investigating to understand their functions and contributions to neurologic phenotypes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent, high-level megakaryocyte expression.

    Science.gov (United States)

    Thornton, Michael A; Zhang, Chunyan; Kowalska, Maria A; Poncz, Mortimer

    2002-11-15

    The alphaIIb/beta3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of alphaIIb, and studies of the alphaIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the alphaIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) alphaIIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the alphaIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the h(alpha)IIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the h(alpha)IIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of h(alpha)IIb mRNA that were about 30% of the native m(alpha)IIb mRNA level. These constructs also resulted in detectable h(alpha)IIb/m(beta)3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the alphaIIb gene for tissue specificity, but also characterizes the distal organization of the alphaIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the alphaIIb gene at high levels during megakaryopoiesis.

  19. Relationship between tumor gene expression and recurrence in four independent studies of patients with stage II/III colon cancer treated with surgery alone or surgery plus adjuvant fluorouracil plus leucovorin.

    Science.gov (United States)

    O'Connell, Michael J; Lavery, Ian; Yothers, Greg; Paik, Soonmyung; Clark-Langone, Kim M; Lopatin, Margarita; Watson, Drew; Baehner, Frederick L; Shak, Steven; Baker, Joffre; Cowens, J Wayne; Wolmark, Norman

    2010-09-01

    These studies were conducted to determine the relationship between quantitative tumor gene expression and risk of cancer recurrence in patients with stage II or III colon cancer treated with surgery alone or surgery plus fluorouracil (FU) and leucovorin (LV) to develop multigene algorithms to quantify the risk of recurrence as well as the likelihood of differential treatment benefit of FU/LV adjuvant chemotherapy for individual patients. We performed quantitative reverse transcription polymerase chain reaction (RT-qPCR) on RNA extracted from fixed, paraffin-embedded (FPE) tumor blocks from patients with stage II or III colon cancer who were treated with surgery alone (n = 270 from National Surgical Adjuvant Breast and Bowel Project [NSABP] C-01/C-02 and n = 765 from Cleveland Clinic [CC]) or surgery plus FU/LV (n = 308 from NSABP C-04 and n = 508 from NSABP C-06). Overall, 761 candidate genes were studied in C-01/C-02 and C-04, and a subset of 375 genes was studied in CC/C-06. A combined analysis of the four studies identified 48 genes significantly associated with risk of recurrence and 66 genes significantly associated with FU/LV benefit (with four genes in common). Seven recurrence-risk genes, six FU/LV-benefit genes, and five reference genes were selected, and algorithms were developed to identify groups of patients with low, intermediate, and high likelihood of recurrence and benefit from FU/LV. RT-qPCR of FPE colon cancer tissue applied to four large independent populations has been used to develop multigene algorithms for estimating recurrence risk and benefit from FU/LV. These algorithms are being independently validated, and their clinical utility is being evaluated in the Quick and Simple and Reliable (QUASAR) study.

  20. TALE-PvuII fusion proteins--novel tools for gene targeting.

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  1. The WRKY Transcription Factor Genes in Lotus japonicus.

    Science.gov (United States)

    Song, Hui; Wang, Pengfei; Nan, Zhibiao; Wang, Xingjun

    2014-01-01

    WRKY transcription factor genes play critical roles in plant growth and development, as well as stress responses. WRKY genes have been examined in various higher plants, but they have not been characterized in Lotus japonicus. The recent release of the L. japonicus whole genome sequence provides an opportunity for a genome wide analysis of WRKY genes in this species. In this study, we identified 61 WRKY genes in the L. japonicus genome. Based on the WRKY protein structure, L. japonicus WRKY (LjWRKY) genes can be classified into three groups (I-III). Investigations of gene copy number and gene clusters indicate that only one gene duplication event occurred on chromosome 4 and no clustered genes were detected on chromosomes 3 or 6. Researchers previously believed that group II and III WRKY domains were derived from the C-terminal WRKY domain of group I. Our results suggest that some WRKY genes in group II originated from the N-terminal domain of group I WRKY genes. Additional evidence to support this hypothesis was obtained by Medicago truncatula WRKY (MtWRKY) protein motif analysis. We found that LjWRKY and MtWRKY group III genes are under purifying selection, suggesting that WRKY genes will become increasingly structured and functionally conserved.

  2. Amphiphilic graft copolymer based on poly(styrene-co-maleic anhydride with low molecular weight polyethylenimine for efficient gene delivery

    Directory of Open Access Journals (Sweden)

    Duan XP

    2012-09-01

    Full Text Available Xiaopin Duan,1,2 Jisheng Xiao,2 Qi Yin,2 Zhiwen Zhang,2 Shirui Mao,1 Yaping Li21School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 2Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, ChinaBackground and methods: A new amphiphilic comb-shaped copolymer (SP was synthesized by conjugating poly(styrene-co-maleic anhydride with low molecular weight polyethyleneimine for gene delivery. Fourier transform infrared spectrum, 1H nuclear magnetic resonance, and gel permeation chromatography were used to characterize the graft copolymer.Results: The buffering capability of SP was similar to that of polyethyleneimine within the endosomal pH range. The copolymer could condense DNA effectively to form complexes with a positive charge (13–30 mV and a small particle size (130–200 nm at N/P ratios between 5 and 20, and protect DNA from degradation by DNase I. In addition, SP showed much lower cytotoxicity than polyethyleneimine 25,000. Importantly, the gene transfection activity and cellular uptake of SP-DNA complexes were all markedly higher than that of complexes of polyethyleneimine 25,000 and DNA in MCF-7 and MCF-7/ADR cell lines.Conclusion: This work highlights the promise of SP as a safe and efficient synthetic vector for DNA delivery.Keywords: poly(styrene-co-maleic anhydride, polyethylenimine, DNA, gene delivery

  3. Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins.

    Science.gov (United States)

    Sallaberry-Pincheira, Nicole; González-Acuña, Daniel; Padilla, Pamela; Dantas, Gisele P M; Luna-Jorquera, Guillermo; Frere, Esteban; Valdés-Velásquez, Armando; Vianna, Juliana A

    2016-10-01

    The evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans-species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long-term survival of the species.

  4. Maximizing biomarker discovery by minimizing gene signatures

    Directory of Open Access Journals (Sweden)

    Chang Chang

    2011-12-01

    Full Text Available Abstract Background The use of gene signatures can potentially be of considerable value in the field of clinical diagnosis. However, gene signatures defined with different methods can be quite various even when applied the same disease and the same endpoint. Previous studies have shown that the correct selection of subsets of genes from microarray data is key for the accurate classification of disease phenotypes, and a number of methods have been proposed for the purpose. However, these methods refine the subsets by only considering each single feature, and they do not confirm the association between the genes identified in each gene signature and the phenotype of the disease. We proposed an innovative new method termed Minimize Feature's Size (MFS based on multiple level similarity analyses and association between the genes and disease for breast cancer endpoints by comparing classifier models generated from the second phase of MicroArray Quality Control (MAQC-II, trying to develop effective meta-analysis strategies to transform the MAQC-II signatures into a robust and reliable set of biomarker for clinical applications. Results We analyzed the similarity of the multiple gene signatures in an endpoint and between the two endpoints of breast cancer at probe and gene levels, the results indicate that disease-related genes can be preferably selected as the components of gene signature, and that the gene signatures for the two endpoints could be interchangeable. The minimized signatures were built at probe level by using MFS for each endpoint. By applying the approach, we generated a much smaller set of gene signature with the similar predictive power compared with those gene signatures from MAQC-II. Conclusions Our results indicate that gene signatures of both large and small sizes could perform equally well in clinical applications. Besides, consistency and biological significances can be detected among different gene signatures, reflecting the

  5. Rekombinant humant deoxyribonuklease til andet end cystisk fibrose

    DEFF Research Database (Denmark)

    Kristensen, Kim

    2010-01-01

    be associated with increased need for supplemental oxygen. In adults with idiopathic bronchiectasis, treatment with rhDNase leads to more pulmonary exacerbations and a greater decline in pulmonary function tests. There are no controlled studies on rhDNase in primary ciliary dyskinesia or atelectasis.......Recombinant human DNase (rhDNase) reduces viscosity of sputum. Effect has been documented in cystic fibrosis and postoperatively in paediatric heart disease. Single dose treatment with rhDNase in paediatric asthma has no effect. In respiratory syncytial virus infection, treatment with rhDNase may...

  6. Treatment of groundwater containing Mn(II), Fe(II), As(III) and Sb(III) by bioaugmented quartz-sand filters.

    Science.gov (United States)

    Bai, Yaohui; Chang, Yangyang; Liang, Jinsong; Chen, Chen; Qu, Jiuhui

    2016-12-01

    High concentrations of iron (Fe(II)) and manganese (Mn(II)) often occur simultaneously in groundwater. Previously, we demonstrated that Fe(II) and Mn(II) could be oxidized to biogenic Fe-Mn oxides (BFMO) via aeration and microbial oxidation, and the formed BFMO could further oxidize and adsorb other pollutants (e.g., arsenic (As(III)) and antimony (Sb(III))). To apply this finding to groundwater remediation, we established four quartz-sand columns for treating groundwater containing Fe(II), Mn(II), As(III), and Sb(III). A Mn-oxidizing bacterium (Pseudomonas sp. QJX-1) was inoculated into two parallel bioaugmented columns. Long-term treatment (120 d) showed that bioaugmentation accelerated the formation of Fe-Mn oxides, resulting in an increase in As and Sb removal. The bioaugmented columns also exhibited higher overall treatment effect and anti-shock load capacity than that of the non-bioaugmented columns. To clarify the causal relationship between the microbial community and treatment effect, we compared the biomass of active bacteria (reverse-transcribed real-time PCR), bacterial community composition (Miseq 16S rRNA sequencing) and community function (metagenomic sequencing) between the bioaugmented and non-bioaugmented columns. Results indicated that the QJX1 strain grew steadily and attached onto the filter material surface in the bioaugmented columns. In general, the inoculated strain did not significantly alter the composition of the indigenous bacterial community, but did improve the relative abundances of xenobiotic metabolism genes and Mn oxidation gene. Thus, bioaugmentation intensified microbial degradation/utilization for the direct removal of pollutants and increased the formation of Fe-Mn oxides for the indirect removal of pollutants. Our study provides an alternative method for the treatment of groundwater containing high Fe(II), Mn(II) and As/Sb. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Enhanced radiation response in radioresistant MCF-7 cells by targeting peroxiredoxin II

    Directory of Open Access Journals (Sweden)

    Diaz AJG

    2013-10-01

    Full Text Available Anthony Joseph Gomez Diaz,1 Daniel Tamae,2 Yun Yen,3 JianJian Li,4 Tieli Wang1 1Department of Chemistry and Biochemistry, California State University at Dominguez Hills, Carson, CA, 2Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, 3Department of Clinical and Molecular Pharmacology, Beckman Research Institute of City of Hope National Medical Center, Duarte, CA, 4Department of Radiation Oncology, University of California Davis, Sacramento, CA, USA Abstract: In our previous study, we identified that a protein target, peroxiredoxin II (PrxII, is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells, and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology's property of targeting to specific cancer-related genes. Keywords: siRNA, PrxII, radiation resistance, Ca2+, MCF+FIR3

  8. Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

    Energy Technology Data Exchange (ETDEWEB)

    Serwer, Philip, E-mail: serwer@uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Wright, Elena T. [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Liu, Zheng; Jiang, Wen [Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-05-15

    DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.

  9. DOP-PCR

    African Journals Online (AJOL)

    Dr Manal Shalaby

    2012-02-07

    Feb 7, 2012 ... 2A genes from these eukaryotic organisms. Moreover, we amplified 1070 and ... finally a number for each individual protein. Although, the ... Total RNA was extracted using the RNeasy Mini Kit (Qiagen), with a DNase digestion step, according to the manufacturer's instructions. Elution was performed with 50 ...

  10. Angiotensin II facilitates breast cancer cell migration and metastasis.

    Directory of Open Access Journals (Sweden)

    Sylvie Rodrigues-Ferreira

    Full Text Available Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

  11. Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

    Science.gov (United States)

    Jindra, Peter T.; Tripathi, Sudipta; Tian, Chaorui; Iacomini, John; Bagley, Jessamyn

    2012-01-01

    Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction. PMID:22833118

  12. Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

    Science.gov (United States)

    Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

    2000-07-01

    A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

  13. Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

    Science.gov (United States)

    Piazza, Carol Lyn; Smith, Dorie

    2018-01-01

    Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

  14. Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast*

    Science.gov (United States)

    Ansari, Suraiya A.; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z.; Rode, Kara A.; Barber, Wesley T.; Ellis, Laura C.; LaPorta, Erika; Orzechowski, Amanda M.; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H.

    2014-01-01

    Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

  15. The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN

    Science.gov (United States)

    Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  16. Virulence Gene Pool Detected in Bovine Group C Streptococcus dysgalactiae subsp. dysgalactiae Isolates by Use of a Group A S. pyogenes Virulence Microarray ▿

    Science.gov (United States)

    Rato, Márcia G.; Nerlich, Andreas; Bergmann, René; Bexiga, Ricardo; Nunes, Sandro F.; Vilela, Cristina L.; Santos-Sanches, Ilda; Chhatwal, Gursharan S.

    2011-01-01

    A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans. PMID:21525223

  17. Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells.

    Science.gov (United States)

    Cavalieri, Vincenzo; Bernardo, Maria Di; Spinelli, Giovanni

    2007-01-01

    PlOtp (Orthopedia), a homeodomain-containing transcription factor, has been recently characterized as a key regulator of the morphogenesis of the skeletal system in the embryo of the sea urchin Paracentrotus lividus. Otp acts as a positive regulator in a subset of oral ectodermal cells which transmit short-range signals to the underlying primary mesenchyme cells where skeletal synthesis is initiated. To shed some light on the molecular mechanisms involved in such a process, we begun a functional analysis of the cis-regulatory sequences of the Otp gene. Congruent with the spatial expression profile of the endogenous Otp gene, we found that while a DNA region from -494 to +358 is shown to drive in vivo GFP reporter expression in the oral ectoderm, but also in the foregut, a larger region spanning from -2044 to +358 is needed to give firmly established tissue specificity. Microinjection of PCR-amplified DNA constructs, truncated in the 5' regulatory region, and determination of GFP mRNA level in injected embryos allowed the identification of a 5'-flanking fragment of 184bp in length, essential for expression of the transgene in the oral ectoderm of pluteus stage embryos. Finally, we conducted DNAse I-footprinting assays in nuclear extracts for the 184bp region and detected two protected sequences. Data bank search indicates that these sites contain consensus binding sites for transcription factors.

  18. Genetics Home Reference: mucolipidosis II alpha/beta

    Science.gov (United States)

    ... Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-hurler polydystrophy) are caused by mutations in ... N-acetylglucosamine-1-phosphotransferase gene (GNPTAB) in a French Canadian founder population. Clin Genet. 2008 Mar;73( ...

  19. Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

    Directory of Open Access Journals (Sweden)

    Kathrin Davari

    2017-04-01

    Full Text Available Summary: Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation. : Davari et al. visualize global changes in RNA Pol II binding, transcription, splicing, and translation. T cells change their functional program by rapid de novo recruitment of RNA Pol II and coupled changes in transcription and translation. This coincides with fluctuations in RNA Pol II phosphorylation and a temporary reduction in cotranscriptional splicing. Keywords: RNA Pol II, cotranscriptional splicing, T cell activation, ribosome profiling, 4sU, H3K36, Ser-5 RNA Pol II, Ser-2 RNA Pol II, immune response, immediate-early genes

  20. Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II in Erlang Mountainous Chickens

    Directory of Open Access Journals (Sweden)

    H. D. Yin

    2014-03-01

    Full Text Available Cellular retinol-binding protein II (CRBP II belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05 in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05 in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.

  1. Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription.

    Science.gov (United States)

    Bauer, David L V; Tellier, Michael; Martínez-Alonso, Mónica; Nojima, Takayuki; Proudfoot, Nick J; Murphy, Shona; Fodor, Ervin

    2018-05-15

    Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Piper betle Induced Cytoprotective Genes and Proteins via the Nrf2/ARE Pathway in Aging Mice.

    Science.gov (United States)

    Aliahmat, Nor Syahida; Abdul Sani, Nur Fathiah; Wan Hasan, Wan Nuraini; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2016-01-01

    The objective of this study was to elucidate the underlying antioxidant mechanism of aqueous extract of Piper betle (PB) in aging rats. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE pathway involving phase II detoxifying and antioxidant enzymes plays an important role in the antioxidant system by reducing electrophiles and reactive oxygen species through induction of phase II enzymes and proteins. Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis. PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes. PB was testified for the first time to induce cytoprotective genes through the Nrf2/ARE signaling pathway, thus unraveling the antioxidant mechanism of PB during the aging process. © 2016 S. Karger AG, Basel.

