Publications
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The Pse31I restriction endonuclease from the bacterial strain Peribacillus species 31, an isozomere of Eco31I, recognizes the non-palindromic DNA sequence 5’-GGTCTC-3’ (1/5) // DNA-Recognizing Enzymes, Vol. 2025(1), DOI: 10.26213/3034-4301.2025.4.1.001 (2025). (In Russian)
Abstract
We have identified a bacterial strain, Peribacillus species 31, which produces a novel restriction endonuclease named Pse31I. The enzyme recognizes the six-nucleotide non-palindromic DNA sequence 5’-GGTCTC-3’ and cleaves it downstream of the recognition site at positions 1/5: 5’-GGTCTC(N)1^/3’-CCAGAG(N)5^-5’. Thus, Pse31I is a true isozomere of the Eco31I restriction enzyme. The producing strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. Pse31I exhibits maximum activity at 37°C and is inactivated by incubation at 55 and 65°C. The enzyme does not cleave sites containing methylated cytosine in the upper strand (5’-GGTCT(5mC)-3’) and/or in the lower strand (3’-(5mC)CAGAG) .
The optimal conditions for enzyme activity are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.
A PecI restriction endonuclease preparation with a concentration of 10,000 units/mL was obtained through purification in four chromatographic stages. The optimal conditions for the enzyme reaction are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C. -
The BmoI restriction endonuclease from the bacterial strain Brevundimonas mongoliensis 53 recognizes the DNA sequence 5’-GG^GWCCC-3’. // DNA-Recognizing Enzymes, vol. 2025(2), DOI: 10.26213/3034-4301.2025.5.2.002 (2025). (In Russian)
Abstract
We have identified a bacterial strain, Brevundimonas mongoliensis 53, which produces a novel large-cleavage restriction endonuclease named BmoI. The BmoI enzyme recognizes the seven-nucleotide palindromic DNA sequence 5’-GG↓GWCCC-3’ (W = A or T) and cleaves it as indicated by the arrow, forming three-nucleotide
5’-overhanging “sticky” ends. BmoI is an isozomere of the previously described restriction endonuclease SanDI. The producer strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of the BmoI restriction endonuclease with a concentration of 2,000 units/mL was obtained by purification through three chromatographic stages. The optimal conditions for enzyme activity are SE buffer W (10 mM Tris-HCl (pH 8.5), 10 mM MgCl₂, 100 mM NaCl, 1 mM DTT) and a reaction temperature of 37°C. -
The EmiI restriction endonuclease, the Ksp632I isoisomer, recognizes the non-palindromic DNA sequence 5’-CTCTTC(1/4)-3’. // DNA-Recognizing Enzymes, Vol. 2025(4), DOI: 10.26213/3034-4301.2025.7.4.002 (2025). (In Russian)
Abstract
We have identified a bacterial strain, Exiguobacterium mexicanum 6, which produces a novel restriction endonuclease named EmiI. The EmiI enzyme recognizes the six-nucleotide non-palindromic DNA sequence 5’-CTCTTC-3’ and cleaves it downstream of the recognition site at positions 1/4: 5’-CTCTTC(N)1↓-3’/3’-GAGAAG(N)4↓-5’, forming 5’-protruding trinucleotide ends. Thus, EmiI is an isozysomer of the restriction enzymes Ksp632I and Bst6I. The producer strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of the EmiI restriction endonuclease with a concentration of 5,000 units/mL was obtained by purification through three chromatographic steps. The optimal conditions for enzyme activity are SE Buffer Y (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.
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Determination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Results from GlaI Hydrolysates and Data from GlaI-PCR Analysis // Epigenetic DNA Diagnostics, Vol. 2025(1), DOI: 10.26213/3034-4298.2025.6.1.001 (2025). (In Russian)
Abstract
The 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves 5′-R(5mC)GY-3′ / 3′-YG(5mC)R-5′ sites in DNA, which are formed de novo in the human genome by the action of the DNA methyltransferase DNMT3. Due to its specificity, the GlaI enzyme is effectively used to determine the methylation status of RCGY sites in the human genome. Genomic mapping of R(5mC)GY sites is performed using next-generation sequencing (NGS) of DNA hydrolysates obtained by GlaI digestion, whereas analysis of individual DNA fragments can be performed using GlaI-PCR.
