The research of the maximum wind speed in Tomsk and calculations of dynamic load on antenna systems

The work is concerned with calculations and analysis of the maximum wind speed in Tomsk city. The data for analysis were taken from the TOR-station located in the north-eastern part of the city. The TOR-station sensors to measure a speed and a direction of wind are installed on the 10-meter meteorological mast. Wind is measured by M-63, which uses the standard approach and the program with one-minute averaging for wind gusts recording as well. According to the measured results in the research performed, the estimation of the dynamic and wind load on different types of antenna systems was performed. The work shows the calculations of wind load on ten types of antenna systems, distinguished by their different constructions and antenna areas. For implementation of calculations, we used methods developed in the Central Research and Development Institute of Building Constructions named after V.A. Kucherenko. The research results could be used for design engineering of the static antenna systems and mobile tracking systems for the distant objects

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation.

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo

DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance in Bacillus subtilis

To properly segregate their chromosomes, organisms tightly regulate the organization and dynamics of their DNA. Aspects of the process by which DNA is translocated during sporulation are not yet fully understood, such as what factors indirectly influence the activity of the motor protein SpoIIIE. In this work, we have shown that a DNA-membrane tether mediated by RacA contributes to the activity of SpoIIIE. Loss of RacA nearly doubles the time of translocation, despite the physically distinct locations these proteins and their activities occupy within the cell. This is a rare example of an explicit effect that DNA-membrane connections can have on cell physiology and demonstrates that distant changes to the state of the chromosome can influence motor proteins which act upon it.Chromosome segregation in sporulating Bacillus subtilis involves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation. Here we demonstrate that, in addition to positioning and condensing the chromosome, RacA contributes to efficient transport of DNA by the chromosome segregation motor SpoIIIE. When RacA is deleted, one-quarter of cells fail to capture DNA in the nascent spore, yet 70% of cells fail to form viable spores without RacA. This discrepancy indicates that RacA possesses a role in sporulation beyond DNA capture and condensation. We observed that the mutant cells had reduced chromosome translocation into the forespore across the entire length of the chromosome, requiring nearly twice as much time to move a given DNA locus. Additionally, functional abolition of the RacA-DivIVA interaction reduced translocation to a similar degree as in a racA deletion strain, demonstrating the importance of the RacA-mediated tether in translocation and chromosome packaging during sporulation. We propose that the DNA-membrane anchor facilitates efficient translocation by SpoIIIE, not through direct protein-protein contacts but by virtue of physical effects on the chromosome that arise from anchoring DNA at a distance

Missense mutations in <i>spoIIIEΔγ</i> rescue sporulation and chromosome transport <i>in vivo</i>.

<p>A. Suppressor mutations rescue spore formation. Intragenic mutations identified by suppressor selection were remade in a <i>spoIIIEΔγ</i> allele by site-directed mutagenesis and expressed from the <i>spoIIIE</i> promoter at an ectopic locus (<i>ycgO</i>) in a <i>ΔspoIIIE</i> strain. Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. The number of heat-resistant spores per ml is indicated for strains harboring full-length (“f.l.”) <i>spoIIIE</i> (bKM776), <i>spoIIIEΔγ</i> (BOSE2042), and 11 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2286), S264I (BOSE2540), E310K (BOSE2411), E312A (BOSE2121), Y316D (BOSE2284), P319S (BOSE2321), A343V (BOSE2288), E347G (BOSE2323), P492Q (BOSE2120), H493Y (BOSE2538), and T617A (BOSE2123). Each number is the average of at least 3 replicates. Error bars indicate one standard deviation in each direction. B. Suppressor mutations rescue chromosome transport <i>in vivo</i>. Sporulation was induced by resuspension and DNA transport was evaluated using a previously-established fluorescent microscopy assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref012" target="_blank">12</a>]. <i>yfp</i> and <i>cfp</i> genes are expressed from a forespore-specific promoter (<i>PspoIIQ</i>). <i>yfp</i> is integrated near the origin (<i>yycR</i>), and its expression indicates that asymmetric septation is complete. <i>cfp</i> is integrated near the terminus (<i>pelB</i>), and its expression indicates that the terminus has been transported into the forespore. Percent of termini in forespores is the percent of YFP+ cells that are also CFP+. Data are shown for full-length (“f.l.”) <i>spoIIIE</i> (bBB128), <i>spoIIIEΔγ</i> (bBB412), and 5 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2331), E312A (BOSE2201), A343V (BOSE2332), P492Q (BOSE2200), and T617A (BOSE2202). Each data point represents the average of ≥ 3 replicates, with ≥ 500 forespores scored for each.</p

Mutations that rescue the sporulation defect of <i>spoIIIEΔγ</i> strains do not rescue a vegetative translocation defect.