  3. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria

    International Nuclear Information System (INIS)

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina

    2015-01-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3′)-IIa/nptII and aph(3′)-IIIa/nptIII – frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides – was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31–856) and 85% for nptIII (1190 copies/g soil; 13–61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0–3.3%) were positive for nptIII, none for nptII (0–0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. - Highlights: • ARM genes may act as environmental pollutants under certain conditions. • Vital criteria for rating are low endemic presence and anthropogenic ARG immission. • Agricultural soils were rarely positive for nptII with few gene copy numbers. • Most fields were nptIII positive with variable but also increased allele frequency. • NptII/III qualify as pollutants in the tested settings with low endemic abundances. - ARM genes may be considered as environmental pollutants if anthropogenic activities raise their abundance above naturally occurring background levels in exposed ecosystems.

  4. Interplay of bistable kinetics of gene expression during cellular growth

    International Nuclear Information System (INIS)

    Zhdanov, Vladimir P

    2009-01-01

    In cells, the bistable kinetics of gene expression can be observed on the level of (i) one gene with positive feedback between protein and mRNA production, (ii) two genes with negative mutual feedback between protein and mRNA production, or (iii) in more complex cases. We analyse the interplay of two genes of type (ii) governed by a gene of type (i) during cellular growth. In particular, using kinetic Monte Carlo simulations, we show that in the case where gene 1, operating in the bistable regime, regulates mutually inhibiting genes 2 and 3, also operating in the bistable regime, the latter genes may eventually be trapped either to the state with high transcriptional activity of gene 2 and low activity of gene 3 or to the state with high transcriptional activity of gene 3 and low activity of gene 2. The probability to get to one of these states depends on the values of the model parameters. If genes 2 and 3 are kinetically equivalent, the probability is equal to 0.5. Thus, our model illustrates how different intracellular states can be chosen at random with predetermined probabilities. This type of kinetics of gene expression may be behind complex processes occurring in cells, e.g., behind the choice of the fate by stem cells

  5. Dilatative uropathy as a manifestation of neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin-II gene.

    Science.gov (United States)

    Lindenthal, V; Mainberger, A; Morris-Rosendahl, D J; Löning, L; Mayer, W; Müller, H L

    2013-12-01

    Polydypsia and polyuria are frequent symptoms in patients with sellar masses caused by neurohypophyseal diabetes insipidus. Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a disorder caused by mutations in the arginine vasopressin (AVP) -neurophysin II (NPII) gene, should be considered as a rare differential diagnosis. A delayed diagnosis bears the risk of life-threatening electrolyte imbalances and permanent urinary tract damage, leading to impaired quality of life.We present a Caucasian kindred of at least 4 generations with FNDI.Clinical histories, endocrine para-meters, and results of molecular analyses of the AVP gene are presented with a review of the literature on diabetes insipidus (DI) related urinary tract dilatation.Polyuria and polydipsia were only reported based on explicit and thorough interrogation after more than 4 years of clinical follow-up. A novel heterozygous mutation in the AVP gene was found in all examined symptomatic subjects (c.1-33_c.4del37nt). A literature review revealed that non-obstructive hydronephrosis (NOH) is a rare but known complication of DI.Since increased fluid intake is often a typical familial pattern in adFNDI, it is frequently missed as being pathologic in affected patients, therefore a detailed clinical history of drinking volumes is of critical importance. AVP gene testing is an important component in the confirmation of the diagnosis. Otherwise unexplainable NOH should lead to further investigations and evaluation of rare diseases like FNDI. © Georg Thieme Verlag KG Stuttgart · New York.

  6. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

    Science.gov (United States)

    Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

    2004-06-01

    The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (PHHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

  7. TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

  8. Deciphering the fine nucleotide diversity of full HLA class I and class II genes in a well-documented population from sub-Saharan Africa.

    Science.gov (United States)

    Goeury, T; Creary, L E; Brunet, L; Galan, M; Pasquier, M; Kervaire, B; Langaney, A; Tiercy, J-M; Fernández-Viña, M A; Nunes, J M; Sanchez-Mazas, A

    2018-01-01

    With the aim to understand how next-generation sequencing (NGS) improves both our assessment of genetic variation within populations and our knowledge on HLA molecular evolution, we sequenced and analysed 8 HLA loci in a well-documented population from sub-Saharan Africa (Mandenka). The results of full-gene NGS-MiSeq sequencing compared with those obtained by traditional typing techniques or limited sequencing strategies showed that segregating sites located outside exon 2 are crucial to describe not only class I but also class II population diversity. A comprehensive analysis of exons 2, 3, 4 and 5 nucleotide diversity at the 8 HLA loci revealed remarkable differences among these gene regions, notably a greater variation concentrated in the antigen recognition sites of class I exons 3 and some class II exons 2, likely associated with their peptide-presentation function, a lower diversity of HLA-C exon 3, possibly related to its role as a KIR ligand, and a peculiar molecular diversity of HLA-A exon 2, revealing demographic signals. Based on full-length HLA sequences, we also propose that the most frequent DRB1 allele in the studied population, DRB1*13:04, emerged from an allelic conversion involving 3 potential alleles as donors and DRB1*11:02:01 as recipient. Finally, our analysis revealed a high occurrence of the DRB1*13:04-DQA1*05:05:01-DQB1*03:19 haplotype, possibly resulting from a selective sweep due to protection to Onchorcerca volvulus, a prevalent pathogen in West Africa. This study unveils highly relevant information on the molecular evolution of HLA genes in relation to their immune function, calling for similar analyses in other populations living in contrasting environments. © 2017 The Authors HLA: Immune Response Genetics Published by John Wiley & Sons Ltd.

  9. Primary structure and mapping of the hupA gene of Salmonella typhimurium.

    OpenAIRE

    Higgins, N P; Hillyard, D

    1988-01-01

    In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. C...

  10. Novel genetic markers of the carbonic anhydrase II gene associated with egg production and reproduction traits in Tsaiya ducks.

    Science.gov (United States)

    Chang, M-T; Cheng, Y-S; Huang, M-C

    2013-02-01

    In our previous cDNA microarray study, we found that the carbonic anhydrase II (CA2) gene is one of the differentially expressed transcripts in the duck isthmus epithelium during egg formation period. The aim of this study was to identify the single-nucleotide polymorphisms (SNPs) in the CA2 gene of Tsaiya ducks. The relationship of SNP genotype with egg production and reproduction traits was also investigated. A total of 317 ducks from two lines, a control line with no selection and a selected line, were employed for testing. Three SNPs (C37T, A62G and A65G) in the 3'-untranslated region of the CA2 gene were found. SNP-trait association analysis showed that SNP C37T and A62G were associated with duck egg weight besides fertility. The ducks with the CT and AG genotypes had a 1.46 and 1.62 g/egg lower egg weight as compared with ducks with the CC and AA genotypes, respectively (p ducks with CT and AG genotypes had 5.20% and 4.22% higher fertility than those with CC and AA genotypes, respectively (p duck fertility, and the diplotype H1H4 was dominant for duck fertility. These findings might provide the basis for balanced selection and may be used in marker-assisted selection to improve egg weight and fertility simultaneously in the Tsaiya ducks. © 2012 Blackwell Verlag GmbH.

  11. A gene encoding starch branching enzyme I (SBEI) in apple (Malusxdomestica, Rosaceae) and its phylogenetic relationship to Sbe genes from other angiosperms.

    Science.gov (United States)

    Han, Yuepeng; Gasic, Ksenija; Sun, Fengjie; Xu, Mingliang; Korban, Schuyler S

    2007-06-01

    An apple starch-branching enzyme SbeI gene (GenBank Accession No. DQ115404) has been isolated, cloned, and sequenced. The SbeI is a single copy gene in the apple genome, consisting of 14 exons and 13 introns, and covering 6075bp. As detected by RT-PCR, the apple SbeI is expressed at very low levels during early stages of fruit development; while, the highest levels of mRNA transcripts are observed at approximately 44 days post-pollination. Besides fruits, the apple SbeI is also expressed in buds and flowers, and very weakly in leaves. The genomic structure of SbeI in apple is strikingly similar to those reported so far in grasses (Poaceae), with exons 4 through 13 being of identical lengths in both apple and grasses. Moreover, structure similarities in exon lengths have also been detected in SbeII genes of both grasses and eudicots. These findings prompted the investigation of the evolutionary process of the Sbe gene family in angiosperms. A total of 26 Sbe sequences, representing an array of monocots and eudicots, are investigated in this study. Phylogenetic analysis has suggested that Sbe genes have duplicated into SbeI and SbeII prior to the divergence of moncots from eudicots. The SbeII gene is further duplicated into SbeIIa and SbeIIb prior to the radiation of grasses; however, it is not yet clear whether this duplication event has occurred before or after the radiation of the eudicots.

  12. Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast.

    Science.gov (United States)

    Ansari, Suraiya A; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z; Rode, Kara A; Barber, Wesley T; Ellis, Laura C; LaPorta, Erika; Orzechowski, Amanda M; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H

    2014-05-23

    Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Human insulin-like growth factor II leader 2 mediates internal initiation of translation

    DEFF Research Database (Denmark)

    Pedersen, Susanne; Christiansen, Jan; Hansen, T.O.

    2002-01-01

    Insulin-like growth factor II (IGF-II) is a fetal growth factor, which belongs to the family of insulin-like peptides. During fetal life, the IGF-II gene generates three mRNAs with different 5' untranslated regions (UTRs), but identical coding regions and 3' UTRs. We have shown previously that IG...

  14. AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I.

    Science.gov (United States)

    Bøe Wolff, A S; Oftedal, B; Johansson, S; Bruland, O; Løvås, K; Meager, A; Pedersen, C; Husebye, E S; Knappskog, P M

    2008-03-01

    Autoimmune Addison's disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed.

  15. Audiological findings in Usher syndrome types IIa and II (non-IIa).

    Science.gov (United States)

    Sadeghi, Mehdi; Cohn, Edward S; Kelly, William J; Kimberling, William J; Tranebjoerg, Lisbeth; Möller, Claes

    2004-03-01

    The aim was to define the natural history of hearing loss in Usher syndrome type IIa compared to non-IIa. People with Usher syndrome type II show moderate-to-severe hearing loss, normal balance and retinitis pigmentosa. Several genes cause Usher syndrome type II. Our subjects formed two genetic groups: (1) subjects with Usher syndrome type IIa with a mutation and/or linkage to the Usher IIa gene; (2) subjects with the Usher II phenotype with no mutation and/or linkage to the Usher IIa gene. Four hundred and two audiograms of 80 Usher IIa subjects were compared with 435 audiograms of 87 non-IIa subjects. Serial audiograms with intervals of > or = 5 years were examined for progression in 109 individuals Those with Usher syndrome type IIa had significantly worse hearing thresholds than those with non-IIa Usher syndrome after the second decade. The hearing loss in Usher syndrome type IIa was found to be more progressive, and the progression started earlier than in non-IIa Usher syndrome. This suggests an auditory phenotype for Usher syndrome type IIa that is different from that of other types of Usher syndrome II. Thus, this is to our knowledge one of the first studies showing a genotype-phenotype auditory correlation.

  16. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

    Science.gov (United States)

    Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

    2010-12-01

    Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

  17. Rhizobia with 16S rRNA and nifH similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC genes are symbionts of New Zealand Carmichaelinae.

    Directory of Open Access Journals (Sweden)

    Heng Wee Tan

    Full Text Available New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

  18. Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

    Science.gov (United States)

    Batchu, Navish Kumar; Khater, Shradha; Patil, Sonal; Nagle, Vinod; Das, Gautam; Bhadra, Bhaskar; Sapre, Ajit; Dasgupta, Santanu

    2018-03-05

    A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Behavioral science and the study of gene-nutrition and gene-physical activity interactions in obesity research.

    Science.gov (United States)

    Faith, Myles S

    2008-12-01

    This report summarizes emerging opportunities for behavioral science to help advance the field of gene-environment and gene-behavior interactions, based on presentations at The National Cancer Institute (NCI) Workshop, "Gene-Nutrition and Gene-Physical Activity Interactions in the Etiology of Obesity." Three opportunities are highlighted: (i) designing potent behavioral "challenges" in experiments, (ii) determining viable behavioral phenotypes for genetics studies, and (iii) identifying specific measures of the environment or environmental exposures. Additional points are underscored, including the need to incorporate novel findings from neuroimaging studies regarding motivation and drive for eating and physical activity. Advances in behavioral science theory and methods can play an important role in advancing understanding of gene-brain-behavior relationships in obesity onset.

  20. Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis.

    Science.gov (United States)

    Wang, P Jeremy; Page, David C

    2002-09-15

    TAF(II)250, the largest subunit of the general transcription factor TFIID, is expressed from the human X chromosome, at least in somatic cells. In male meiosis, however, the sex chromosomes are transcriptionally silenced, while the autosomes remain active. How then are protein-encoding genes transcribed during human male meiosis? Here we present a novel autosomal human gene, TAF1L, which is homologous to TAF(II)250 and is expressed specifically in the testis, apparently in germ cells. We hypothesize that during male meiosis, transcription of protein-encoding genes relies upon TAF1L as a functional substitute for TAF(II)250. Like TAF(II)250, the human TAF1L protein can bind directly to TATA-binding protein, an essential component of TFIID. Most importantly, transfection with human TAF1L rescued the temperature-sensitive lethality of a hamster cell line mutant in TAF(II)250. TAF1L lacks introns and evidently arose by retroposition of a processed TAF(II)250 mRNA during primate evolution. The observation that TAF1L can functionally replace TAF(II)250 provides experimental support for the hypothesis that during male meiosis, autosomes provide cellular functions usually supplied by the X chromosome in somatic cells.

  1. Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma

    Directory of Open Access Journals (Sweden)

    Rasha Abd Alraouf Neama

    2017-01-01

    Full Text Available Background: The human epidermal growth factor receptor 2(HER2 proto-oncogene is overexpressed or amplified in approximately 15%–25% of invasive breast cancers. Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A gene encoding an enzyme that is a major target of anthracyclines. Hence, the determination of genetic alteration (amplification or deletion of both genes is considered as an important predictive factor that determines the response of breast cancer patients to treatment. The aims of this study are to determinate TOP2A status gene amplification in a set of Iraqi patients with breast cancer that have had an equivocal (2+ and positive HER2/neu by immunohistochemistry (IHC and to compare the results with estrogen receptor (ER and progesterone receptor (PR and HER2/neu status. Patients and Methods: A cross-sectional prospective study done on 53 patients with invasive breast carcinoma. Twenty-six out of total 53 cases were positive HER2/neu (3+, the remaining 27 equivocal HER2-IHC (2+ cases reanalyzed using dual-color chromogenic in situ hybridization (ZytoVision probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH, TOP2A gene status determination was done for all cases. Results: There is a direct significant correlation between TOP2A gene amplification and HER2/neu positivity, P < 0.05 in that 15 (39.4% out of 38 positive HER2/neu cases were associated with topoisomerase gene amplification. Regarding relation of topoisomerase gene to hormone receptor status (ER and PR, there was a significant negative relationship between the gene and ER receptor status. The higher level of gene amplification was noticed in ER and PR negative cases in about 13 (43.3% and 14 (48.2% for ER and PR, respectively. Conclusion: TOP2A gene status has a significantly positive correlation with HER2/neu status while it has a significantly negative

  2. Enhancer SINEs Link Pol III to Pol II Transcription in Neurons

    Directory of Open Access Journals (Sweden)

    Cristina Policarpi

    2017-12-01

    Full Text Available Summary: Spatiotemporal regulation of gene expression depends on the cooperation of multiple mechanisms, including the functional interaction of promoters with distally located enhancers. Here, we show that, in cortical neurons, a subset of short interspersed nuclear elements (SINEs located in the proximity of activity-regulated genes bears features of enhancers. Enhancer SINEs (eSINEs recruit the Pol III cofactor complex TFIIIC in a stimulus-dependent manner and are transcribed by Pol III in response to neuronal depolarization. Characterization of an eSINE located in proximity to the Fos gene (FosRSINE1 indicated that the FosRSINE1-encoded transcript interacts with Pol II at the Fos promoter and mediates Fos relocation to Pol II factories, providing an unprecedented molecular link between Pol III and Pol II transcription. Strikingly, knockdown of the FosRSINE1 transcript induces defects of both cortical radial migration in vivo and activity-dependent dendritogenesis in vitro, demonstrating that FosRSINE1 acts as a strong enhancer of Fos expression in diverse physiological contexts. : Spatiotemporal regulation of gene expression requires the interaction between promoters and distally located enhancers. Policarpi et al. identify a subset of SINEs that functions as enhancers for activity-dependent neuronal genes. The enhancer SINE FosRSINE1 regulates Fos transcription and is necessary for both activity-dependent dendritogenesis and proper brain development. Keywords: neuroscience, epigenetics, transcription, enhancers, SINEs, neuronal activity, neuronal development

  3. Switchable photosystem-II designer algae for photobiological hydrogen production

    Science.gov (United States)

    Lee, James Weifu

    2010-01-05

    A switchable photosystem-II designer algae for photobiological hydrogen production. The designer transgenic algae includes at least two transgenes for enhanced photobiological H.sub.2 production wherein a first transgene serves as a genetic switch that can controls photosystem II (PSII) oxygen evolution and a second transgene encodes for creation of free proton channels in the algal photosynthetic membrane. In one embodiment, the algae includes a DNA construct having polymerase chain reaction forward primer (302), a inducible promoter (304), a PSII-iRNA sequence (306), a terminator (308), and a PCR reverse primer (310). In other embodiments, the PSII-iRNA sequence (306) is replaced with a CF.sub.1-iRNA sequence (312), a streptomycin-production gene (314), a targeting sequence (316) followed by a proton-channel producing gene (318), or a PSII-producing gene (320). In one embodiment, a photo-bioreactor and gas-product separation and utilization system produce photobiological H.sub.2 from the switchable PSII designer alga.