In this study, we compared the results of NGS analysis of GlaI-digested DNA hydrolysates with those of GlaI-PCR analysis of the regulatory regions of 11 tumor-suppressor genes in the DNA of the human cell lines L68 (normal lung fibroblasts), Raji (Burkitt’s lymphoma), and U937 (histiocytic lymphoma). It was shown that in L68 cell DNA, most of the regulatory regions studied are unmethylated, whereas in the DNA of the malignant cell lines Raji and U937, methylation of these regions is observed. With the exception of one region, for all other fragments studied, the methylation status determined by GlaI-PCR analysis fully correlates with the number of R(5mC)GY sites identified by NGS. The only discrepancy between the data from the two methods was identified in the regulatory region of the RASSF1A gene in L68 cell DNA, which is explained by the presence of allele-specific methylation, confirmed by an additional real-time GlaI-PCR analysis. Given the presence of allelic methylation in the regulatory region of the RASSF1A gene, we can conclude that there is complete agreement between the NGS data for GlaI hydrolysates and the GlaI-PCR analysis of the regulatory regions of the studied tumor suppressor genes in the human cell lines L68, Raji, and U937.
Thus, NGS analysis of GlaI-hydrolyzed DNA and GlaI-PCR analysis are complementary methods that enable the reliable identification of abnormally methylated genomic regions in normal and tumor cells, making them promising tools for epigenetic research and DNA diagnostics. -
Abstract
We have identified a bacterial strain, Paracoccus species 12, that produces a novel restriction endonuclease named PecI. This enzyme recognizes the six-nucleotide palindromic DNA sequence 5’-TTA^TAA-3’ and cleaves it to form “blunt” ends, as indicated by the arrow. Thus, PecI is a true isozym of the PsiI restriction endonuclease.
A PecI restriction endonuclease preparation with a concentration of 10,000 units/mL was obtained through a four-step chromatographic purification process. The optimal reaction conditions for the enzyme are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C. -
Abstract
A single-nucleotide polymorphism (SNP) involves the substitution of one nucleotide for another, which often results in the appearance (or disappearance) of a recognition site for a specific restriction enzyme. As a result, when a DNA fragment is amplified using primers flanking the SNP site (which contains either the N1 or N2 nucleotide) and the resulting amplicon is subsequently digested with the relevant restriction enzyme, the DNA fragmentation patterns will differ for the three possible variants in diploid DNA (genotypes N1/N1, N1/N2, and N2/N2). This method for determining restriction fragment length polymorphism (RFLP) is widely used in genetic research. Previously, we developed GlaI- and FatI-PCR analysis methods, in which real-time PCR is performed instead of electrophoresis, and demonstrated their applicability for detecting the T/C SNP. In this study, we propose a new method for detecting the G/C single-nucleotide polymorphism using Bst2UI-PCR analysis. GlaI- and Bst2UI-PCR were used to determine the frequency of G/C polymorphism variants at position chr20:37352001 (according to the GRCh38.p14 genome assembly) in DNA preparations isolated from the blood cells of 161 individuals. The study included: 1) isolation of leukocyte DNA from blood cells; 2) performing GlaI- and Bst2UI-PCR analysis of the DNA fragment chr20:37351957–37352083; 3) determination of cytosine and guanine at position chr20:37352001 in the analyzed DNA preparations; 4) comparative analysis of the obtained results. It was shown that 68 donors (42.2%) have a heterozygous G/C allele at chr20:37352001, while 89 donors (55.3%) are homozygous. It has been shown that 68 donors (42.2%) have a heterozygous G/C genotype at locus chr20:37352001, 89 donors (55.3%) are homozygous for G, and four donors (2.5%) are homozygous for C. Thus, given that blood cells have a diploid set of chromosomes, the G-to-C substitution occurs in 76 of the 322 analyzed variants (23.6%). Furthermore, the results indicate that cytosine, which is complementary to G at position chr20:37352001, is present in the methylated form (5-methylcytosine) in the majority of DNA molecules in both homozygotes and heterozygotes. The proposed Bst2UI-PCR method expands the capabilities for SNP detection using real-time PCR.
