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Free DNA (free + dna)
Selected AbstractsSulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonationENVIRONMENTAL MICROBIOLOGY, Issue 5 2009Julie Leloup Summary In order to better understand the main factors that influence the distribution of sulfate-reducing bacteria (SRB), their population size and their metabolic activity in high- and low-sulfate zones, we studied the SRB diversity in 3- to 5-m-deep sediment cores, which comprised the entire sulfate reduction zone and the upper methanogenic zone. By combining EMA (ethidium monoazide that can only enter damaged/dead cells and may also bind to free DNA) treatment with real-time PCR, we determined the distributions of total intact bacteria (16S rDNA genes) and intact SRB (dsrAB gene), their relative population sizes, and the proportion of dead cells or free DNA with depth. The abundance of SRB corresponded in average to 13% of the total bacterial community in the sulfate zone, 22% in the sulfate,methane transition zone and 8% in the methane zone. Compared with the total bacterial community, there were relatively less dead/damaged cells and free DNA present than among the SRB and this fraction did not change systematically with depth. By DGGE analysis, based on the amplification of the dsrA gene (400 bp), we found that the richness of SRB did not change with depth through the geochemical zones; but the clustering was related to the chemical zonation. A full-length clone library of the dsrAB gene (1900 bp) was constructed from four different depths (20, 110, 280 and 500 cm), and showed that the dsrAB genes in the near-surface sediment (20 cm) was mainly composed of sequences close to the Desulfobacteraceae, including marine complete and incomplete oxidizers such as Desulfosarcina, Desulfobacterium and Desulfococcus. The three other libraries were predominantly composed of Gram-positive SRB. [source] Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27FEBS JOURNAL, Issue 18 2006Cornelia Schwarzenlander Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 µg DNA·(mg protein),1·min,1, demonstrating an extremely efficient binding and uptake rate of 40 kb·s,1·cell,1. Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments. [source] Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilusFEMS MICROBIOLOGY REVIEWS, Issue 3 2009Beate Averhoff Abstract Natural transformation permits the transport of DNA through bacterial membranes and represents a dominant mode for the transfer of genetic information between bacteria and between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal, or lateral, gene transfer, has been a major force for genome plasticity over evolutionary history, and is largely responsible for the spread of fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Here, we present a survey of the natural transformation machinery of the thermophile Thermus thermophilus HB27. A tentative model of the transformation machinery comprising of components similar to proteins of type IV pili and type II secretion systems is presented. A comparative discussion of the subunits and the structure of the DNA translocator and the underlying mechanism of transfer of free DNA in T. thermophilus highlights conserved and unique features of the DNA translocator in T. thermophilus. We hypothesize that the extraordinary broad substrate specificity and the high efficiency of the T. thermophilus DNA uptake system is of major importance for thermoadaptation and interdomain DNA transfer in hot environments. [source] PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNAGENES TO CELLS, Issue 10 2002Tetsuo Iida Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase ,. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp. [source] Gas-phase theoretical prediction of the metal affinity of copper(I) ion for DNA and RNA basesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2003Nino Russo Abstract The most stable tautomeric forms of free DNA and RNA bases were considered as substrates for the interaction of Cu+ ion. Several suitable attachment sites were selected that involved mono- and bi-coordination of the cation. B3LYP/6,311 + G(2df,2p) bond energies showed that copper ion has the major affinity for guanine and cytosine bases. The proposed values of Cu+ ion affinity are 59.9, 60.0, 80.2, 88.0 and 69.0 kcal mol,1 for uracil, thymine, cytosine, guanine and adenine, respectively. The preference for the mono- or bi-coordination depends on the particular tautomer for each base. Copyright © 2003 John Wiley & Sons, Ltd. [source] Hydroxyurea therapy lowers circulating DNA levels in sickle cell anemia,AMERICAN JOURNAL OF HEMATOLOGY, Issue 9 2008Pinar Ulug Hydroxyurea reduces the frequency of acute pain in sickle cell disease (SCD). We sought to determine if hydroxyurea therapy affects cell free DNA (cfDNA) levels in SCD. cfDNA levels fell in all 10 patients studied; before hydroxyurea, mean was 1,879 (95% CI 1,104,3,199) GE/mL; after hydroxyurea, mean was 780 (95% CI, 634,959) GE/mL (P = 0.002). Mean cfDNA level in the 10 HbSS adults prior to starting hydroxyurea was also significantly higher than that in 115 HbSS case controls who had never taken hydroxyurea (1,879 vs 975 GE/mL, P = 0.02). cfDNA levels may be useful in monitoring response to hydroxyurea therapy in SCD. Am. J. Hematol., 2008. © 2008 Wiley-Liss, Inc. [source] Circulating tumour-associated plasma DNA represents an independent and informative predictor of prostate cancerBJU INTERNATIONAL, Issue 3 2006FELIX K.-H. OBJECTIVE To investigate whether preoperative plasma levels of free DNA can discriminate between men with localized prostate cancer and benign prostatic hyperplasia (BPH). PATIENTS AND METHODS In all, 161 referred patients suspicious for prostate cancer either by an elevated prostate-specific antigen (PSA) level and/or abnormal digital rectal examination (DRE) were included in this prospective study. Peripheral plasma was taken before prostate biopsy and genomic DNA was extracted from the plasma using the a commercial kit and a vacuum chamber. After controlling for age, PSA level, the percentage free/total (f/t) PSA and prostate volume, the median prostate cancer plasma DNA concentration served as diagnostic threshold in uni- and multivariate logistic regression models. Multivariate models were subjected to 200 bootstraps for internal validation and to reduce over-fit bias. RESULTS Subgroups consisted of 142 men with clinically localized prostate cancer and 19 with BPH. The median plasma concentration of cell-free DNA was 267 ng/mL in men with BPH vs 709 ng/mL in men with prostate cancer. In univariate analyses, plasma DNA concentration was a statistically significant and informative predictor (P = 0.032 and predictive accuracy 0.643). In multivariate analyses, it remained statistically significant after controlling for age, tPSA, f/tPSA and prostate volume, increasing the predictive accuracy by 5.6%. CONCLUSIONS Our data suggest that plasma DNA level is a highly accurate and informative predictor in uni- and multivariate models for the presence of prostate cancer on needle biopsy. The predictive accuracy was substantially increased by adding plasma DNA level. However, larger-scale studies are needed to further confirm its clinical impact on prostate cancer detection. [source] Cyclen-Based Side-Chain Homopolymer Self-Assembly with Plasmid DNA: Protection of DNA from Enzymatic DegradationCHEMISTRY & BIODIVERSITY, Issue 5 2009Kun Li Abstract In this study, a 1,4,7,10-tetraazacyclododecane (cyclen)-based side-chain homopolymer was developed for the first time; this polymer can self-assembly with plasmid DNA to form polyelectrolyte complexes (polyplex), which can protect DNA from enzymatic degradation. Moreover, the polyplex can disassembly and release free DNA when NaCl solution is added to this system. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used for imaging of the surface structure of the polyplex, and results indicated that the polyplex structures respond to the polymer concentration. Circular dichroism (CD) spectrum suggested that the DNA configuration in the polyplex was retained. [source] Dnase1l3 deficiency in lupus-prone MRL and NZB/W F1 miceCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2003A. WILBER SUMMARY Loss of deoxyribonuclease I (Dnase1) function is associated with systemic lupus erythematosus (SLE) in humans and mice; however, no coding mutations in Dnase1 are found in polygenic murine models. Instead, both MRL- lpr strains and NZB/W F1 hybrids are homozygous for T89I missense in the macrophage-DNASE, desoxyribonuclease I-like 3 (Dnase1l3). By in vitro expression studies, this substitution decreases this enzyme's nuclease activity against free DNA by only approximately twofold; however, the mutation has a greater effect on the capacity of media conditioned with Dnase1l3 to confer a barrier to liposomal gene transfection to HeLa cells. The 89I substitution decreases the Dnase1l3 barrier function in vitro by eightfold (P < 0·01). In splenocytes and BM-derived macrophages of SLE mice, while cellular Dnase1l3 levels are induced relative to C57BL/6 (control) mice, levels of FD-nuclease activity are similar. Finally, media conditioned by MRL and NZB/W F1 macrophages, relative to control, contains a weak interferon-gamma (IFN- ,) inducible Dnase1l3-associated barrier to transfection. This barrier function is hypothesized to reflect the inability of SLE mice to degrade membrane-enveloped DNA-associated antigens, such as apoptotic bodies, which are predicted to stimulate the characteristic autoimmunity of SLE. Our results for these two generally independent models strongly suggest that Dnase1l3 deficiency increases the susceptibility of these mice to polygenic SLE. [source] | ||||||||||||||||