  4. Reconciliation of Gene and Species Trees

    Directory of Open Access Journals (Sweden)

    L. Y. Rusin

    2014-01-01

    Full Text Available The first part of the paper briefly overviews the problem of gene and species trees reconciliation with the focus on defining and algorithmic construction of the evolutionary scenario. Basic ideas are discussed for the aspects of mapping definitions, costs of the mapping and evolutionary scenario, imposing time scales on a scenario, incorporating horizontal gene transfers, binarization and reconciliation of polytomous trees, and construction of species trees and scenarios. The review does not intend to cover the vast diversity of literature published on these subjects. Instead, the authors strived to overview the problem of the evolutionary scenario as a central concept in many areas of evolutionary research. The second part provides detailed mathematical proofs for the solutions of two problems: (i inferring a gene evolution along a species tree accounting for various types of evolutionary events and (ii trees reconciliation into a single species tree when only gene duplications and losses are allowed. All proposed algorithms have a cubic time complexity and are mathematically proved to find exact solutions. Solving algorithms for problem (ii can be naturally extended to incorporate horizontal transfers, other evolutionary events, and time scales on the species tree.

  5. Subchronic inhalation of soluble manganese induces expression of hypoxia-associated angiogenic genes in adult mouse lungs

    International Nuclear Information System (INIS)

    Bredow, Sebastian; Falgout, Melanie M.; March, Thomas H.; Yingling, Christin M.; Malkoski, Stephen P.; Aden, James; Bedrick, Edward J.; Lewis, Johnnye L.; Divine, Kevin K.

    2007-01-01

    Although the lung constitutes the major exposure route for airborne manganese (Mn), little is known about the potential pulmonary effects and the underlying molecular mechanisms. Transition metals can mimic a hypoxia-like response, activating the hypoxia inducible factor-1 (HIF-1) transcription factor family. Through binding to the hypoxia-response element (HRE), these factors regulate expression of many genes, including vascular endothelial growth factor (VEGF). Increases in VEGF, an important biomarker of angiogenesis, have been linked to respiratory diseases, including pulmonary hypertension. The objective of this study was to evaluate pulmonary hypoxia-associated angiogenic gene expression in response to exposure of soluble Mn(II) and to assess the genes' role as intermediaries of potential pulmonary Mn toxicity. In vitro, 0.25 mM Mn(II) altered morphology and slowed the growth of human pulmonary epithelial cell lines. Acute doses between 0.05 and 1 mM stimulated VEGF promoter activity up to 3.7-fold in transient transfection assays. Deletion of the HRE within the promoter had no effect on Mn(II)-induced VEGF expression but decreased cobalt [Co(II)]-induced activity 2-fold, suggesting that HIF-1 may not be involved in Mn(II)-induced VEGF gene transcription. Nose-only inhalation to 2 mg Mn(II)/m 3 for 5 days at 6 h/day produced no significant pulmonary inflammation but induced a 2-fold increase in pulmonary VEGF mRNA levels in adult mice and significantly altered expression of genes associated with murine angiogenesis. These findings suggest that even short-term exposures to soluble, occupationally relevant Mn(II) concentrations may alter pulmonary gene expression in pathways that ultimately could affect the lungs' susceptibility to respiratory disease

  6. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. ... acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP .... Helix II plays a dominant role in the interaction ... main distinguishing features of plant ACPs in plastids and ..... synthase component; J. Biol.

  7. Purifying Selection and Birth-and-Death Evolution in the Class II Hydrophobin Gene Families of the Ascomycete Trichoderma/Hypocrea

    Energy Technology Data Exchange (ETDEWEB)

    kubicek, Christian P.; Baker, Scott E.; Gamauf, Christian; Kenerley, Chuck; Druzhinina, Irina S.

    2008-01-10

    Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi, where their main function is to confer hydrophobicity to fungal surfaces in contact with air and during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or of themselves resulting in morphogenetic signals. Based on their hydropathy patterns and their solubility characteristics, they are classified in class I and class II hydrophobins, the latter being found only in ascomycetes. Here we have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three fully sequenced genomes (H. jecorina=T. reesei, H. atroviridis=T. atroviride; H. virens=T. virens) and a total of 14.000 ESTs of six others (T. asperellum, H. lixii=T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, which is the highest number found in any other ascomycete so far. They all showed the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these HFBs contained an extended N-terminus rich in either praline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades contain duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2-4) from each species, and most of them were from Pyrenomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other pyrenomycetes occured in shared clades. Our study shows

  8. Establishment of a 12-gene expression signature to predict colon cancer prognosis

    Directory of Open Access Journals (Sweden)

    Dalong Sun

    2018-06-01

    Full Text Available A robust and accurate gene expression signature is essential to assist oncologists to determine which subset of patients at similar Tumor-Lymph Node-Metastasis (TNM stage has high recurrence risk and could benefit from adjuvant therapies. Here we applied a two-step supervised machine-learning method and established a 12-gene expression signature to precisely predict colon adenocarcinoma (COAD prognosis by using COAD RNA-seq transcriptome data from The Cancer Genome Atlas (TCGA. The predictive performance of the 12-gene signature was validated with two independent gene expression microarray datasets: GSE39582 includes 566 COAD cases for the development of six molecular subtypes with distinct clinical, molecular and survival characteristics; GSE17538 is a dataset containing 232 colon cancer patients for the generation of a metastasis gene expression profile to predict recurrence and death in COAD patients. The signature could effectively separate the poor prognosis patients from good prognosis group (disease specific survival (DSS: Kaplan Meier (KM Log Rank p = 0.0034; overall survival (OS: KM Log Rank p = 0.0336 in GSE17538. For patients with proficient mismatch repair system (pMMR in GSE39582, the signature could also effectively distinguish high risk group from low risk group (OS: KM Log Rank p = 0.005; Relapse free survival (RFS: KM Log Rank p = 0.022. Interestingly, advanced stage patients were significantly enriched in high 12-gene score group (Fisher’s exact test p = 0.0003. After stage stratification, the signature could still distinguish poor prognosis patients in GSE17538 from good prognosis within stage II (Log Rank p = 0.01 and stage II & III (Log Rank p = 0.017 in the outcome of DFS. Within stage III or II/III pMMR patients treated with Adjuvant Chemotherapies (ACT and patients with higher 12-gene score showed poorer prognosis (III, OS: KM Log Rank p = 0.046; III & II, OS: KM Log Rank p = 0.041. Among stage II/III pMMR patients

  9. Altered deoxyribonuclease activity in cancer cells and its role in non toxic adjuvant cancer therapy with mixed vitamins C and K3.

    Science.gov (United States)

    Taper, Henryk S

    2008-01-01

    The alterations of deoxyribonuclease DNase activity in cancer cells were the basis of the utilization of mixed vitamins C and K3 in a nontoxic, adjuvant cancer therapy. In order to localize exactly the altered activities of DNase in cancer cells, histochemical methods were utilized. The deficiency of alkaline and acid DNase activity appeared to be characteristic for non-necrotic cells of malignant human and animal tumors. This enzymatic deficiency appeared in experimental carcinogenesis before the phenotypic signs of malignancy. Tumor promoters directly reduced the activity of both DNases. The incidence of spontaneous malignant human and animal tumors appeared to be inversely proportional to the intensity of the activity of both DNases in normal cells and tissues from which these tumors were derived. The fact that alkaline and acid DNase activity was reactivated during the spontaneous and therapeutically induced necrosis of cancer cells suggests that this enzymatic deficiency of DNase activity in cancer cells was due to the action of specific inhibitors of DNases. Characteristic variations of serum alkaline DNase activity in positive responders to therapy, examined in more than 800 cancer-bearing patients, may be the basis for the development of a useful test for therapeutic prognosis and for monitoring of cancer bearing patients. Acid DNase was selectively reactivated in malignant tumor cells by vitamin C (sodium ascorbate), whereas alkaline DNase was reactivated by vitamin K3. Joint vitamin C and K3 administration produced in vitro and in vivo tumor growth inhibition, potentiation and sensitization of chemo- and/or radiotherapy and a decrease in the number of metastases in animals with experimental tumors. Joint vitamin C and K3 administration may be considered as a possible new, non-toxic, adjuvant cancer therapy, which can be easily introduced into the classic protocols of clinical cancer therapy without any supplementary risk for patients.

  10. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2014-05-01

    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  11. Isolation of an X-ray-responsive element in the promoter region of tissue-type plasminogen activator: Potential uses of X-ray-responsive elements for gene therapy

    International Nuclear Information System (INIS)

    Boothman, D.A.; Lee, I.W.; Sahijdak, W.M.

    1994-01-01

    Tissue-type plasminogen activator (t-PA) was induced over 50-fold after X irradiation in radioresistant human melanoma cells. Activities of t-PA were induced 14-fold in ataxia telangiectasia, 9-fold in Bloom's syndrome and 6-fold in Fanconi's anemia cells, compared to normal human fibroblasts. X-ray-inducible synthesis of the protease, t-PA, may play a role(s) in damage-inducible repair processes in mammalian cells, similar to the SOS repair systems in lower eukaryotes and prokaryotes. DNA band shift and DNase I footprinting assays were used to determine binding if transcription factors to a previously unknown X-ray-responsive element (XRE) in the t-PA promoter. The major goals of our research with XREs are to understand (a) which transcription factor(s) regulates to-PA induction after X-rays, and (b) the role(s) of t-PA in DNA repair, apoptosis or other responses to X rays. The purpose of this paper is to discuss the potential use of an XRE, such as the one in the t-PA promoter, for gene radiotherapy. Several gene therapy strategies are proposed. 22 refs., 3 figs

  12. Influence of prenatal application of angiotensin II and postnatal salt diet on GABAergic and oxytocin system in rat brain steam and cerebellum

    International Nuclear Information System (INIS)

    Jackova, L.; Olexova, L.; Svitok, P.; Senko, T.; Stefanik, P.

    2015-01-01

    Our goal was to determinate how gene expression of GABA transporter 1 (GAT1), glutamate decarboxylase 67 (GAD67) and oxytocin receptor (OTR) is influenced with prenatal exposition to angiotensin II (Ang II) and postnatal salt diet in nucleus tractus solitarii (NTS) and cerebellum in rats. In NTS we observed strong tendency in different reaction of OTR gene expression between Ang II prenatal treatment and control rats after high salt diet. We observed significant influence of sex on GAD67 gene expression in cerebellum. Also, sex in combination with salt diet is significant factor in expression of GAT1 gene in cerebellum. (authors)

  13. Ultraviolet irradiation of herpes simplex virus (type 1): delayed transcription and comparative sensitivites of virus functions.

    Science.gov (United States)

    Eglin, R P; Gugerli, P; Wildy, P

    1980-07-01

    The delay in the replication of herpes simplex virus surviving u.v. irradiation occurs after the uncoating of virus, as judged by sensitivity to DNase. It occurs before translation, judged by the kinetics of appearance of various virus-specific proteins, and before transcription, judged by the detection of virus-specific RNA by in situ hybridization. Since the delays in both transcription and translation are reversed by photoreactivation, the simplest hypothesis is that pyrimidine dimers directly obstruct transcription;unless these are broken by photoreactivating enzymes, there will be transcriptional delay until reactivating processes have repaired the lesion. The u.v. sensitivities of the abilities to induce various enzymes (thymidine kinase, DNase and DNA polymerase) were only about four times less than that of infectivity. The The ability to induce the three enzymes was three times less sensitive than that of the structural antigen (Band II).

  14. Ultraviolet irradiation of herpes simplex virus (type 1): delayed transcription and comparative sensitivities of virus functions

    International Nuclear Information System (INIS)

    Eglin, R.P.; Gugerli, P.; Wildy, P.

    1980-01-01

    The delay in the replication of herpes simplex virus surviving u.v. irradiation occurs after the uncoating of virus, as judged by sensitivity to DNase. It occurs before translation, judged by the kinetics of appearance of various virus-specific proteins, and before transcription, judged by the detection of virus-specific RNA by in situ hybridization. Since the delays in both transcription and translation are reversed by photoreactivation, the simplest hypothesis is that pyrimidine dimers directly obstruct transcription; unless these are broken by photoreactivating enzymes, there will be transcriptional delay until reactivating processes have repaired the lesion. The u.v. sensitivities of the abilities to induce various enzymes (thymidine kinase, DNase and DNA polymerase) were only about four times less than that of infectivity. The ability to induce the three enzymes was three times less sensitive than that of the structural antigen (Band II). (U.K.)

  15. Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

    Directory of Open Access Journals (Sweden)

    Ravid Rivka

    2008-05-01

    Full Text Available Abstract Background Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD, Parkinson's disease (PD and dementia with Lewy bodies (DLB. Quantitative real-time PCR (RT qPCR is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA] in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays. Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusion This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

  16. Phylogenetic Analysis, Lineage-Specific Expansion and Functional Divergence of seed dormancy 4-Like Genes in Plants.

    Directory of Open Access Journals (Sweden)

    Saminathan Subburaj

    Full Text Available The rice gene seed dormancy 4 (OsSdr4 functions in seed dormancy and is a major factor associated with pre-harvest sprouting (PHS. Although previous studies of this protein family were reported for rice and other species, knowledge of the evolution of genes homologous to OsSdr4 in plants remains inadequate. Fifty four Sdr4-like (hereafter designated Sdr4L genes were identified in nine plant lineages including 36 species. Phylogenetic analysis placed these genes in eight subfamilies (I-VIII. Genes from the same lineage clustered together, supported by analysis of conserved motifs and exon-intron patterns. Segmental duplications were present in both dicot and monocot clusters, while tandemly duplicated genes occurred only in monocot clusters indicating that both tandem and segmental duplications contributed to expansion of the grass I and II subfamilies. Estimation of the approximate ages of the duplication events indicated that ancestral Sdr4 genes evolved from a common angiosperm ancestor, about 160 million years ago (MYA. Moreover, diversification of Sdr4L genes in mono and dicot plants was mainly associated with genome-wide duplication and speciation events. Functional divergence was observed in all subfamily pairs, except IV/VIIIa. Further analysis indicated that functional constraints between subfamily pairs I/II, I/VIIIb, II/VI, II/VIIIb, II/IV, and VI/VIIIb were statistically significant. Site and branch-site model analyses of positive selection suggested that these genes were under strong adaptive selection pressure. Critical amino acids detected for both functional divergence and positive selection were mostly located in the loops, pointing to functional importance of these regions in this protein family. In addition, differential expression studies by transcriptome atlas of 11 Sdr4L genes showed that the duplicated genes may have undergone divergence in expression between plant species. Our findings showed that Sdr4L genes are

  17. Nonrandom association of a type II procollagen genotype with achondroplasia

    OpenAIRE

    1986-01-01

    Achondroplasia is an autosomal dominant disorder that involves defective endochondral bone formation. Type II collagen is the predominant collagen of cartilage. We found a HindIII polymorphic site in the normal Caucasian population by using the type II procollagen gene probe pgHCol(II)A. The presence of this site yields a 7.0-kilobase (kb) band; its absence yields a 14.0-kb band. We found a significant deviation in genotype distribution and allele frequencies in a population of unrelated indi...