<p>Growth of strains in various concentrations of the replication-stress-inducing antibiotic novobiocin (nov) was evaluated. Cells were grown to mid-exponential phase in LB, and then diluted to OD₆₀₀ 0.0125 in LB containing 960 ng ml^-1 nov (A), 480 ng ml^-1 (B), or no nov (C). OD600 measurements are plotted versus hours after dilution. All strains harbor Δ<i>spoIIIE</i>::<i>neo</i>. As indicated, samples were from strains with no ectopic <i>spoIIIE</i> (bDR1066), <i>spoIIIE</i> (bKM776), <i>spoIIIEΔγ</i> (BOSE2042), or a <i>spoIIIEΔγ</i> mutant: P260L (BOSE2286), E312A (BOSE2121), Y316D (BOSE2284), A343V (BOSE2288), E347G (BOSE2323), P492Q (BOSE2120), T617A (BOSE2123). Representative data from one of at least two replicates are plotted.</p

Intragenic <i>spoIIIEΔγ</i> suppressor mutations alter residues in the linker and motor domains.

<p>A. Positions of intragenic suppressor mutations are indicated on a schematic of the <i>spoIIIEΔγ</i> linear sequence. The γ domain is not shown, but is C-terminal to the β subdomain in full-length SpoIIIE. Numbers in parentheses indicate the number of times each mutation was isolated. Underlined mutations were identified only after the P492 codon was mutated from ccg to cct to lessen the chances of obtaining P492Q. In this study, SpoIIIE codons are numbered for the protein beginning with the sequence MSVAKKKRKS. This presumes an earlier translation start and thus the codons are numbered +2 relative to some annotations of SpoIIIE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref050" target="_blank">50</a>]. (B-E). Positions of intragenic suppressor mutations are shown in a 3D-model of the SpoIIIE motor domain, obtained by threading the SpoIIIE sequence onto a FtsK crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref044" target="_blank">44</a>]. B. Two of the six subunits of a SpoIIIE hexamer are shown. The Walker A and Walker B sites are shown in red and ADP is shown in brown. P319 (pink), A343 (orange), E347 (yellow), P492 (green), H493 (cyan), D586 (blue), and T617 (purple) are shown as space-filled residues. C. A343 (orange) and E347 (yellow) lie on the same face of a helix in the α domain. D. P319 (pink), P492 (green) and H493 (cyan) are near each other. E. D586 (blue) and T617 (purple) are near the Walker B motif (red) in the β domain.</p

Spore formation in various genetic backgrounds.

<p>Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. The average of at least 3 replicates is plotted. Error bars indicate one standard deviation in each direction. A. Intragenic mutants are partially or fully dominant to <i>spoIIIEΔγ</i>. Strains expressing both <i>spoIIIEΔγ</i> and an intragenic <i>spoIIIEΔγ</i> suppressor mutant exhibit sporulation efficiencies that are intermediate between those of strains with only the <i>spoIIIEΔγ</i> or the mutant allele, or that are similar to that of the mutant allele. All displayed strains express the indicated <i>spoIIIE</i> allele from the <i>spoIIIE</i> promoter at the ectopic locus <i>ycgO</i>. White bars indicate strains that also express <i>spoIIIEΔγ</i> from the <i>spoIIIE</i> promoter at the ectopic locus <i>yhdGH</i>. Asterisks mark pairs of sporulation efficiencies that were significantly different from each other by t-tests (p<0.05). Sporulation efficiencies are shown for <i>spoIIIEΔγ</i> (BOSE2042; BOSE2301) and 10 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2286; BOSE2311), S264I (BOSE2540; BOSE3089), E312A (BOSE2121; BOSE2305), Y316D (BOSE2284; BOSE2309), P319S (BOSE2321; BOSE3083), A343V (BOSE2288; BOSE2313), E347G (BOSE2323; BOSE3085), P492Q (BOSE2120; BOSE2303), H493Y (BOSE2538; BOSE3087), and T617A (BOSE2123; BOSE2307). B. Missense mutations that suppress the <i>spoIIIEΔγ</i> phenotype do not affect the function of full-length SpoIIIE. Site-directed mutagenesis was used to introduce each indicated mutation into a <i>spoIIIE</i> allele that was then expressed under its native promoter at <i>ycgO</i> in <i>ΔspoIIIE</i> strains. Each mutant sporulated as well as cells with wild-type <i>spoIIIE</i> (bKM776). Seven mutants were tested: P260L (BOSE2298), E312A (BOSE2290), Y316D (BOSE2296), E347G (BOSE2325), P492Q (BOSE2294), D586N (BOSE3091), and T617A (BOSE2292). All eight sporulation efficiencies were not significantly different from each other by single factor ANOVA (p = 0.503).</p

GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells.

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins