Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase. 相似文献
In the bottom sediments from a number of the Barents Sea sites, including coastal areas of the Novaya Zemlya, Franz Josef Land, and Svalbard archipelagos, sulphate reduction rates were measured and the phylogenetic composition of sulphate-reducing bacterial (SRB) communities was analysed for the first time. Molecular genetic analysis of the sequences of the 16S rRNA and dsrB genes (the latter encodes the β-subunit of dissimilatory (bi)sulphite reductase) revealed significant differences in the composition of bacterial communities in different sampling stations and sediment horizons of the Barents Sea depending on the physicochemical conditions. The major bacteria involved in reduction of sulphur compounds in Arctic marine bottom sediments belonged to Desulfobulbaceae, Desulfobacteraceae, Desulfovibrionaceae, Desulfuromonadaceae, and Desulfarculaceae families, as well as to uncultured clades SAR324 and Sva0485. Desulfobulbaceae and Desulfuromonadaceae predominated in the oxidised (Eh = 154–226 mV) upper layers of the sediments (up to 9% and 5.9% from all reads of the 16S rRNA gene sequences in the sample, correspondingly), while in deeper, more reduced layers (Eh = ?210 to ?105 mV) the share of Desulfobacteraceae in the SRB community was also significant (up to 5%). The highest relative abundance of members of Desulfarculaceae family (3.1%) was revealed in reduced layers of sandy-clayey sediments from the Barents Sea area affected by currents of transformed (mixed, with changed physicochemical characteristics) Atlantic waters.
Biochemistry (Moscow) - Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area,... 相似文献
Heat stress is one of the best-studied cellular stress factors; however, little is known about its delayed effects. Here, we demonstrate that heat stress induces p21-dependent cellular senescence-like cell cycle arrest. Notably, only early S-phase cells undergo such an arrest in response to heat stress. The encounter of DNA replication forks with topoisomerase I-generated single-stranded DNA breaks resulted in the generation of persistent double-stranded DNA breaks was found to be a primary cause of heat stress-induced cellular senescence in these cells. This investigation of heat stress-induced cellular senescence elucidates the mechanisms underlying the exclusive sensitivity of early S-phase cells to ultra-low doses of agents that induce single-stranded DNA breaks. 相似文献
A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.
Results
MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).
Conclusions
Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains. 相似文献
To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154. 相似文献
Abstract The Japanese scallop (Mizuhopectenyessoensis) is one of the main fishery products in Japan, but with the expansion of culture operations of the Japanese scallop, various
problems have been encountered including high mortality, poor growth, poor seed production, and so on. Moreover, there is
concern that many years of cultivation may have affected the genetic structure of the scallop population. To approach these
problems and concerns, we developed microsatellite markers as a molecular tool for population genetic studies. By using 4
microsatellite markers as well as a mitochondrial marker, we investigated the genetic structure of samples from the islands
of Hokkaido (14 populations) and Honshu (Tohoku, 3 populations) in Japan, and south Primorye (4 populations) in Russia. All
the populations sampled had high genetic diversity (average expected heterozygosity, 0.7011 to 0.7622; haplotype diversity,
0.6090 to 0.8848), and almost all showed a tendency of homozygote excess, which was significant in 2 populations. Hierarchical
analysis of molecular variance tests based on the microsatellite and mitochondrial markers indicated that the 3 geographic
regions were genetically divergent from one another, with little evidence of divergence within regions. Homogeneity in allele
frequency distributions between natural and cultured scallops and allele frequency stability over a period of 2 decades indicated
that the culturing operations have probably not had a substantial effect on the genetic structure of the populations. 相似文献