  18. MSCs with ACE II gene affect apoptosis pathway of acute lung injury induced by bleomycin.

    Science.gov (United States)

    Zhang, Xiaomiao; Gao, Fengying; Li, Qian; Dong, Zhixia; Sun, Bo; Hou, Lili; Li, Zhuozhe; Liu, Zhenwei

    2015-02-01

    The aim of this study was to evaluate the effect and related mechanisms of Mesenchymal stem cells (MSCs) and Angiotensin converting enzyme II (ACE II) on acute lung injury (ALI). MSCs were separated from umbilical cord cells, and the changes of phenotype before and after ACE II silence were observed using Flow Cytometer. ALI model was induced by 10 mg/mL bleomycin in 60 Balb/c mice, and the rest 8 mice were regarded as the baseline group. The mice were randomly divided into four groups (n = 15): control, ACE II, stem, and stem + ACE II. The apoptotic index (AI) was calculated using TUNEL, and the detection of protein and mRNA of Bax, Bak and p53, Bcl-2, Grp78, CHOP and Caspase 12 were used by western-blot and RT-PCR, respectively. The umbilical cord cells differentiated into stable MSCs about 14 days, and ACE II transfection reached a peak at the 5th day after transfection. ACE II silence did not affect the phenotype of MSCs. All the proteins and mRNAs expression except Bcl-2 in the stem and stem + ACE II were significantly lower than those in control from 8 h (p ACE II performed a better effect than single stem in most indexes, including AI (p ACE II can significantly suppress apoptosis in ALI mice, and may be an effective clinical treatment for ALI.

  19. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. (Cincinnati College of Medicine, OH (USA))

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  20. The effect of interaction between Lipoprotein Lipase and ApoVLDL-II ...

    African Journals Online (AJOL)

    Body weight, abdominal fat weight and serum biochemical levels were determined from lean and fat chicken breeds at 12 weeks of age. Single nucleotide polymorphism (SNP) in apoVLDL-II and lipoprotein lipase genes was screened by PCR-SSCP and detected by direct sequencing. Lipoprotein lipase gene frequency ...

  1. Association of Angiotensin-Converting Enzyme Intron 16 Insertion/Deletion and Angiotensin II Type 1 Receptor A1166C Gene Polymorphisms with Preeclampsia in South East of Iran

    Directory of Open Access Journals (Sweden)

    Saeedeh Salimi

    2011-01-01

    Full Text Available Some evidence suggests that a variety of genetic factors contributed in pathogenesis of the preeclampsia. The aim of this study was to assess the association between the angiotensin-converting enzyme (ACE I/D and angiotensin II type1 receptor A1166C polymorphisms with preeclampsia. This study was performed in 125 preeclamptic pregnant women and 132 controls. The I/D Polymorphism of the ACE gene was assessed by polymerase chain reaction and the A1166C Polymorphism of the AT1R gene was determined by restriction fragment length polymorphism. The genotype and allele frequencies of I/D polymorphism differed between two groups. The risk of preeclampsia was 3.2-fold in pregnant women with D allele (OR, 3.2 [95% CI, 1.1 to 3.8]; P=0.01. The distribution of the AT1R gene A1166C polymorphism was similar in affected and control groups. Our results supported that presence of the I/D polymorphism of ACE gene is a marker for the increased risk of preeclampsia.

  2. Ubiquitylation and degradation of elongating RNA polymerase II

    DEFF Research Database (Denmark)

    Wilson, Marcus D; Harreman, Michelle; Svejstrup, Jesper Q

    2013-01-01

    During its journey across a gene, RNA polymerase II has to contend with a number of obstacles to its progression, including nucleosomes, DNA-binding proteins, DNA damage, and sequences that are intrinsically difficult to transcribe. Not surprisingly, a large number of elongation factors have....... In this review, we describe the mechanisms and factors responsible for the last resort mechanism of transcriptional elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation....

  3. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    International Nuclear Information System (INIS)

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong; Wan, Min; Li, Yong-Qiang; Zhang, Rong-Ying; Zhao, Yuan-Di

    2012-01-01

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  4. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Wan, Min [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Li, Yong-Qiang [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhang, Rong-Ying [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhao, Yuan-Di, E-mail: zydi@mail.hust.edu.cn [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China)

    2012-11-15

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  5. Sulforaphane Prevents Angiotensin II-Induced Testicular Cell Death via Activation of NRF2

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    Yonggang Wang

    2017-01-01

    Full Text Available Although angiotensin II (Ang II was reported to facilitate sperm motility and intratesticular sperm transport, recent findings shed light on the efficacy of Ang II in stimulating inflammatory events in testicular peritubular cells, effect of which may play a role in male infertility. It is still unknown whether Ang II can induce testicular apoptotic cell death, which may be a more direct action of Ang II in male infertility. Therefore, the present study aims to determine whether Ang II can induce testicular apoptotic cell death and whether this action can be prevented by sulforaphane (SFN via activating nuclear factor (erythroid-derived 2-like 2 (NRF2, the governor of antioxidant-redox signalling. Eight-week-old male C57BL/6J wild type (WT and Nrf2 gene knockout mice were treated with Ang II, in the presence or absence of SFN. In WT mice, SFN activated testicular NRF2 expression and function, along with a marked attenuation in Ang II-induced testicular oxidative stress, inflammation, endoplasmic reticulum stress, and apoptotic cell death. Deletion of the Nrf2 gene led to a complete abolishment of these efficacies of SFN. The present study indicated that Ang II may result in testicular apoptotic cell death, which can be prevented by SFN via the activation of NRF2.

  6. Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

    Science.gov (United States)

    Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu; He, Chuan

    2012-04-01

    Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

  7. MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants.

    Science.gov (United States)

    Gramzow, Lydia; Weilandt, Lisa; Theißen, Günter

    2014-11-01

    MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11-14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14-16 Type II MADS-box genes. The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box genes for the development of gymnosperms. This study is

  8. El socratismo de Diógenes Laercio. Vidas de los filósofos ilustres (libro II

    Directory of Open Access Journals (Sweden)

    Luis Gerena Carrillo

    2015-08-01

    Full Text Available En este trabajo se trata de mostrar que en el libro II de las Vidas de los filósofos ilustres, Diógenes Laercio establece los rasgos que distinguen como tales a los principales socráticos menores (Jenofonte, Esquines Aristipo y Euclides. De acuerdo con esto, se sostiene que para Laercio los socráticos son aquellos que escribieron diálogos socráticos, pero principalmente quienes elaboraron una propuesta de vida buena a partir de su relación con Sócrates y, en este sentido, sustentada en supuestos filosóficos que se pueden considerar socráticos, especialmente el supuesto de que el conocimiento es el bien. Esta lectura de Laercio es importante porque nos permite pensar a los socráticos como un movimiento con rasgos comunes, que desarrolla la ética, la cual, según Laercio, introduce Sócrates como una disciplina distinta de la física y la dialéctica, pero que constituye una nueva forma de filosofía: una cuyo problema es cómo vivir bien, pero que elabora sus propuestas a partir del diálogo y la confrontación, entendiéndose esto por conocimiento.   Palabras clave: Sócrates, socratismo, filosofía socrática   The Socrates of Diogenes Laertius. Lives of Eminent Philosophers (book II This paper aims is to show that in book II of the Lives of Eminent Philosophers, Diogenes Laertius establishes the distinctive traits of the minor Socratics (Xenophon, Aeschines, Aristippus and Euclid. Accordingly for Laertius, the socratics wrote socratic dialogues, and mainly those who made a proposal of a good life from their relation with Socrates and based on philosophical suppositions that can be considered Socratic, specially that the knowledge is good. This interpretation of Laertius is important because we think of the Socratics as a movement with common traits, that developed the ethics, which according to Laertius, Socrates introduced as a distinct discipline of physics and dialectic, but it is a new form of philosophy: one in which the

  9. Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

    Science.gov (United States)

    Giovanella, Patricia; Cabral, Lucélia; Bento, Fátima Menezes; Gianello, Clesio; Camargo, Flávio Anastácio Oliveira

    2016-01-25

    This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. The potential mechanism of Bursal-derived BPP-II on the antibody production and avian pre-B cell.

    Science.gov (United States)

    Feng, Xiuli; Cao, Ruibing; Zhou, Bin; Liu, Qingtao; Liu, Ke; Liu, Xiaodong; Zhang, Yuanpeng; Gu, Jinyan; Miao, Denian; Chen, Puyan

    2013-03-01

    The bursa of Fabricius is critical for the normal development of the B lymphocytes responsible for antibody production. However, the mechanism of the bursal-derived bioactive factor on B cell development is little reported. In this paper, chicks were immunized with BPP-II and AIV vaccine or AIV antigen, and antibody and IL-4 production were detected. The results showed that BPP-II played strongly inducing roles on the humoral immune responses. To investigate the gene expression at transcriptional level, avian pre-B lymphocyte DT40 cells were treated with BPP-II, and were analyzed with the gene microarray. The results proved that BPP-II treatment regulated 11 pathways, in which homologous recombination is a vital mechanism which is involved in antibody Ig gene conversion and diversification during B cell development. These results suggested Bursal-derived biological active factor BPP-II might be involved in the antibody production processes and B cell development, which is vital to the humoral central immune organ, the bursa of Fabricius. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. The Anti-Adipogenic Potential of COUP-TFII Is Mediated by Downregulation of the Notch Target Gene Hey1.

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    Ilse Scroyen

    Full Text Available Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII belongs to the steroid/thyroid hormone receptor superfamily and may contribute to the pathogenesis of obesity. It has not conclusively been established, however, whether its role is pro- or anti-adipogenic.Gene silencing of Coup-tfII in 3T3-F442A preadipocytes resulted in enhanced differentiation into mature adipocytes. This was associated with upregulation of the Notch signaling target gene Hey1. A functional role of Hey1 was confirmed by gene silencing in 3T3-F442A preadipocytes, resulting in impaired differentiation. In vivo, de novo fat pad formation in NUDE mice was significantly stimulated following injection of preadipocytes with Coup-tfII gene silencing, but impaired with Hey1 gene silencing. Moreover, expression of Coup-tfII was lower and that of Hey1 higher in isolated adipocytes of obese as compared to lean adipose tissue.These in vitro and in vivo data support an anti-adipogenic role of COUP-TFII via downregulating the Notch signaling target gene Hey1.

  12. Impact of estrogen receptor α gene and oxytocin receptor gene polymorphisms on female sexuality

    Directory of Open Access Journals (Sweden)

    Anastasia K Armeni

    2017-02-01

    Full Text Available Over the past decades, research attention has increasingly been paid to the neurobiological component of sexual behavior. The aim of the present study was to investigate the correlation of estrogen receptor α (ERA gene polymorphism (rs2234693-PvuII (T→C substitution and oxytocin receptor gene polymorphism (rs53576 (G→A substitution with sexuality parameters of young, healthy women. One hundred thirty-three Greek heterosexual women, students in higher education institutions, 20–25 years of age, sexually active, with normal menstrual cycles (28–35 days, were recruited in the study. Exclusion criteria were chronic and/or major psychiatric diseases, use of oral contraceptive pills (OCs, polycystic ovary syndrome (PCOS, thyroid diseases as well as drugs that are implicated in hypothalamus–pituitary–gonadal axis. T allele (wildtype of rs2234693 (PvuII polymorphism of ERA gene was correlated with increased levels of arousal and lubrication, whereas A allele (polymorphic of rs53576 (OXTR polymorphism was correlated with increased arousal levels. The simultaneous presence of both T allele of rs2234693 (PvuII and A allele of rs53576 (OXTR polymorphisms (T + A group was correlated with increased arousal, orgasm levels as well as female sexual function index full score. To our knowledge, this is the first study to investigate the interaction between ERA and OXTR with regard to sexual function in women. Female sexuality is a complex behavioral trait that encompasses both biological and psychological components. It seems that variability in female sexual response stems from genetic variability that characterizes endocrine, neurotransmitter and central nervous system influences.

  13. Endothelial Nitric Oxide Synthase Gene Polymorphism (G894T and Diabetes Mellitus (Type II among South Indians

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    T. Angeline

    2011-01-01

    Full Text Available The objective of the study is to find out whether the endothelial nitric oxide synthase (eNOS G894T single-nucleotide polymorphism is associated with type 2 diabetes mellitus in South Indian (Tamil population. A total number of 260 subjects comprising 100 type 2 diabetic mellitus patients and 160 healthy individuals with no documented history of diabetes were included for the study. DNA was isolated, and eNOS G894T genotyping was performed using the polymerase chain reaction followed by restriction enzyme analysis using Ban II. The genotype distribution in patients and controls were compatible with the Hardy-Weinberg expectations (P>0.05. Odds ratio indicates that the occurrence of mutant genotype (GT/TT was 7.2 times (95% CI = 4.09–12.71 more frequent in the cases than in controls. Thus, the present study demonstrates that there is an association of endothelial nitric oxide synthase gene (G894T polymorphism with diabetes mellitus among South Indians.

  14. Genetic heterogeneity of Usher syndrome type II.

    OpenAIRE

    Pieke Dahl, S; Kimberling, WJ; Gorin, MB; Weston, MD; Furman, JM; Pikus, A; Moller, C

    1993-01-01

    Usher syndrome is an autosomal recessive disorder characterised by retinitis pigmentosa and congenital sensorineural hearing loss. A gene for Usher syndrome type II (USH2) has been localised to chromosome 1q32-q41. DNA from a family with four of seven sibs affected with clinical characteristics of Usher syndrome type II was genotyped using markers spanning the 1q32-1q41 region. These included D1S70 and D1S81, which are believed to flank USH2. Genotypic results and subsequent linkage analysis ...

  15. Structure and function of the human metallothionein gene family: Final technical report

    International Nuclear Information System (INIS)

    Karin, M.

    1986-01-01

    The full nucleotide sequence of two additional human metallothionein (hMT) genes has been determined. These genes, hMT-I/sub B/ and hMT-I/sub F/, are located within the MT-I gene cluster we have described originally. The hMT-I/sub F/ gene is the first hMT-I gene whose amino acid sequence is in complete agreement with the published sequence of the human MT-I proteins. Therefore it is likely to be an active gene encoding a functional protein. However, since we have just completed the sequence analysis, we have not characterized this gene further yet. The hMT-I/sub B/ gene is closely linked to the hMT-I/sub A/ gene, and two pseudogenes, hMT-I/sub C/ and hMT-I/sub D/ separate the two. From its nucleotide sequence hMT-I/sub B/ seems to be an active gene, encoding a functional protein even though it differs in four positions from the published sequence of human MT-I proteins. This gene is expressed in a human hepatoma cell line, HepG2, and its expression is stimulated by Cd ++ . Using gene fusions to the viral thymidine-kinase gene we find that hMT-I/sub B/, like the hMT-I/sub A/ and hMT-II/sub A/ genes, contains a heavy metal responsive promoterregulatory element within its 5' flanking region. We analyzed the level of hMT-I/sub B/ mRNA in a variety of human cell lines by the S1 nuclease technique, and compared it to the expression of the hMT-II/sub A/ gene. While the hMT-II/sub A/ gene was expressed in all of the cell lines analyzed, the hMT-I/sub B/ gene was expressed in liver and kidney derived cell lines cells. This suggest that the expression of the hMT-I/sub B/ gene is controlled in a tissue specific manner. 13 refs

  16. QUANTIFICATION OF TRANSGENIC PLANT MARKER GENE PERSISTENCE IN THE FIELD

    Science.gov (United States)

    Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, com...

  17. Polymorphisms of the low-density lipoprotein receptor gene in Brazilian individuals with heterozygous familial hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    L.A. Salazar

    2000-11-01

    Full Text Available Familial hypercholesterolemia (FH is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low-density lipoprotein (LDL level. The disease is caused by several different mutations in the LDL receptor gene. Although early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infarction, the techniques available for determining the number of the functional LDL receptor molecules are difficult to carry out and expensive. Polymorphisms associated with this gene may be used for unequivocal diagnosis of FH in several populations. The aim of our study was to evaluate the genotype distribution and relative allele frequencies of three polymorphisms of the LDL receptor gene, HincII1773 (exon 12, AvaII (exon 13 and PvuII (intron 15, in 50 unrelated Brazilian individuals with a diagnosis of heterozygous FH and in 130 normolipidemic controls. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. The polymorphisms were detected by PCR-RFLP. The FH subjects showed a higher frequency of A+A+ (AvaII, H+H+ (HincII1773 and P1P1 (PvuII homozygous genotypes when compared to the control group (P<0.05. In addition, FH probands presented a high frequency of A+ (0.58, H+ (0.61 and P1 (0.78 alleles when compared to normolipidemic individuals (0.45, 0.45 and 0.64, respectively. The strong association observed between these alleles and FH suggests that AvaII, HincII1773 and PvuII polymorphisms could be useful to monitor the inheritance of FH in Brazilian families.

  18. Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(DJ Recombination

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    Louise S. Matheson

    2017-11-01

    Full Text Available V(DJ recombination is essential for the generation of diverse antigen receptor (AgR repertoires. In B cells, immunoglobulin kappa (Igκ light chain recombination follows immunoglobulin heavy chain (Igh recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(DJ recombination and provide avenues for further investigation of chromatin signatures that may underpin V(DJ-mediated chromosomal translocations.

  19. HCV proteins and immunoglobulin variable gene (IgV) subfamilies in HCV-induced type II mixed cryoglobulinemia: a concurrent pathogenetic role.

