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V. A. Chernukhin V. V. Kuznetsov D. A. Gonchar Yu. G. Kashirina N. A. Netesova S. Kh. Degtyarev 《Biochemistry. Biokhimii?a》2010,75(1):63-71
Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison
of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I
has nearly fourfold lower turnover number (0.24 min−1) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one.
In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally
modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate
of the canonical 5′-GGATG-3′ recognition site. 相似文献
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A. G. Degtyarev 《Contemporary Problems of Ecology》2010,3(1):90-99
In 1982—1995 and 2007 the Bewick’s swan was monitored. The air record routes were 48 000 km long. An increase in population,
widening of the range in the north and west directions are recorded. The data on dynamics of population, territorial location,
social structure, and breeding parameters are given. The ecological problems related to the increase in the swan population
are discussed. 相似文献
15.
Sergei Kh. Degtyarev Olga A. Belichenko Natalya A. Lebedeva Vladimir S. Dedkov Murat A. Abdurashitov 《Nucleic acids research》2000,28(11):e56
The recognition sequence and cleavage positions of a new restriction endonuclease BtrI isolated from Bacillus stearothermophilus SE-U62 have been determined. BtrI belongs to a rare type IIQ of restriction endonucleases, which recognise non-palindromic nucleotide sequences and cleave DNA symmetrically within them. 相似文献
16.
Changes in the content of dammarane-type triterpene alcohols in the leaves of silver birch (Betula pendula Roth) are studied for the first time along a zonal-climatic transect from the northern to southern borders of the species
areal in the Trans-Ural region. It is shown that populations of northern and southern regions differ concerning the triterpene
content in leaves. A correlation between the content of triterpene alcohols in silver birch leaves and some climatic parameters
is revealed. At the same time, the range of the intrapopulation variability of triterpene content is significantly less than
that of the interpopulation variability, which indicates a high potential of population selection on the basis of this biochemical
parameter. 相似文献
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Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. 相似文献
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J. E. Tomilova V. V. Kuznetsov M. A. Abdurashitov N. A. Netesova S. Kh. Degtyarev 《Molecular Biology》2010,44(4):606-615
The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in
Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P
R
/P
L
from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature
optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters
of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k
cat) is 1.8 ± 0.05 min−1. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases
has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence. 相似文献
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Tomilova Yu. E. Abdurashitov M. A. Golikova L. N. Netesova N. A. Degtyarev S. Kh. 《Molecular Biology》2004,38(6):850-856
The Streptococcus faecalis ND547 and Bacillus stearothermophilus 19 genes that code for DNA methyltransferases (MTases, M.) of restriction–modification (RM) systems with the same recognition sequence, 5-GCATC-3 were cloned and sequenced. The Bst19I RM system includes two MTases, M1.Bst19I and M2.Bst19I. The SfaNI RM system has only one MTase, M.SfaNI, whose N and C domains are homologous to M2.Bst19I and M1.Bst19I, respectively. Both M1.Bst19I and M2.Bst19I and the two domains of M.SfaNI contain conserved elements, which are arranged in the order characteristic of class N6-adenine MTases. The enzymes of the SfaNI and Bst19I RM systems proved to be highly homologous to their FokI and BstF5I counterparts, which was explained by the presence of the common tetranucleotide 5-GATG-3 in their recognition sites. Based on sequence homology, the spatial arrangement of highly conserved amino acid residues was determined using the known three-dimensional model of M.DpnIIA, which belongs to the same MTase class. 相似文献