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Detection of the 5mC/T polymorphism at position Chr1:245618129 in human blood DNA samples using GlaI- and FatI-PCR analysis // Epigenetic DNA Diagnostics, DOI: 10.26213/SE.2019.69.42836 (2021). (In Russian)
Abstract
The frequencies of the 5mC/T polymorphism variant at position chr1:245618129 (according to the GRCh38.p13 genome assembly) were determined using GlaI- and FatI-PCR analysis in DNA samples isolated from the blood cells of 92 individuals. The study included (1) isolation of leukocyte DNA from blood cells, (2) GlaI- and FatI-PCR analysis of the DNA fragment chr1: 245617889–245618464, (3) the determination of 5-methylcytosine and thymine at position chr1: 245618129 in the analyzed DNA samples, and (4) a comparative analysis of the obtained results.
It was shown that 43 donors (46.74%) have a heterozygous C/T genotype at position chr1: 245618129, 28 donors (30.43%) are homozygous for T, and 21 donors (22.83%) are homozygous for C. Thus, given that blood cells have a diploid set of chromosomes, the substitution of C for T occurs in 99 of the 184 analyzed variants (53.8%) . Furthermore, the results indicate that cytosine at position chr1:245618129 is present in the methylated form (5-methylcytosine) in the majority of DNA molecules. -
Assessment of the Risk of Breast and Stomach Cancer by Determining the 5mC/T Polymorphism Variants in the KIF26B Gene Intron at Position chr1:245618129 // Epigenetic DNA Diagnostics, DOI: 10.26213/SE.2019.78.40307 (2021). (In Russian)
Abstract
The frequencies of the 5mC/T polymorphism variant at position chr1: 245618129 (according to the GRCh38.p13 genome assembly) in DNA samples isolated from blood cells of 51 patients with breast cancer (BC) and 63 patients with gastric cancer (GC). The obtained data were compared with previously established values for 92 healthy blood donors.
It was shown that the cytosine base at this position is predominantly in the methylated form in all DNA samples. The frequency of the C/C diploid genotype at this locus in patients with BC and GC is 9.8% and 7.94%, respectively. This differs significantly from the frequency of C homozygotes in healthy women (26.09%) and healthy donors of both sexes (22.83%). Thus, the presence of the C/C genotype at position chr1:245618129 is a favorable trait, indicating a 2.7- to 2.9-fold reduced risk of developing breast cancer and colorectal cancer compared to population-average values. At the same time, the prevalence of C/T heterozygotes and T/T homozygotes among patients with colorectal cancer and breast cancer exceeds the corresponding figures for the control groups of healthy individuals by 5.5–10.7%. -
Detection of the 5mC/T polymorphism in the AluSx repeat (CHR16: 75033884) in human blood DNA preparations using GlaI- and FatI-PCR analysis // Epigenetic DNA Diagnostics, DOI: 10.26213/SE.2021.11.76.001 (2021). (In Russian)
Abstract
The frequencies of the 5mC/T polymorphism variants in the AluSx repeat at position chr16:75033884 (according to the GRCh38.p13 genome assembly) were determined using GlaI- and FatI-PCR analysis in DNA preparations isolated from the blood cells of 92 individuals. The study included (1) isolation of leukocyte DNA from blood cells, (2) GlaI- and FatI-PCR analysis of the DNA fragment chr16: 75033860–75033957, (3) the determination of 5-methylcytosine and thymine at position (chromosome 16: 75033884) in the analyzed DNA samples, and (4) a comparative analysis of the obtained results.
It was shown that 53 donors (57.61%) have a heterozygous C/T genotype at chr16: 75033884, 19 donors (20.65%) are homozygous for T, and 20 donors (21.74%) are homozygous for C. Thus, given that blood cells have a diploid set of chromosomes, the substitution of C for T occurs in 91 of the 184 analyzed variants (49.46%). Furthermore, the results indicate that cytosine at position chr16:75033884 is present in the methylated form (5-methylcytosine) in the majority of DNA molecules, which is consistent with the finding that CG dinucleotides are predominantly methylated in Alu repeats. -
Investigation of RCGY Site Methylation in the Regulatory Region of the FAM19A4 Gene in Human Cell Line DNA Preparations Using GLAD-PCR Analysis // Epigenetic DNA Diagnostics, DOI: 10.26213/SE.2021.97.62.001 (2021). (In Russian)
Abstract
Using the GLAD-PCR method, methylation of RCGY sites in the regulatory region of the FAM19A4 gene (Chr 3: 68932451–68932800) was determined in DNA preparations from the human malignant cell lines Raji, Jurkat, HeLa, and U-937, as well as control DNA from the L68 lung fibroblast cell line.
It was shown that the octanucleotide GCGCGCGC (Chr 3: 68932500), located in the first exon of the FAM19A4 gene, is cleaved to approximately the same extent in the DNA of all four human malignant cell lines, whereas in L68 DNA, this site is hydrolyzed 10–15 times less frequently. These results are consistent with previously reported data from epigenomic sequencing of L68, Raji, and U-937 DNA.
Methylation of CG dinucleotides at positions 68932727 and/or 68932740 in the FAM19A4 gene promoter region occurs only in tumor DNA and is not observed in L68 DNA, while methylation of the CG dinucleotide at position 68932704 is observed only in HeLa DNA. -
Abstract
Real-time PCR using a TaqMan probe (TaqMan PCR) is one of the primary methods for analyzing human DNA structure and is widely used in medicine. In this study, we performed real-time PCR on four fragments of human genomic DNA (chr1: 90717178–90717300, chr9: 97464604–97464756, chr11: 65647215–65647307, chr17: 4560027–4560175), using human blood DNA preparations as templates, obtained both by the standard phenol deproteinization method and by column purification. The PCR used both crude DNA preparations and DNA that had been treated with a restriction enzyme that cleaves the amplifiable fragment. For native DNA preparations purified using columns, PCR on 3 out of 4 DNA fragments yields an overestimated Cq value. For phenol-purified samples, an overestimated Cq value for native DNA preparations is observed for 2 out of 4 DNA fragments. For DNA preparations obtained after column purification, treatment of the DNA with a restriction endonuclease that cleaves the amplified fragment results in normal Cq values for all four analyzed DNA fragments. Thus, PCR with native DNA obtained by column purification may result in an overestimation of the Cq value, which can be corrected by first hydrolyzing the DNA with a restriction enzyme that cleaves the amplicon.
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GlaD-PCR Analysis of DNA Methylation Sites in Regulatory Regions of Tumor Suppressor Genes in Gastric Cancer // Acta Naturae, 2020, Vol. 12, No. 3(46): 124–133 (2020). (In Russian)
Abstract
During the early stages of carcinogenesis, tumor DNA undergoes aberrant methylation within the regulatory regions of multiple tumor suppressor genes at RCGY sites, which serve as substrates for the DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered the first step toward the development of epigenetic PCR-based assays for the early diagnosis of cancer. Previously, we developed the GLAD-PCR method, which enables detection of an R(5mC)GY site at a defined position in the human genome even in the presence of a large excess of DNA molecules containing the corresponding unmethylated RCGY site.
The aim of the present study was to identify aberrantly methylated R(5mC)GY sites in the regulatory regions of the tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA isolated from gastric cancer tissues using GLAD-PCR analysis.
Analysis of DNA samples obtained from surgically resected gastric tumors (n = 29) and histologically normal tissues (n = 25) demonstrated the high diagnostic potential of an epigenetic biomarker panel consisting of R(5mC)GY sites located in the regulatory regions of the irx1, cacna2d3, and epha7 genes. The combined sensitivity and specificity of the marker panel were 96.6% and 100.0%, respectively. -
Comparison of the substrate specificity of GlaI recombinant site-specific methyl-directed DNA endonuclease and the native enzyme isolated from Glacial ice bacterium strain // DNA processing enzymes , DOI: 10.26213/SE.2019.81.28075, (2019). (in Russian)
Abstract
This study describes the cloning of the site-specific methyl-dependent DNA endonuclease GlaI and compares the substrate specificity of the recombinant and native enzymes. An analysis of the properties of the recombinant GlaI endonuclease showed that the substrate specificity of the native and recombinant enzymes does not differ; however, due to the higher amount of enzyme in the recombinant strain, its concentration in the final preparation is more than 10 times higher than that of the native enzyme.