    Science.gov (United States)

    Sautto, Giuseppe; Mancini, Nicasio; Solforosi, Laura; Diotti, Roberta A; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

  20. The great diversity of major histocompatibility complex class II genes in Philippine native cattle

    Science.gov (United States)

    Takeshima, S.N.; Miyasaka, T.; Polat, M.; Kikuya, M.; Matsumoto, Y.; Mingala, C.N.; Villanueva, M.A.; Salces, A.J.; Onuma, M.; Aida, Y.

    2014-01-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle. PMID:25606401

  1. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp.

    Science.gov (United States)

    Xu, J; Johnson, R C

    1995-06-01

    Escherichia coli aldB was identified as a gene that is negatively regulated by Fis but positively regulated by RpoS. The complete DNA sequence determined in this study indicates that aldB encodes a 56.3-kDa protein which shares a high degree of homology with an acetaldehyde dehydrogenase encoded by acoD of Alcaligenes eutrophus and an aldehyde dehydrogenase encoded by aldA of Vibrio cholerae and significant homology with a group of other aldehyde dehydrogenases from prokaryotes and eukaryotes. Expression of aldB is maximally induced during the transition from exponential phase to stationary phase. Its message levels are elevated three- to fourfold by a fis mutation and abolished by an rpoS mutation. In addition, the expression of an aldB-lacZ fusion was decreased about 20-fold in the absence of crp. DNase I footprinting analysis showed that five Fis binding sites and one Crp binding site are located within the aldB promoter region, suggesting that Fis and Crp are acting directly to control aldB transcription. AldB expression is induced by ethanol, but in contrast to that of most of the RpoS-dependent genes, the expression of aldB is not altered by an increase in medium osmolarity.

  2. Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation.

    Directory of Open Access Journals (Sweden)

    Yoshiharu Yamaichi

    2011-07-01

    Full Text Available There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products that surround the origin of replication (oriCII of Vibrio cholerae chromosome II (chrII are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.

  3. Prevalence of Methicillin-Resistant Staphylococcus Aureus Carrying Panton-Valentine Leukocidin Gene in Cutaneous Infections in the City of Isfahan

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Pourmand

    2012-03-01

    Full Text Available Background: Methicillin-Resistant Staphylococcus aureus (MRSA is a major cause of Nosocomial and community infections that are becoming increasingly difficult to combat, because of emerging resistance to all classes of antibiotics. Moreover Panton-Valentine leukocidin (PVL is an important virulence factor in S. aureus and causes white blood cell destruction, necrosis and accelerated apoptosis. The aim of this study was to determine the frequency of PVL-positive MRSA in cutaneous infections, for epidemiological purposes and also to determine antibiotic resistance of the isolates.Methods: Collectively, 56 isolates of S. aureus were obtained from Isfahan University of Medical sciences affiliated hospitals and confirmed with biochemical tests (coagulase, mannitol fermentation, and DNase. Then polymerase chain reaction (PCR was used to detect pvl gene. Coagulase gene was used as internal control. The antibiotic susceptibility of all isolates to methicillin was determined using disk diffusion method.Results: Out of 56 isolates 14.3% were PVL positive that 37.5% were from abscess and 62.5% were from wound. Among all of these isolates 67.8% were MRSA and also 75% of PVL-positive isolates were MRSA.Conclusion: The prevalence of PVL positive MRSA in cutaneous isolates is high. Future works are necessary for a more complete understanding of distribution of these virulent isolates in nasal carriers to decrease the risk of infections.

  4. Anti-DNase B

    Science.gov (United States)

    ... care provider about the meaning of your specific test results. What Abnormal Results ... another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than ...

  5. Modes of salmonid MHC class I and II evolution differ from the primate paradigm

    NARCIS (Netherlands)

    Shum, B.P.; Guethlein, L.; Flodin, L.R.; Adkison, M.A.; Hedrick, R.P.; Nehring, R.B.; Stet, R.J.M.; Secombes, C.; Parham, P.

    2001-01-01

    Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15-20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II β-chain gene DAB from 15 rainbow and 10 brown trout. Both genes

  6. Genome-wide analysis of KAP1, the 7SK snRNP complex, and RNA polymerase II

    Directory of Open Access Journals (Sweden)

    Ryan P. McNamara

    2016-03-01

    Full Text Available The transition of RNA polymerase II (Pol II from transcription initiation into productive elongation in eukaryotic cells is regulated by the P-TEFb kinase, which phosphorylates the C-terminal domain of paused Pol II at promoter-proximal regions. Our recent study found that P-TEFb (in an inhibited state bound to the 7SK snRNP complex interacts with the KAP1/TRIM28 transcriptional regulator, and that KAP1 and the 7SK snRNP co-occupy most gene promoters containing paused Pol II. Here we provide a detailed experimental description and analysis of the ChIP-seq datasets that have been deposited into Gene Expression Omnibus (GEO: GS72622, so that independent groups can replicate and expand upon these findings. We propose these datasets would provide valuable information for researchers studying mechanisms of transcriptional regulation including Pol II pausing and pause release. Keywords: P-TEFb/7SK snRNP, KAP1, RNA polymerase II, ChIP-seq, Transcription elongation

  7. Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination.

    Science.gov (United States)

    Matheson, Louise S; Bolland, Daniel J; Chovanec, Peter; Krueger, Felix; Andrews, Simon; Koohy, Hashem; Corcoran, Anne E

    2017-01-01

    V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa ( Igκ ) light chain recombination follows immunoglobulin heavy chain ( Igh ) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh , as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.

  8. Heterologous Expression of Xylanase II from Aspergillus usamii in Pichia pastoris

    OpenAIRE

    Zhou, Chenyan; Wang, Yongtao; Wu, Minchen; Wang, Wu; Li, Dongfeng

    2009-01-01

    To efficiently produce xylanase for food processing industry, a gene encoding xylanase II (XynII) from Aspergillus usamii has been cloned into the vector pPIC9K and integrated into the genome of Pichia pastoris KM71 by electroporation. By means of minimal dextrose (MD) plates and PCR, the recombinant P. pastoris strains (His+Muts) have been obtained. Activity assay and SDS-PAGE demonstrate that XynII was extracellularly expressed in P. pastoris with the induction of methanol. In shake flask c...

  9. Comprehensive screening of the USH2A gene in Usher syndrome type II and non-syndromic recessive retinitis pigmentosa.

    Science.gov (United States)

    Seyedahmadi, Babak Jian; Rivolta, Carlo; Keene, Julia A; Berson, Eliot L; Dryja, Thaddeus P

    2004-08-01

    A screen of the entire coding region of the USH2A gene in 129 unrelated patients with Usher syndrome type II (USH2) and in 146 unrelated patients with non-syndromic autosomal recessive retinitis pigmentosa (ARRP) uncovered 54 different sequence variations, including 18 likely pathogenic mutations (13 frameshift, three nonsense, and two missense), 12 changes of uncertain pathogenicity (11 missense changes and one in-frame deletion), and 24 non-pathogenic rare variants or polymorphisms. Of the 18 likely pathogenic mutations, nine were novel. Among the USH2 patients, 50 (39%) had one or two likely pathogenic mutations. The most common mutant allele in USH2 patients was E767fs, which was found in 29 patients, including one homozygote. Among the ARRP patients, we found 17 (12%) with one or two likely pathogenic mutations. The most common mutant allele in ARRP patients was C759F and it was found in 10 patients. The C759F allele was also found in two USH2 patients; in neither of them was a change in the other allele found. The second most common mutant allele in both patient groups was L1447fs (found in 6/50 USH2 patients and 6/17 ARRP patients). Of the 50+17=67 patients with identified USH2A mutations, only one mutation in one allele was found in 41+12=53 (79%); the reason for the high proportion of patients with only one identified mutation is obscure. Our results indicate that USH2A mutations are found in about 7% of all cases of RP in North America, a frequency similar to the RPGR gene (8%) and the rhodopsin gene (10%).

  10. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that e...... in the nucleolus....

  11. Loss of Resistance to Angiotensin II-Induced Hypertension in the Jackson Laboratory Recombination-Activating Gene Null Mouse on the C57BL/6J Background.

    Science.gov (United States)

    Ji, Hong; Pai, Amrita V; West, Crystal A; Wu, Xie; Speth, Robert C; Sandberg, Kathryn

    2017-06-01

    Resistance to angiotensin II (Ang II)-induced hypertension in T-cell-deficient male mice with a targeted mutation in the recombination-activating gene-1 ( Rag1 ) on the C57BL/6J background (B6. Rag1 -/- -M), which was reported by 5 independent laboratories including ours before 2015, has been lost. In mice purchased from Jackson Laboratory in 2015 and 2016, the time course and magnitude increase in mean arterial pressure induced by 2 weeks of Ang II infusion at 490 ng/kg per minute was identical between B6. Rag1 -/- -M and male wild-type littermates. Moreover, there were no differences in the time course or magnitude increase in mean arterial pressure at the lowest dose of Ang II (200 ng/kg per minute) that increased mean arterial pressure. This loss in Ang II resistance is independent of T cells. Angiotensin type 1-receptor binding was 1.4-fold higher in glomeruli isolated from recently purchased B6. Rag1 -/- -M suggesting an increase in renal angiotensin type 1-receptor activity masks the blood pressure protection afforded by the lack of T cells. The phenotypic change in B6. Rag1 -/- -M has implications for investigators using this strain to study mechanisms of T-cell modulation of Ang II-dependent blood pressure control. These findings also serve as a reminder that the universal drive for genetic variation occurs in all animals including inbred mouse strains and that spontaneous mutations leading to phenotypic change can compromise experimental reproducibility over time and place. Finally, these observations illustrate the importance of including experimental details about the location and time period over which animals are bred in publications involving animal studies to promote rigor and reproducibility in the scientific literature. © 2017 American Heart Association, Inc.

  12. Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

    Science.gov (United States)

    Das, Theerthankar; Ibugo, Amaye; Buckle, Edwina; Manefield, Mike; Manos, Jim

    2016-01-01

    Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies. PMID:27161630

  13. Extracellular deoxyribonuclease production by periodontal bacteria.

    Science.gov (United States)

    Palmer, L J; Chapple, I L C; Wright, H J; Roberts, A; Cooper, P R

    2012-08-01

    Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis. © 2011 John Wiley & Sons A/S.

  14. Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes

    Science.gov (United States)

    López-Garriga, Juan; Cadilla, Carmen L.

    2016-01-01

    The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5’end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments. PMID:26824233

  15. Machine Learning-Assisted Network Inference Approach to Identify a New Class of Genes that Coordinate the Functionality of Cancer Networks.

    Science.gov (United States)

    Ghanat Bari, Mehrab; Ung, Choong Yong; Zhang, Cheng; Zhu, Shizhen; Li, Hu

    2017-08-01

    Emerging evidence indicates the existence of a new class of cancer genes that act as "signal linkers" coordinating oncogenic signals between mutated and differentially expressed genes. While frequently mutated oncogenes and differentially expressed genes, which we term Class I cancer genes, are readily detected by most analytical tools, the new class of cancer-related genes, i.e., Class II, escape detection because they are neither mutated nor differentially expressed. Given this hypothesis, we developed a Machine Learning-Assisted Network Inference (MALANI) algorithm, which assesses all genes regardless of expression or mutational status in the context of cancer etiology. We used 8807 expression arrays, corresponding to 9 cancer types, to build more than 2 × 10 8 Support Vector Machine (SVM) models for reconstructing a cancer network. We found that ~3% of ~19,000 not differentially expressed genes are Class II cancer gene candidates. Some Class II genes that we found, such as SLC19A1 and ATAD3B, have been recently reported to associate with cancer outcomes. To our knowledge, this is the first study that utilizes both machine learning and network biology approaches to uncover Class II cancer genes in coordinating functionality in cancer networks and will illuminate our understanding of how genes are modulated in a tissue-specific network contribute to tumorigenesis and therapy development.

  16. Identification and characterization of polymorphisms at the HSA a1-acid glycoprotein (ORM* gene locus in Caucasians

    Directory of Open Access Journals (Sweden)

    Owczarek Catherine M.

    2002-01-01

    Full Text Available Human alpha1-acid glycoprotein (AGP or orosomucoid (ORM is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a panel of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Non-random associations were found between the BamHI, EcoRI, BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the indiviuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms will prove useful for other population and forensic studies.

  17. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  18. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Actin is closely associated with RNA polymerase II and involved in activation of gene transcription

    International Nuclear Information System (INIS)

    Zhu Xiaojuan; Zeng Xianlu; Huang Baiqu; Hao, Shui

    2004-01-01

    Biochemical and morphological studies have demonstrated the presence of actin in the nucleus of different eukaryotic cells, whereas its role remains unclear. In this work, we studied the interaction and the functional relationship between nuclear actin and RNA polymerase II (RNAP II). The immunofluorescence study demonstrated a clear co-localization of nuclear actin with RNAP II in HeLa cells. Meanwhile, actin can be immunoprecipitated by anti-RNAP II antibody, indicating that they could interact with each other. Treatment of cells with α-amanitin induced the formation of actin bundle network in the nucleoplasm. Blocking of the formation of filamentous actin (F-actin) by cytochalasin B modified the distribution of actin. Although the actin content remained unchanged in resting and concanavalinA stimulated mouse lymphocytes, the actin content in the nuclei showed a progressive increase after stimulation. Furthermore, the antibody against actin blocked RNA synthesis in a eukaryotic in vitro transcription system. These observations implicate that nuclear actin interacts with RNAP II and may have function on the RNAP II-mediated transcription

  20. Case reports of juvenile GM1 gangliosidosisis type II caused by mutation in GLB1 gene.

    Science.gov (United States)

    Karimzadeh, Parvaneh; Naderi, Samaneh; Modarresi, Farzaneh; Dastsooz, Hassan; Nemati, Hamid; Farokhashtiani, Tayebeh; Shamsian, Bibi Shahin; Inaloo, Soroor; Faghihi, Mohammad Ali

    2017-07-17

    Type II or juvenile GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder, which is clinically distinct from infantile form of the disease by the lack of characteristic cherry-red spot and hepatosplenomegaly. The disease is characterized by slowly progressive neurodegeneration and mild skeletal changes. Due to the later age of onset and uncharacteristic presentation, diagnosis is frequently puzzled with other ataxic and purely neurological disorders. Up to now, 3-4 types of GM1-gangliosidosis have been reported and among them type I is the most common phenotype with the age of onset around 6 months. Various forms of GM1-gangliosidosis are caused by GLB1 gene mutations but severity of the disease and age of onset are directly related to the position and the nature of deleterious mutations. However, due to its unique genetic cause and overlapping clinical features, some researchers believe that GM1 gangliosidosis represents an overlapped disease spectrum instead of four distinct types. Here, we report a less frequent type of autosomal recessive GM1 gangliosidosis with perplexing clinical presentation in three families in the southwest part of Iran, who are unrelated but all from "Lurs" ethnic background. To identify disease-causing mutations, Whole Exome Sequencing (WES) utilizing next generation sequencing was performed. Four patients from three families were investigated with the age of onset around 3 years old. Clinical presentations were ataxia, gate disturbances and dystonia leading to wheelchair-dependent disability, regression of intellectual abilities, and general developmental regression. They all were born in consanguineous families with no previous documented similar disease in their parents. A homozygote missense mutation in GLB1 gene (c. 601 G > A, p.R201C) was found in all patients. Using Sanger sequencing this identified mutation was confirmed in the proband, their parents, grandparents, and extended family members, confirming

  1. Association of IL-6 and MMP-3 gene polymorphisms with ...

    Indian Academy of Sciences (India)

    comprehensively and systematically performed this meta-analysis to detect whether the two gene polymorphisms are corre- .... control studies; (ii) 'failure to provide the genes' distribution details ..... biological activity of corresponding proteins such as IL-6 .... pathic scoliosis is related to inadequate calcium intake and weight.

  2. A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice.

    Science.gov (United States)

    Takahashi, T; Guron, C; Shetty, S; Matsui, H; Raghow, R

    1997-09-05

    To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient

  3. Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II ...

    Indian Academy of Sciences (India)

    Unknown

    Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II) Schiff base complexes derived from o-phenylenediamine and acetoacetanilide. N RAMAN*, Y PITCHAIKANI RAJA and A KULANDAISAMY. Department of Chemistry, VHNSN College, Virudhunagar 626 001, India e-mail: ra_man@123india.com.

  4. Activation of vitellogenin II gene expression by steroid hormones in the old Japanese quail.

    Science.gov (United States)

    Gupta, S; Upadhyay, R; Kanungo, M S

    1998-11-01

    Alterations in the basal transcription rates of eukaryotic genes are believed to involve the binding of trans-acting factor(s) with specific DNA sequences in the promoter. We show here two interrelated events for the VTGII gene of the old, non-egg laying Japanese quail: alterations in the structure of the chromatin encompassing the gene, and binding of trans-acting factors to the promoter of the gene. Estradiol/progesterone alone or together cause alterations in the conformation of the chromatin of the promoter region of the gene. This may allow free access of nuclear protein(s) to the cis-acting elements, ERE, PRE and NF1, in the promoter of the gene and cause activation of transcription.

  5. Distribution of genes associated with yield potential and water ...

    Indian Academy of Sciences (India)

    Supplementary data: Distribution of genes associated with yield potential and water-saving in. Chinese Zone II wheat detected by developed functional markers. Zhenxian Gao, Zhanliang Shi, Aimin Zhang and Jinkao Guo. J. Genet. 94, 35–42. Table 1. Functional markers for high-yield or water-saving genes in wheat and ...

  6. HCV Proteins and Immunoglobulin Variable Gene (IgV Subfamilies in HCV-Induced Type II Mixed Cryoglobulinemia: A Concurrent Pathogenetic Role

    Directory of Open Access Journals (Sweden)

    Giuseppe Sautto

    2012-01-01

    Full Text Available The association between hepatitis C virus (HCV infection and type II mixed cryoglobulinemia (MCII is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV gene subfamilies frequently endowed with rheumatoid factor (RF activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

  7. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin

    2015-01-01

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  8. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun

    2015-09-27

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  9. redD and actII-ORF4, Pathway-Specific Regulatory Genes for Antibiotic Production in Streptomyces coelicolor A3(2), Are Transcribed In Vitro by an RNA Polymerase Holoenzyme Containing σhrdD

    NARCIS (Netherlands)

    Fujii, T.; Gramajo, H.C.; Takano, E.; Bibb, M.J.

    1996-01-01

    redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing σhrdD. Disruption of hrdD had no effect on antibiotic production, indicating that

  10. Biologically active new Fe(II, Co(II, Ni(II, Cu(II, Zn(II and Cd(II complexes of N-(2-thienylmethylenemethanamine

    Directory of Open Access Journals (Sweden)

    C. SPÎNU

    2008-04-01

    Full Text Available Iron(II, cobalt(II, nickel (II, copper (II, zinc(II and cadmium(II complexes of the type ML2Cl2, where M is a metal and L is the Schiff base N-(2-thienylmethylenemethanamine (TNAM formed by the condensation of 2-thiophenecarboxaldehyde and methylamine, were prepared and characterized by elemental analysis as well as magnetic and spectroscopic measurements. The elemental analyses suggest the stoichiometry to be 1:2 (metal:ligand. Magnetic susceptibility data coupled with electronic, ESR and Mössbauer spectra suggest a distorted octahedral structure for the Fe(II, Co(II and Ni(II complexes, a square-planar geometry for the Cu(II compound and a tetrahedral geometry for the Zn(II and Cd(II complexes. The infrared and NMR spectra of the complexes agree with co-ordination to the central metal atom through nitrogen and sulphur atoms. Conductance measurements suggest the non-electrolytic nature of the complexes, except for the Cu(II, Zn(II and Cd(II complexes, which are 1:2 electrolytes. The Schiff base and its metal chelates were screened for their biological activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and the metal chelates were found to possess better antibacterial activity than that of the uncomplexed Schiff base.

  11. Reassignment of Drosophila willistoni Genome Scaffolds to Chromosome II Arms.

    Science.gov (United States)

    Garcia, Carolina; Delprat, Alejandra; Ruiz, Alfredo; Valente, Vera L S

    2015-10-04

    Drosophila willistoni is a geographically widespread Neotropical species. The genome of strain Gd-H4-1 from Guadeloupe Island (Caribbean) was sequenced in 2007 as part of the 12 Drosophila Genomes Project. The assembled scaffolds were joined based on conserved linkage and assigned to polytene chromosomes based on a handful of genetic and physical markers. This paucity of markers was particularly striking in the metacentric chromosome II, comprised two similarly sized arms, IIL and IIR, traditionally considered homologous to Muller elements C and B, respectively. In this paper we present the cytological mapping of 22 new gene markers to increase the number of markers mapped by in situ hybridization and to test the assignment of scaffolds to the polytene chromosome II arms. For this purpose, we generated, by polymerase chain reaction amplification, one or two gene probes from each scaffold assigned to the chromosome II arms and mapped these probes to the Gd-H4-1 strain's polytene chromosomes by nonfluorescent in situ hybridization. Our findings show that chromosome arms IIL and IIR correspond to Muller elements B and C, respectively, directly contrasting the current homology assignments in D. willistoni and constituting a major reassignment of the scaffolds to chromosome II arms. Copyright © 2015 Garcia et al.

  12. Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica.

    Science.gov (United States)

    Nishita, Y; Abramov, A V; Kosintsev, P A; Lin, L-K; Watanabe, S; Yamazaki, K; Kaneko, Y; Masuda, R

    2015-12-01

    Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Transactivation mediated by B-Myb is dependent on TAF(II)250.

    Science.gov (United States)

    Bartusel, Thorsten; Klempnauer, Karl-Heinz

    2003-05-15

    B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. We have employed the hamster cell line ts13 as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells.

  14. Low molecular weight thiols and thioredoxins are important players in Hg(II) resistance in Thermus thermophilus HB27.

    Science.gov (United States)

    Norambuena, J; Wang, Y; Hanson, T; Boyd, J M; Barkay, T

    2017-11-17

    Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low molecular weight and protein thiols may be important cell components used in Hg resistance. To date, the role of low molecular weight thiols in Hg-detoxification remains understudied. The mercury resistance ( mer ) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low molecular weight thiol metabolism. This mer operon encodes for an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low molecular weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in presence of either a thiol oxidizing agent or a thiol alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus spp. mer operons. The presence of a thiol related gene was also detected in some alphaprotobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II) buffering agents while Hg is reduced by MerA. Importance The survival of microorganisms in presence of toxic metals is central to life's sustainability. The affinity of thiol groups to toxic heavy metals drives microbe-metal interactions and modulate metal toxicity. Mercury detoxification ( mer ) genes likely originated early in microbial evolution among geothermal environments. Little is

  15. Selection of reference genes for expression studies with fish myogenic cell cultures

    Directory of Open Access Journals (Sweden)

    Johnston Ian A

    2009-08-01

    Full Text Available Abstract Background Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.. The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Results Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1α, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. Conclusion The geometric average of any three of Hprt1, Ef1α, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

  16. Selection of reference genes for expression studies with fish myogenic cell cultures.

    Science.gov (United States)

    Bower, Neil I; Johnston, Ian A

    2009-08-10

    Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

  17. Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii

    Directory of Open Access Journals (Sweden)

    Plemenitaš Ana

    2007-08-01

    Full Text Available Abstract Background Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. Results A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. Conclusion Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at

  18. Complexes of cobalt(II), nickel(II), copper(II), zinc(II), cadmium(II) and dioxouranium(II) with thiophene-2-aldehydethiosemicarbazone

    International Nuclear Information System (INIS)

    Singh, Balwan; Misra, Harihar

    1986-01-01

    Metal complexes of thiosemicarbazides have been known for their pharmacological applications. Significant antitubercular, fungicidal and antiviral activities have been reported for thiosemicarbazides and their derivatives. The present study describes the systhesis and characterisation of complexes of Co II , Cu II , Zn II ,Cd II and UO II with thiosemicarbazone obtained by condensing thiophene-2-aldehyde with thiosemicarbazide. 17 refs., 2 tables. (author)

  19. Altered gene activity correlated with long-term memory formation of conditioned taste aversion in Lymnaea.

    Science.gov (United States)

    Azami, Sachiyo; Wagatsuma, Akiko; Sadamoto, Hisayo; Hatakeyama, Dai; Usami, Takeshi; Fujie, Manabu; Koyanagi, Ryo; Azumi, Kaoru; Fujito, Yutaka; Lukowiak, Ken; Ito, Etsuro

    2006-11-15

    The pond snail Lymnaea stagnalis is capable of learning conditioned taste aversion (CTA) and then consolidating that learning into long-term memory (LTM) that persists for at least 1 month. LTM requires de novo protein synthesis and altered gene activity. Changes in gene activity in Lymnaea that are correlated with, much less causative, memory formation have not yet been identified. As a first step toward rectifying this situation, we constructed a cDNA microarray with mRNAs extracted from the central nervous system (CNS) of Lymnaea. We then, using this microarray assay, identified genes whose activity either increased or decreased following CTA memory consolidation. We also identified genes whose expression levels were altered after inhibition of the cyclic AMP response element-binding protein (CREB) that is hypothesized to be a key transcription factor for CTA memory. We found that the molluscan insulin-related peptide II (MIP II) was up-regulated during CTA-LTM, whereas the gene encoding pedal peptide preprohormone (Pep) was down-regulated by CREB2 RNA interference. We next examined mRNAs of MIP II and Pep using real-time RT-PCR with SYBR Green. The MIP II mRNA level in the CNS of snails exhibiting "good" memory for CTA was confirmed to be significantly higher than that from the CNS of snails exhibiting "poor" memory. In contrast, there was no significant difference in expression levels of the Pep mRNA between "good" and "poor" performers. These data suggest that in Lymnaea MIP II may play a role in the consolidation process that forms LTM following CTA training.

  20. Human gene therapy and imaging: cardiology

    International Nuclear Information System (INIS)

    Wu, Joseph C.; Yla-Herttuala, Seppo

    2005-01-01

    This review discusses the basics of cardiovascular gene therapy, the results of recent human clinical trials, and the rapid progress in imaging techniques in cardiology. Improved understanding of the molecular and genetic basis of coronary heart disease has made gene therapy a potential new alternative for the treatment of cardiovascular diseases. Experimental studies have established the proof-of-principle that gene transfer to the cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of feasibility and safety of cardiovascular gene therapy. However, phase II/III clinical trials have so far been rather disappointing and one of the major problems in cardiovascular gene therapy has been the inability to verify gene expression in the target tissue. New imaging techniques could significantly contribute to the development of better gene therapeutic approaches. Although the exact choice of imaging modality will depend on the biological question asked, further improvement in image resolution and detection sensitivity will be needed for all modalities as we move from imaging of organs and tissues to imaging of cells and genes. (orig.)

  1. Evaluation of hyaluronic acid-combined ternary complexes for serum-resistant and targeted gene delivery system.

    Science.gov (United States)

    Hong, Woong-Gil; Jeong, Gyeong-Won; Nah, Jae-Woon

    2018-04-19

    Branched polyethylenimine (bPEI) was well known as high transfection agent, which has many amine group. However, utilization of bPEI was limited due to high toxicity. To solve these problems, bPEI was introduced to low molecular weight water-soluble chitosan (LMWSC) with coupling agent. In addition, hyaluronic acid (HA), one of natural anion polymer, was introduced to binary complex of pDNA/bPEI-grafted LMWSC (LMPEI) to target the specific cancer cell and impart the serum resistant. Ternary complexes of pDNA/LMPEI/HA were prepared by electrostatic charge interaction and their binding affinity and DNase protection assay were conducted by gel retardation assay. Particle size of ternary complexes showed that had each 482 ± 245.4 (pDNA/LMPEI2%/HA, 1:16:1, w/w/w) and 410 ± 78.5 nm (pDNA/LMPEI4%/HA, 1:16:2, w/w/w). Moreover, to demonstrate serum-resistant effect of ternary complexes, particle size of them was measured according to incubated time (0-10 h) under serum condition. Transfection assay of ternary complexes showed that their transfection efficiency in CD44-receptor overexpressed HCT116 cell was higher than CD44-receptor negative CT26 cell. Additionally, intracellular uptake of ternary complexes with propidium iodide (PI)-labeled pDNA was observed to confirm targeting effect and cellular internalization by fluorescence microscopy. These results suggest that ternary complexes are superb gene carrier with excellent serum-resistant and high gene transfection. Copyright © 2017. Published by Elsevier B.V.

  2. Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

    Science.gov (United States)

    Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

    1999-09-01

    We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

  3. Clinical and genetic investigation of families with type II Waardenburg syndrome.

    Science.gov (United States)

    Chen, Yong; Yang, Fuwei; Zheng, Hexin; Zhou, Jianda; Zhu, Ganghua; Hu, Peng; Wu, Weijing

    2016-03-01

    The present study aimed to investigate the molecular pathology of Waardenburg syndrome type II in three families, in order to provide genetic diagnosis and hereditary counseling for family members. Relevant clinical examinations were conducted on the probands of the three pedigrees. Peripheral blood samples of the probands and related family members were collected and genomic DNA was extracted. The coding sequences of paired box 3 (PAX3), microphthalmia‑associated transcription factor (MITF), sex‑determining region Y‑box 10 (SOX10) and snail family zinc finger 2 (SNAI2) were analyzed by polymerase chain reaction and DNA sequencing. The heterozygous mutation, c.649_651delAGA in exon 7 of the MITF gene was detected in the proband and all patients of pedigree 1; however, no pathological mutation of the relevant genes (MITF, SNAI2, SOX10 or PAX3) was detected in pedigrees 2 and 3. The heterozygous mutation c.649_651delAGA in exon 7 of the MITF gene is therefore considered the disease‑causing mutation in pedigree 1. However, there are novel disease‑causing genes in Waardenburg syndrome type II, which require further research.

  4. MspI and PvuII polymorphisms in the Na,K-ATPase. alpha. subunit related gene ATP1AL1

    Energy Technology Data Exchange (ETDEWEB)

    Shull, M.M.; Pugh, D.G.; Lingrel, J.B. (Univ. of Cincinnati, OH (USA))

    1990-01-11

    ATP1AL1 78-1-3 is a 0.56 kb genomic EcoRI-XbaI fragment from within the Na,K-ATPase {alpha} subunit related gene, previously referred to as {alpha}D on chromosome 13. The fragment was subcloned into pIBI31. MspI identifies a two-allele polymorphism (M1: 2.8 kb, M2: 2.5 kb). PvuII, which cuts within the probe sequence, detects two two-allele polymorphism (A1: 6.0 kb, A2: 5.7 kb, B1: 1.3 kb, B2: 1.1 kb). A1 and A2 appear to result from an insertion/deletion polymorphism that is also identified by MspI. ATP1AL1 78-1-3 has been assigned to chromosome 13q by somatic cell hybrid analysis. Codominant segregation of the RELPs was observed in 2 two-generation families.

  5. Experiment list: SRX189413 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available M1008583: Duke DnaseSeq Cerebellum OC source_name=Cerebellum_OC || biomaterial_provider=NICHD || datatype=Dn...neric_male,p-value cutoff: 0.05 || replicate=1,2 || cell=Cerebellum_OC || cell or...SRX189413 hg19 DNase-seq DNase-Seq Neural Adult brains NA 394358920,0.0,11.4,115 GS

  6. Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking.

    Science.gov (United States)

    Joshee, Nirmal; Bastola, Dhundy R; Cheng, Pi-Wan

    2002-11-01

    We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.

  7. EcoGene 3.0.

    Science.gov (United States)

    Zhou, Jindan; Rudd, Kenneth E

    2013-01-01

    EcoGene (http://ecogene.org) is a database and website devoted to continuously improving the structural and functional annotation of Escherichia coli K-12, one of the most well understood model organisms, represented by the MG1655(Seq) genome sequence and annotations. Major improvements to EcoGene in the past decade include (i) graphic presentations of genome map features; (ii) ability to design Boolean queries and Venn diagrams from EcoArray, EcoTopics or user-provided GeneSets; (iii) the genome-wide clone and deletion primer design tool, PrimerPairs; (iv) sequence searches using a customized EcoBLAST; (v) a Cross Reference table of synonymous gene and protein identifiers; (vi) proteome-wide indexing with GO terms; (vii) EcoTools access to >2000 complete bacterial genomes in EcoGene-RefSeq; (viii) establishment of a MySql relational database; and (ix) use of web content management systems. The biomedical literature is surveyed daily to provide citation and gene function updates. As of September 2012, the review of 37 397 abstracts and articles led to creation of 98 425 PubMed-Gene links and 5415 PubMed-Topic links. Annotation updates to Genbank U00096 are transmitted from EcoGene to NCBI. Experimental verifications include confirmation of a CTG start codon, pseudogene restoration and quality assurance of the Keio strain collection.

  8. EcoGene 3.0

    Science.gov (United States)

    Zhou, Jindan; Rudd, Kenneth E.

    2013-01-01

    EcoGene (http://ecogene.org) is a database and website devoted to continuously improving the structural and functional annotation of Escherichia coli K-12, one of the most well understood model organisms, represented by the MG1655(Seq) genome sequence and annotations. Major improvements to EcoGene in the past decade include (i) graphic presentations of genome map features; (ii) ability to design Boolean queries and Venn diagrams from EcoArray, EcoTopics or user-provided GeneSets; (iii) the genome-wide clone and deletion primer design tool, PrimerPairs; (iv) sequence searches using a customized EcoBLAST; (v) a Cross Reference table of synonymous gene and protein identifiers; (vi) proteome-wide indexing with GO terms; (vii) EcoTools access to >2000 complete bacterial genomes in EcoGene-RefSeq; (viii) establishment of a MySql relational database; and (ix) use of web content management systems. The biomedical literature is surveyed daily to provide citation and gene function updates. As of September 2012, the review of 37 397 abstracts and articles led to creation of 98 425 PubMed-Gene links and 5415 PubMed-Topic links. Annotation updates to Genbank U00096 are transmitted from EcoGene to NCBI. Experimental verifications include confirmation of a CTG start codon, pseudogene restoration and quality assurance of the Keio strain collection. PMID:23197660

  9. [Gene deletion and functional analysis of the heptyl glycosyltransferase (waaF) gene in Vibrio parahemolyticus O-antigen cluster].

