We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and ...subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a single-sequenced sample.
Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is ...well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).
To determine why herbaceous productivity in tropical and subtropical savannas is often significantly higher under crowns of isolated trees than in adjacent grass-lands, experimental plots were ...established in three concentric zones, crown, tree-root, and grassland, surrounding isolated trees of Acacia tortilis in low-rainfall and high-rainfall savannas in Tsavo National Park, Kenya. Plots were fertilized (to determine the importance of nutrient enrichment by trees), shaded (to determine the importance of crown shade), fertilized and shaded (to identify fertilizer x shade interactions), or trenched (tree roots entering plots were severed to determine the importance of belowground competition between overstory trees and understory herbaceous plants). In addition, vertical root distributions of trees and herbaceous species were determined, and root systems of A. tortilis saplings were excavated. At both sites fertilization significantly increased herbaceous productivity in tree-root and grassland zones, but not in canopy zones; artificial shade had no effect on productivity at the low-rainfall site but increased productivity in the tree-root zone at the high-rainfall site; and severing tree roots had no effect on herbaceous productivity at the low-rainfall site, but increased productivity in the crown and tree-root zones at the high-rainfall site. Roots of herbaceous and woody species co-occurred within the same soil horizons, but tree roots extended farther into grasslands at the low-rainfall site than at the high-rainfall site. These studies suggest that savanna trees completed more intensely with understory plants at wetters sites, where their roots terminated in or near crown zones, than at drier sites, where their roots extended farther into open grassland. Nutrients added by trees to crown zones in the form of tree litter and animal droppings increased understory productivity by fertilizing nutrient-limited soils. Shade contributed more to regrowth after severe defoliation than to growth under more normal conditions.
Eukaryotic replication origins are defined by the ORC‐dependent loading of the Mcm2‐7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2‐7 relative to ORC assembled ...onto chromatin in G1. These excess Mcm2‐7 complexes exhibit little co‐localization with ORC or replication foci and can function as dormant origins. We dissected the mechanisms regulating the assembly and distribution of the Mcm2‐7 complex in the Drosophila genome. We found that in the absence of cyclin E/Cdk2 activity, there was a 10‐fold decrease in chromatin‐associated Mcm2‐7 relative to the levels found at the G1/S transition. The minimal amounts of Mcm2‐7 loaded in the absence of cyclin E/Cdk2 activity were strictly localized to ORC binding sites. In contrast, cyclin E/Cdk2 activity was required for maximal loading of Mcm2‐7 and a dramatic genome‐wide reorganization of the distribution of Mcm2‐7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 activity over the course of G1 is not only critical for Mcm2‐7 loading, but also for the distribution of the Mcm2‐7 helicase prior to S‐phase entry.
Synopsis
Origin recognition complex (ORC)‐dependent chromatin loading of the Mcm2‐7 replicative helicase occurs in excess, resulting in formation of copious dormant replication origins. Genomics analyses show that in Drosophila, initial minimal Mcm2‐7 loading increases in a cyclin E/Cdk2‐dependent manner and involves transcription‐related redistribution beyond ORC binding sites.
Mcm2‐7 loading occurs at ORC binding sites in the absence of cyclin E/Cdk2 activity.
Loading the full complement of Mcm2‐7 during G1 requires cyclin E/Cdk2 activity.
Chromatin‐associated Mcm2‐7 levels peak immediately prior to S‐phase.
Active transcription shapes a genome‐wide redistribution of the Mcm2‐7 complex by the time of G1/S transition.
Pre‐replicative chromatin loading of the full Mcm2‐7 complement depends on cyclin E/Cdk2 activity and involves transcription‐dependent redistribution beyond ORC binding sites.
Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we ...assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.
The use of wind power plants is growing rapidly, despite the continued growth in the extraction of traditional fossil resources. In this paper, the analysis of the power curves of real wind turbine ...models and the polynomials common among the scientific community describing the dependence of the wind turbine output power on wind is carried out. The article discusses 82 models of wind power plants with a horizontal axis of rotation, rated power up to 100 kW and with a method of limiting the speed of rotation in the operating mode pitch control. The calculation of the potential generation of each wind turbine at an oil and gas field for a period of twenty years with an interval of one hour is carried out, the Installed Capacity Utilization Factor is calculated, after which the values are compared with the results of calculations based on polynomials of other authors. The results obtained can be widely applied in modeling the operating modes of wind power plants and will improve the accuracy of feasibility studies for the implementation of autonomous power supply systems.
The paper reviews challenges associated with questions of efficiency of thermal methods of impact on productive oil strata. The concept of using electrothermal complexes with WEG power supply for the ...indicated purposes was proposed and justified, their operating principles, main advantages and disadvantages, as well as a schematechnical solution for the implementation of the intensification of oil extraction, were considered. A mathematical model for finding the operating characteristics of WEG is presented and its main energy parameters are determined. The adequacy of the mathematical model is confirmed by laboratory simulation stand tests with nominal parameters.
FACT (
fa
cilitates
c
hromatin
t
ranscription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation of spt16 results in a global loss of nucleosomes as ...well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037
S
pt16-suppressed
n
oncoding
t
ranscripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.
The article discusses the experience of operating a wind power complex with a low-power wind power installa- tion (5 kW), the use of which is promising for powering remote oil production facilities, ...exploration and other types of mining operations. The structure of the studied complex and its characteristics, technical problems that have arisen during operation for 6 years are given. The elements of the wind energy complex – the battery charge regulator and the inverter-converter are considered. The consequences of the mechanical regulator failure of battery charge are con- sidered and recommendations for its replacement are presented. The issues of diagnostics and repair of one of the main elements of the complex – the inverter-converter, its component – DC link are highlighted in detail. Oscil- lograms of the output voltage of the inverter-converter are presented for different capacities of the DC link and the images of the repaired inverter-converter are given. Recommendations are given on choosing an inverter-converter and setting up the operating modes of the wind energycomplex.
The authors proposed a scheme of autonomous power supply for oil leakage detection systems in pipelines by applying the photovoltaic modules and thermoelectric generators as a power source. ...Laboratory experimental studies were carried out, the operating energy characteristics of the complex were obtained. A sufficient number of generating units has been determined for uninterrupted power supply to the data collection point of the oil leakage detection system.
PDF