    Science.gov (United States)

    Zhao, Feng; Meng, Songsong; Zhou, Deqing

    2016-02-04

    To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

  10. c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

    Science.gov (United States)

    Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

    2001-07-06

    c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

  11. Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Wang Xiaoyi

    2009-06-01

    Full Text Available Abstract Background The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.

  12. Identification of Early Response Genes in Human Peripheral Leukocytes Infected with Orientia tsutsugamushi: The Emergent of a Unique Gene Expression Profile for Diagnosis of O. tsutsugamush Infection

    Science.gov (United States)

    2010-01-01

    all found in Homo sapiens and the biological processes were assigned based on human protein reference database (HPRD, www.hprd.org). Gene names in...the following: i) whether infection by O. tsutsugamushi is accompanied by distinct gene expression profiles; ii) which features of the host

  13. Mn(II), Zn(II) and VO(II) Schiff

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 113; Issue 3. Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II) Schiff base complexes derived from o-phenylenediamine and acetoacetanilide. N Raman Y Pitchaikani Raja A Kulandaisamy. Inorganic Volume 113 Issue 3 June 2001 pp 183-189 ...

  14. Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Han, Li; Huang, Xueshi [Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819 (China); He, Jing, E-mail: hejingjj@mail.hzau.edu.cn [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China)

    2016-04-22

    Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

  15. Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

    International Nuclear Information System (INIS)

    Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan; Han, Li; Huang, Xueshi; He, Jing

    2016-01-01

    Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

  16. ORF Sequence: ch_oct10_gene_aa_db [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available GTGMKSFLEKLDEATKEFETQYKKWINDRREAIKKQRENEKLQKWNEISNIFKSDGVELNRDAQTPCIPEHLVEGFEESNESEDLSEIDQIEQVMLNPKGRLNFV* ... ch_oct10_gene_aa_db Chro.50191 >Chro.50191 hypothetical protein MSNSFLRDLKFVGVSSFL

  17. An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease.

    Science.gov (United States)

    Pan, Rong; Zhou, Yuan; He, Hai-Jin; He, Rong-Qiao

    2011-04-01

    The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5'-phosphate dsDNA (5'P DNA) and degrades it without sequence specificity, but does not recognize 5'OH DNA. As is the case for most DNases, Mg(2+) was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

    Science.gov (United States)

    Wong, J M; Ingles, C J

    2001-02-01

    Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

  19. Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

    Science.gov (United States)

    Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

    2004-06-01

    Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

  20. Mutations in the VLGR1 Gene Implicate G-Protein Signaling in the Pathogenesis of Usher Syndrome Type II

    Science.gov (United States)

    Weston, Michael D.; Luijendijk, Mirjam W. J.; Humphrey, Kurt D.; Möller, Claes; Kimberling, William J.

    2004-01-01

    Usher syndrome type II (USH2) is a genetically heterogeneous autosomal recessive disorder with at least three genetic subtypes (USH2A, USH2B, and USH2C) and is classified phenotypically as congenital hearing loss and progressive retinitis pigmentosa. The VLGR1 (MASS1) gene in the 5q14.3-q21.1 USH2C locus was considered a likely candidate on the basis of its protein motif structure and expressed-sequence-tag representation from both cochlear and retinal subtracted libraries. Denaturing high-performance liquid chromatography and direct sequencing of polymerase-chain-reaction products amplified from 10 genetically independent patients with USH2C and 156 other patients with USH2 identified four isoform-specific VLGR1 mutations (Q2301X, I2906FS, M2931FS, and T6244X) from three families with USH2C, as well as two sporadic cases. All patients with VLGR1 mutations are female, a significant deviation from random expectations. The ligand(s) for the VLGR1 protein is unknown, but on the basis of its potential extracellular and intracellular protein-protein interaction domains and its wide mRNA expression profile, it is probable that VLGR1 serves diverse cellular and signaling processes. VLGR1 mutations have been previously identified in both humans and mice and are associated with a reflex-seizure phenotype in both species. The identification of additional VLGR1 mutations to test whether a phenotype/genotype correlation exists, akin to that shown for other Usher syndrome disease genes, is warranted. PMID:14740321

  1. Expression of the KNOTTED HOMEOBOX Genes in the Cactaceae Cambial Zone Suggests Their Involvement in Wood Development.

    Science.gov (United States)

    Reyes-Rivera, Jorge; Rodríguez-Alonso, Gustavo; Petrone, Emilio; Vasco, Alejandra; Vergara-Silva, Francisco; Shishkova, Svetlana; Terrazas, Teresa

    2017-01-01

    The vascular cambium is a lateral meristem that produces secondary xylem (i.e., wood) and phloem. Different Cactaceae species develop different types of secondary xylem; however, little is known about the mechanisms underlying wood formation in the Cactaceae. The KNOTTED HOMEOBOX (KNOX) gene family encodes transcription factors that regulate plant development. The role of class I KNOX genes in the regulation of the shoot apical meristem, inflorescence architecture, and secondary growth is established in a few model species, while the functions of class II KNOX genes are less well understood, although the Arabidopsis thaliana class II KNOX protein KNAT7 is known to regulate secondary cell wall biosynthesis. To explore the involvement of the KNOX genes in the enormous variability of wood in Cactaceae, we identified orthologous genes expressed in species with fibrous ( Pereskia lychnidiflora and Pilosocereus alensis ), non-fibrous ( Ariocarpus retusus ), and dimorphic ( Ferocactus pilosus ) wood. Both class I and class II KNOX genes were expressed in the cactus cambial zone, including one or two class I paralogs of KNAT1 , as well as one or two class II paralogs of KNAT3 - KNAT4 - KNAT5 . While the KNOX gene SHOOTMERISTEMLESS ( STM) and its ortholog ARK1 are expressed during secondary growth in the Arabidopsis and Populus stem, respectively, we did not find STM orthologs in the Cactaceae cambial zone, which suggests possible differences in the vascular cambium genetic regulatory network in these species. Importantly, while two class II KNOX paralogs from the KNAT7 clade were expressed in the cambial zone of A. retusus and F. pilosus , we did not detect KNAT7 ortholog expression in the cambial zone of P. lychnidiflora . Differences in the transcriptional repressor activity of secondary cell wall biosynthesis by the KNAT7 orthologs could therefore explain the differences in wood development in the cactus species.

  2. Molecular characterization of the porcine surfactant, pulmonary-associated protein C gene

    DEFF Research Database (Denmark)

    Cirera, S.; Nygård, A.B.; Jensen, H.E.

    2006-01-01

    The surfactant, pulmonary-associated protein C (SFTPC) is a peptide secreted by the alveolar type II pneumocytes of the lung. We have characterized the porcine SFTPC gene at genomic, transcriptional, and protein levels. The porcine SFTPC is a single-copy gene on pig chromosome 14. Two transcripts...

  3. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    Science.gov (United States)

    2011-07-01

    melanoma, renal carcinoma and breast cancer cell lines. Furthermore, we show that neither HIF-1a protein levels nor hypoxic response through an HRE ...constitutively expressed gene to control for equivalent DNase digestion between the cell lines examined. HRE -Luciferase assay Cell lines ,85% confluent in 60...mm dishes were transfected with an HRE -luciferase reporter vector [19] (2.5 mg) and Renilla luciferase (1.5 mg) according to Lipofectamine 2000

  4. An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation.

    Science.gov (United States)

    Sabbattini, Pierangela; Sjoberg, Marcela; Nikic, Svetlana; Frangini, Alberto; Holmqvist, Per-Henrik; Kunowska, Natalia; Carroll, Tom; Brookes, Emily; Arthur, Simon J; Pombo, Ana; Dillon, Niall

    2014-03-01

    Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

  5. Scavenger receptor AI/II truncation, lung function and COPD: a large population-based study

    DEFF Research Database (Denmark)

    Thomsen, M; Nordestgaard, B G; Tybjærg-Hansen, Anne

    2011-01-01

    The scavenger receptor A-I/II (SRA-I/II) on alveolar macrophages is involved in recognition and clearance of modified lipids and inhaled particulates. A rare variant of the SRA-I/II gene, Arg293X, truncates the distal collagen-like domain, which is essential for ligand recognition. We tested...... whether the Arg293X variant is associated with reduced lung function and risk of chronic obstructive pulmonary disease (COPD) in the general population....

  6. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2016-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

  7. Controversy Associated With the Common Component of Most Transgenic Plants – Kanamycin Resistance Marker Gene

    Directory of Open Access Journals (Sweden)

    Srećko Jelenić

    2003-01-01

    Full Text Available Plant genetic engineering is a powerful tool for producing crops resistant to pests, diseases and abiotic stress or crops with improved nutritional value or better quality products. Currently over 70 genetically modified (GM crops have been approved for use in different countries. These cover a wide range of plant species with significant number of different modified traits. However, beside the technology used for their improvement, the common component of most GM crops is the neomycin phosphotransferase II gene (nptII, which confers resistance to the antibiotics kanamycin and neomycin. The nptII gene is present in GM crops as a marker gene to select transformed plant cells during the first steps of the transformation process. The use of antibiotic-resistance genes is subject to controversy and intense debate, because of the likelihood that clinical therapy could be compromised due to inactivation of the oral dose of the antibiotic from consumption of food derived from the transgenic plant, and because of the risk of gene transfer from plants to gut and soil microorganisms or to consumer’s cells. The present article discusses these possibilities in the light of current scientific knowledge.

  8. Adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II), and As(V) on bacterially produced metal sulfides.

    Science.gov (United States)

    Jong, Tony; Parry, David L

    2004-07-01

    The adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II) and As(V) onto bacterially produced metal sulfide (BPMS) material was investigated using a batch equilibrium method. It was found that the sulfide material had adsorptive properties comparable with those of other adsorbents with respect to the specific uptake of a range of metals and, the levels to which dissolved metal concentrations in solution can be reduced. The percentage of adsorption increased with increasing pH and adsorbent dose, but decreased with increasing initial dissolved metal concentration. The pH of the solution was the most important parameter controlling adsorption of Cd(II), Cu(II), Fe(II), Ni(II), Pb(II), Zn(II), and As(V) by BPMS. The adsorption data were successfully modeled using the Langmuir adsorption isotherm. Desorption experiments showed that the reversibility of adsorption was low, suggesting high-affinity adsorption governed by chemisorption. The mechanism of adsorption for the divalent metals was thought to be the formation of strong, inner-sphere complexes involving surface hydroxyl groups. However, the mechanism for the adsorption of As(V) by BPMS appears to be distinct from that of surface hydroxyl exchange. These results have important implications to the management of metal sulfide sludge produced by bacterial sulfate reduction.

  9. Primary structure of the tms and prs genes of Bacillus subtilis

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne; Arnvig, Kirsten

    1989-01-01

    The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene...... products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit Mr of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an Mr of 49,554. Comparison of the deduced B. subtilis PRPP...

  10. Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence.

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-03-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40 degrees C compared to cells grown at 30 degrees C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter P(hupA), and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection.

  11. Regulation of the Vibrio vulnificus hupA Gene by Temperature Alteration and Cyclic AMP Receptor Protein and Evaluation of Its Role in Virulence▿

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-01-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40°C compared to cells grown at 30°C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter PhupA, and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection. PMID:19139193

  12. Pulmonary artery hypertension in childhood: The transforming growth factor-β superfamily-related genes

    Directory of Open Access Journals (Sweden)

    Shi-Min Yuan

    2018-04-01

    Full Text Available Pulmonary artery hypertension (PAH is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia, caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH. Key Words: bone morphogenetic proteins, mutation, pulmonary hypertension

  13. Cry3A δ-endotoxin gene mutagenized for enhanced toxicity

    African Journals Online (AJOL)

    Bacillus thuringiensis Cry3A gene was redesigned for high expression in Norwegian spruce and the sequence was slightly modified to allow for simple N- and C- terminal deletions and domain II loop 1 exchange for synthetic oligos. Modified Cry3A toxins from 13 variants of the synthetic gene were expressed in Escherichia ...

  14. The Effect of MHC Class II Transactivator on the Growth and Metastasis of Breast Tumors

    Science.gov (United States)

    1999-06-01

    F. Manca, and R. S. Accolla. 1998. HLA class II expression in uninducible hepatocarcinoma cells after transfection of AIR-1 gene product CIITA...Cestari, A. D’Agostino, ’ A M Megiovanni, F. Manca, and R. S. Accolla. 1998. HLA class II expression in uninducible hepatocarcinoma cells after

  15. Natural Selection in Virulence Genes of Francisella tularensis.

    Science.gov (United States)

    Gunnell, Mark K; Robison, Richard A; Adams, Byron J

    2016-06-01

    A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution

  16. Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity

    Directory of Open Access Journals (Sweden)

    Songjoon Baek

    2017-05-01

    Full Text Available In response to activating signals, transcription factors (TFs bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint. Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot, efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq, all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined.

  17. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.

    Science.gov (United States)

    Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

    2007-11-01

    Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

  18. MHC class II deficiency: Report of a novel mutation and special review.

    Science.gov (United States)

    Farrokhi, S; Shabani, M; Aryan, Z; Zoghi, S; Krolo, A; Boztug, K; Rezaei, N

    The MHC II deficiency is a rare autosomal recessive primary immunodeficiency syndrome with increased susceptibility to respiratory and gastrointestinal infections, failure to thrive and early mortality. This syndrome is caused by mutations in transcription regulators of the MHC II gene and results in development of blind lymphocytes due to the lack of indicatory MHC II molecules. Despite homogeneity of clinical manifestations of patients with MHC II deficiency, the genetic defects underlying this disease are heterogeneous. Herein, we report an Iranian patient with MHC II deficiency harbouring a novel mutation in RFXANK and novel misleading clinical features. He had ataxic gait and dysarthria from 30 months of age. Epidemiology, clinical and immunological features, therapeutic options and prognosis of patients with MHC II are reviewed in this paper. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

  19. Renin-angiotensin system inhibitors, angiotensin I-converting enzyme gene insertion/deletion polymorphism, and cancer: The Rotterdam study

    NARCIS (Netherlands)

    R. van der Knaap (Ronald); C. Siemes (Claire); J.W.W. Coebergh (Jan Willem); P. Tikka-Kleemola (Päivi); A. Hofman (Albert); B.H.Ch. Stricker (Bruno)

    2008-01-01

    textabstractBACKGROUND. Angiotensin I-converting enzyme (ACE) inhibitors, angiotensin II antagonists, and the ACE insertion/deletion (I/D) gene polymorphism all influence serum angiotensin II action. Because angiotensin II levels have been associated with cancer, the objective of the current

  20. Rate of evolution in brain-expressed genes in humans and other primates.

    Directory of Open Access Journals (Sweden)

    Hurng-Yi Wang

    2007-02-01

    Full Text Available Brain-expressed genes are known to evolve slowly in mammals. Nevertheless, since brains of higher primates have evolved rapidly, one might expect acceleration in DNA sequence evolution in their brain-expressed genes. In this study, we carried out full-length cDNA sequencing on the brain transcriptome of an Old World monkey (OWM and then conducted three-way comparisons among (i mouse, OWM, and human, and (ii OWM, chimpanzee, and human. Although brain-expressed genes indeed appear to evolve more rapidly in species with more advanced brains (apes > OWM > mouse, a similar lineage effect is observable for most other genes. The broad inclusion of genes in the reference set to represent the genomic average is therefore critical to this type of analysis. Calibrated against the genomic average, the rate of evolution among brain-expressed genes is probably lower (or at most equal in humans than in chimpanzee and OWM. Interestingly, the trend of slow evolution in coding sequence is no less pronounced among brain-specific genes, vis-à-vis brain-expressed genes in general. The human brain may thus differ from those of our close relatives in two opposite directions: (i faster evolution in gene expression, and (ii a likely slowdown in the evolution of protein sequences. Possible explanations and hypotheses are discussed.

  1. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    Science.gov (United States)

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

    2015-11-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes

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    Hain Torsten

    2012-04-01

    Full Text Available Abstract Background Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99 and 4b (CLIP80459, and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. Results The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. Conclusion Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence

  3. rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

    Science.gov (United States)

    Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

    2012-05-01

    The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

  4. Synthesis and spectroscopic studies of biologically active tetraazamacrocyclic complexes of Mn(II, Co(II, Ni(II, Pd(II and Pt(II

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    Monika Tyagi

    2014-01-01

    Full Text Available Complexes of Mn(II, Co(II, Ni(II, Pd(II and Pt(II were synthesized with the macrocyclic ligand, i.e., 2,3,9,10-tetraketo-1,4,8,11-tetraazacycoletradecane. The ligand was prepared by the [2 + 2] condensation of diethyloxalate and 1,3-diamino propane and characterized by elemental analysis, mass, IR and 1H NMR spectral studies. All the complexes were characterized by elemental analysis, molar conductance, magnetic susceptibility measurements, IR, electronic and electron paramagnetic resonance spectral studies. The molar conductance measurements of Mn(II, Co(II and Ni(II complexes in DMF correspond to non electrolyte nature, whereas Pd(II and Pt(II complexes are 1:2 electrolyte. On the basis of spectral studies an octahedral geometry has been assigned for Mn(II, Co(II and Ni(II complexes, whereas square planar geometry assigned for Pd(II and Pt(II. In vitro the ligand and its metal complexes were evaluated against plant pathogenic fungi (Fusarium odum, Aspergillus niger and Rhizoctonia bataticola and some compounds found to be more active as commercially available fungicide like Chlorothalonil.

  5. IDENTIFIKASI TIPE HLA KELAS II DENGAN TEKNIK PCR

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    Ervi Salwati

    2012-09-01

    Full Text Available HLA (Human Leukocyte Antigen contains a set of genes located together on the short arm of chromosome 6. These genes control immune responses, graft acceptance or rejection and tumor surveillance. These abilities have close relationship with genetic variation (occur in "many forms" or alleles that bind and present antigens to T lymphocytes. Using advanced technology and molecular biology approaches (PCR technique detection of genetic variation in the HLA region (or HLA typing has been performed based on DNA.. PCR is an in vitro technique to amplify the DNA sequence enzymatically. "Sequence Specific Primers" (SSP are designed for this PCR to obtain amplification of specific alleles or groups of alleles. The PCR products are visualized through agarose gel electrophoresis stained with ethidium bromide. The PCR technique requires small amount of whole blood (0.5 - 1 ml, gives rapid, accurate and complete result. This paper discuss identification of HLA class II typing using PCR-SSP technique and show the examples of the results.   Key words: HLA (Human Leukocyte Antigen class II, PCR (Polymerase Chain Reaction

  6. A Probabilistic Genome-Wide Gene Reading Frame Sequence Model

    DEFF Research Database (Denmark)

    Have, Christian Theil; Mørk, Søren

    We introduce a new type of probabilistic sequence model, that model the sequential composition of reading frames of genes in a genome. Our approach extends gene finders with a model of the sequential composition of genes at the genome-level -- effectively producing a sequential genome annotation...... as output. The model can be used to obtain the most probable genome annotation based on a combination of i: a gene finder score of each gene candidate and ii: the sequence of the reading frames of gene candidates through a genome. The model --- as well as a higher order variant --- is developed and tested...... and are evaluated by the effect on prediction performance. Since bacterial gene finding to a large extent is a solved problem it forms an ideal proving ground for evaluating the explicit modeling of larger scale gene sequence composition of genomes. We conclude that the sequential composition of gene reading frames...

  7. Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

    Science.gov (United States)

    Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying

    2018-01-01

    Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

  8. Influence of the experimental design of gene expression studies on the inference of gene regulatory networks: environmental factors

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    Frank Emmert-Streib

    2013-02-01

    Full Text Available The inference of gene regulatory networks gained within recent years a considerable interest in the biology and biomedical community. The purpose of this paper is to investigate the influence that environmental conditions can exhibit on the inference performance of network inference algorithms. Specifically, we study five network inference methods, Aracne, BC3NET, CLR, C3NET and MRNET, and compare the results for three different conditions: (I observational gene expression data: normal environmental condition, (II interventional gene expression data: growth in rich media, (III interventional gene expression data: normal environmental condition interrupted by a positive spike-in stimulation. Overall, we find that different statistical inference methods lead to comparable, but condition-specific results. Further, our results suggest that non-steady-state data enhance the inferability of regulatory networks.

  9. Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Tobey, R.A.

    1989-01-01

    Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. There the authors report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 μg mL -1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression

  10. Role of Kidneys in Sex Differences in Angiotensin II-Induced Hypertension.

    Science.gov (United States)

    Wang, Lei; Wang, Ximing; Qu, Helena Y; Jiang, Shan; Zhang, Jie; Fu, Liying; Buggs, Jacentha; Pang, Bo; Wei, Jin; Liu, Ruisheng

    2017-12-01

    The significance of kidneys in regulation of sodium and water balance and hemodynamics has been demonstrated both in patients and animal models. In the present study, we tested our hypothesis that kidneys play an essential role in control of sex differences in angiotensin II (Ang II)-dependent hypertension. Kidney transplantations (KTXs) were performed between male (M) and female (F) C57BL/6 mice (donor→recipient: F→F, M→M, F→M, and M→F). Radiotelemetry transmitters were implanted for measurement of mean arterial pressure during the infusion of Ang II (600 ng·kg -1 ·min -1 ). Gene expressions and inflammatory responses in the transplanted grafts were assessed. We found that same-sex-KTX mice still exhibited sex differences in Ang II-dependent hypertension (31.3±0.8 mm Hg in M→M versus 12.2±0.6 mm Hg in F→F), which were reduced between males and females when they received kidneys of the opposite sex (32.9±1 mm Hg in M→F versus 22.3±0.7 mm Hg in F→M). The sex differences in gene expressions, including AT 1 R (angiotensin II receptor, type 1), AT 1 R/AT 2 R, ET-1 (endothelin-1), ETA (endothelin receptor type A), NHE3 (sodium-hydrogen exchanger 3), α-ENaC (α-epithelial sodium channel), and γ-ENaC, were unaltered in same-sex KTXs and much lessened in cross-sex KTXs. In addition, the cross-sex KTXs exhibited more robust inflammatory responses reflected by higher expression of IL-6 (interleukin 6), TNFα (tumor necrosis factor α), and KC (keratinocyte-derived chemokine) than same-sex KTX. Our results indicate that kidneys play an essential role in sex differences of Ang II-dependent hypertension. KTX of male kidneys to females augmented the blood pressure response, whereas KTX of female kidneys to males attenuated the blood pressure response. The host's extrarenal systems modulate expressions of many genes and inflammatory response, which may also contribute to the sex differences in blood pressure regulation. © 2017 American Heart

  11. Analysis of the clonal repertoire of gene-corrected cells in gene therapy.

    Science.gov (United States)

    Paruzynski, Anna; Glimm, Hanno; Schmidt, Manfred; Kalle, Christof von

    2012-01-01

    Gene therapy-based clinical phase I/II studies using integrating retroviral vectors could successfully treat different monogenetic inherited diseases. However, with increased efficiency of this therapy, severe side effects occurred in various gene therapy trials. In all cases, integration of the vector close to or within a proto-oncogene contributed substantially to the development of the malignancies. Thus, the in-depth analysis of integration site patterns is of high importance to uncover potential clonal outgrowth and to assess the safety of gene transfer vectors and gene therapy protocols. The standard and nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) in combination with high-throughput sequencing exhibits technologies that allow to comprehensively analyze the clonal repertoire of gene-corrected cells and to assess the safety of the used vector system at an early stage on the molecular level. It enables clarifying the biological consequences of the vector system on the fate of the transduced cell. Furthermore, the downstream performance of real-time PCR allows a quantitative estimation of the clonality of individual cells and their clonal progeny. Here, we present a guideline that should allow researchers to perform comprehensive integration site analysis in preclinical and clinical studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Large granular lymphocytosis in a patient infected with HTLV-II.

    Science.gov (United States)

    Martin, M P; Biggar, R J; Hamlin-Green, G; Staal, S; Mann, D

    1993-08-01

    HTLV-II has been associated with a variety of lymphoproliferative disorders, including atypical hairy cell leukemia, chronic T cell leukemia, T prolymphocytic leukemia, and large granular lymphocytic leukemia. However, a direct or indirect role for HTLV-II in these disorders is not yet firmly established. We studied a patient diagnosed as having leukemia of the large granular lymphocyte (LGL) type who was HTLV-II seropositive, to determine if the expanded cell population was infected. Two populations of CD3-CD16+ LGL were identified; one was CD8+, the other CD8-. Populations of cells with these surface markers as well as normal CD3+CD4+ and CD3+CD8+ cells were separated by flow cytometric methods, DNA extracted, and gene regions of HTLV-II pol and tax amplified, using the polymerase chain reaction, and probed after Southern blotting. HTLV-II was detected in the CD3+CD8+ population, and not in the CD3-CD16+ large granular lymphocyte population. This finding indicates that the role of HTLV-II, if any, in LGL proliferation is indirect.

  13. Overexpression of the S100A2 protein as a prognostic marker for patients with stage II and III colorectal cancer

    Science.gov (United States)

    MASUDA, TAIKI; ISHIKAWA, TOSHIAKI; MOGUSHI, KAORU; OKAZAKI, SATOSHI; ISHIGURO, MEGUMI; IIDA, SATORU; MIZUSHIMA, HIROSHI; TANAKA, HIROSHI; UETAKE, HIROYUKI; SUGIHARA, KENICHI

    2016-01-01

    We aimed to identify a novel prognostic biomarker related to recurrence in stage II and III colorectal cancer (CRC) patients. Stage II and III CRC tissue mRNA expression was profiled using an Affymetrix Gene Chip, and copy number profiles of 125 patients were generated using an Affymetrix 250K Sty array. Genes showing both upregulated expression and copy number gains in cases involving recurrence were extracted as candidate biomarkers. The protein expression of the candidate gene was assessed using immunohistochemical staining of tissue from 161 patients. The relationship between protein expression and clinicopathological features was also examined. We identified 9 candidate genes related to recurrence of stage II and III CRC, whose mRNA expression was significantly higher in CRC than in normal tissue. Of these proteins, the S100 calcium-binding protein A2 (S100A2) has been observed in several human cancers. S100A2 protein overexpression in CRC cells was associated with significantly worse overall survival and relapse-free survival, indicating that S100A2 is an independent risk factor for stage II and III CRC recurrence. S100A2 overexpression in cancer cells could be a biomarker of poor prognosis in stage II and III CRC recurrence and a target for treatment of this disease. PMID:26783118

  14. Plant Core Environmental Stress Response Genes Are Systemically Coordinated during Abiotic Stresses

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    Kenneth W. Berendzen

    2013-04-01

    Full Text Available Studying plant stress responses is an important issue in a world threatened by global warming. Unfortunately, comparative analyses are hampered by varying experimental setups. In contrast, the AtGenExpress abiotic stress experiment displays intercomparability. Importantly, six of the nine stresses (wounding, genotoxic, oxidative, UV-B light, osmotic and salt can be examined for their capacity to generate systemic signals between the shoot and root, which might be essential to regain homeostasis in Arabidopsis thaliana. We classified the systemic responses into two groups: genes that are regulated in the non-treated tissue only are defined as type I responsive and, accordingly, genes that react in both tissues are termed type II responsive. Analysis of type I and II systemic responses suggest distinct functionalities, but also significant overlap between different stresses. Comparison with salicylic acid (SA and methyl-jasmonate (MeJA responsive genes implies that MeJA is involved in the systemic stress response. Certain genes are predominantly responding in only one of the categories, e.g., WRKY genes respond mainly non-systemically. Instead, genes of the plant core environmental stress response (PCESR, e.g., ZAT10, ZAT12, ERD9 or MES9, are part of different response types. Moreover, several PCESR genes switch between the categories in a stress-specific manner.

  15. Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

    Energy Technology Data Exchange (ETDEWEB)

    Carr, Carolyn E. [Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States; Musiani, Francesco [Laboratory; Huang, Hsin-Ting [Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States; Chivers, Peter T. [Departments of Biosciences and Chemistry, Durham University, Durham DH1 3LE, United Kingdom; Ciurli, Stefano [Laboratory; Maroney, Michael J. [Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States

    2017-05-18

    Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR) is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PRcnAB. The DNA binding affinity is weakened when the cognate ions Ni(II) and Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the noncognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity, and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the glutamic acid variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal-site structures of EcRcnR bound to Ni(II), Co(II), and Zn(II). The results are interpreted

  16. Comparison of Dissolved Nickel and Nickel Nanoparticles Toxicity in Larval Zebrafish in Terms of Gene Expression and DNA Damage.

    Science.gov (United States)

    Boran, Halis; Şaffak, Savaş

    2018-01-01

    With the use of nanoparticles (NPs) in many industrial activities and consumer products, it is important to evaluate the effects of their release into the environment. Metal NPs (e.g., Ni-NPs or Cu-NPs) can release metal ions that are toxic to aquatic organisms; however, whether the toxicity is from metal ions rather than unique "nano-scale" effects of the NPs is unresolved. This research investigated Ni-NP toxicity in zebrafish Danio rerio larvae to clarify whether toxic effects are attributable to release of Ni ions. First, the acute (96-h lethal) toxicity of Ni-NPs was determined in comparison to aqueous Ni in fish exposed to Ni(II) by water-soluble NiCl 2 . Subsequently, sublethal experiments with Ni-NPs and Ni(II) were conducted to assess changes in expression of stress-related genes (mt2, rad51, and p53) by quantitative PCR. Acute toxicity of Ni in fish exposed to Ni(II) was higher (96-h LC 50  = 32.6 mg/L) than for fish exposed to Ni-NPs (96-h LC 50  = 122.2 mg/L). Also, DNA strand breaks were higher in Ni(II)- than Ni-NPs-exposed larvae. Induction of stress-related genes in larvae was complex and was not directly related to Ni-NPs and Ni(II) concentration, although there was a significant induction in the mt2 and p53 gene of the larvae exposed to Ni-NPs and Ni(II) relative to controls. Results indicated that while Ni-NPs induced gene expression (presumably by the release of Ni ions), the differences in concentration relationships of gene expression between Ni-NPs and Ni(II) suggest that factors in addition to the release of Ni ions from Ni-NPs influence acute toxicity of Ni-NPs.

  17. Translational approach for gene therapy in epilepsy

    DEFF Research Database (Denmark)

    Ledri, Litsa Nikitidou; Melin, Esbjörn; Christiansen, Søren H.

    2016-01-01

    clinical trial for gene therapy of temporal lobe epilepsy was explored: We investigated (i) whether the post intrahippocampal kainate-induced status epilepticus (SE) model of chronic epilepsy in rats could be clinically relevant; and (ii) whether a translationally designed neuropeptide Y (NPY)/Y2 receptor...

  18. The ACE gene D/I polymorphism as a modulator of severity of cystic fibrosis

    Directory of Open Access Journals (Sweden)

    Marson Fernando A L

    2012-08-01

    Full Text Available Abstract Background Cystic Fibrosis (CF is a monogenic disease with complex expression because of the action of genetic and environmental factors. We investigated whether the ACE gene D/I polymorphism is associated with severity of CF. Methods A cross-sectional study was performed, from 2009 to 2011, at University of Campinas – UNICAMP. We analyzed 180 patients for the most frequent mutations in the CFTR gene, presence of the ACE gene D/I polymorphism and clinical characteristics of CF. Results There was an association of the D/D genotype with early initiation of clinical manifestations (OR: 1.519, CI: 1.074 to 2.146, bacterium Burkholderia cepacia colonization (OR: 3.309, CI: 1.476 to 6.256 and Bhalla score (BS (p = 0.015. The association was observed in subgroups of patients which were defined by their CFTR mutation genotype (all patients; subgroup I: no mutation detected; subgroup II: one CFTR allele identified to mutation class I, II or III; subgroup III: both CFTR alleles identified to mutation class I, II and/or III. Conclusion An association between the D allele in the ACE gene and the severity of CF was found in our study.

  19. Regulation of hypoxia-inducible factor-1α (HIF-1α expression by interleukin-1β (IL-1 β, insulin-like growth factors I (IGF-I and II (IGF-II in human osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Angelica Rossi Sartori-Cintra

    2012-01-01

    Full Text Available OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α can regulate cytokines (catabolic action and/or growth factors (anabolic action in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β and insulin-like growth factors I (IGF-I and II (IGF-II and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.

  20. Bovine mammary gene expression profiling during the onset of lactation.

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    Yuanyuan Gao

    Full Text Available BACKGROUND: Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE on bovine mammary tissue at three time points (on approximately day 35 before parturition (-35 d, day 7 before parturition (-7 d and day 3 after parturition (+3 d. Approximately 6.2 million (M, 5.8 million (M and 6.1 million (M 21-nt cDNA tags were sequenced in the three cDNA libraries (-35 d, -7 d and +3 d, respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥ 2 or ≤-2 and a false discovery rate (FDR of ≤ 0.001, a total of 812 genes were significantly differentially expressed at -7 d compared with -35 d (stage I. Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with -7 d (stage II, and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with -35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. CONCLUSIONS: The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.