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GlaI-PCR analysis of methylation of ACGC site in Chr11: 65647266 in DNA samples from the blood cells of healthy donors and early stage breast cancers // Epigenetic DNA diagnostics, DOI: 10.26213/SE.2019.66.12.001, (2019).
Abstract
Using the GlaI-PCR method, we analyzed methylation of the ACGC site in the SIPA1 gene at position chr11:65647266 (according to the GRCh38 PrimaryAssembly database) in DNA samples obtained from the light fraction of blood cells from donors and patients with early-stage breast cancer. The study included a) isolation of the light blood cell fraction, b) extraction of genomic DNA, c) determination of genomic DNA concentration by real-time PCR, d) determination of the concentration of the unmethylated ACGC site at position chr11: 65647266 in DNA preparations from the light fraction of blood cells; and e) determining the proportion of DNA molecules containing the unmethylated ACGC site, expressed as a percentage of the total number of DNA molecules. GlaI-PCR analysis showed that in more than 82% of donors, the proportion of the unmethylated ACGC site at chr11: 65647266 in DNA from the light fraction of blood cells is 3% or less, whereas in approximately 70% of patients with breast cancer, the percentage of the unmethylated ACGC site is higher, ranging from 3.2% to 6.4%. These data suggest that in early-stage breast cancer, in two-thirds of patients, demethylation of the A(5mC)GC site in the SIPA1 gene at position 65647266 occurs in the DNA of a small fraction of white blood cells (up to 3.5%). The data presented in this study can be used to develop a PCR test system capable of ruling out breast cancer in its early stages. The GlaI-PCR analysis method does not require complex or expensive equipment or reagents, and the test itself can be performed in a standard PCR laboratory as part of annual preventive screenings and blood tests.
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A new restriction endonuclease LmnI recognizes the nonpalindromic DNA sequence 5`-GCTCC(1/-1)-3` // DNA processing enzymes, DOI: 10.26213/SE.2019.99.27373, (2019). (In Russian)
Abstract
The bacterial strain Lysinibacillus manganicus An22, which we isolated from natural sources, produces the restriction endonuclease LmnI, which recognizes the non-palindromic DNA sequence 5’-GCTCC-3’/3’-CGAGG-5’. This sequence represents a new prototype for restriction enzyme recognition sites. A preparation of the LmnI restriction endonuclease at a concentration of 1,000 units/mL was obtained through purification using a four-step chromatographic process. It has been shown that the new enzyme hydrolyzes DNA to form 3’-protruding “sticky” dinucleotide ends, as indicated by the arrows—5’-GCTCCN↓-3’/3’-CGAG↑GN-5’—and belongs to the IIS subtype of restriction endonucleases.
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Application of GLAD-PCR Assay for Study on DNA Methylation in Regulatory Regions of Some Tumor-Suppressor Genes in Lung Cancer // Zhongguo Fei Ai Za Zhi. 2019 Sep 20;22(9):551-561.
Abstract
Hypermethylation of the gene regulatory regions are common for many cancer diseases. In this work we applied GLAD-PCR assay for identificating of the aberrantly methylated RCGY sites in the regulatory regions of some downregulated genes in tissue samples of lung cancer (LC). This list includes EFEMP1, EPHA5, HOXA5, HOXA9, LHX1, MYF6, NID2, OTX1, PAX9, RARB, RASSF1A, RXRG, SIX6, SKOR1 and TERT genes. The results of DNA samples from 40 cancer and 25 normal lung tissues showed a good diagnostic potential of selected RCGY sites in regulatory regions of MYF6, SIX6, RXRG, LHX1, RASSF1A and TERT genes with relatively high sensitivity (80.0 %) and specificity (88.0 %) of LC detection in tumor DNA.
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Comparative analysis of RCGY sites methylation in three human cell lines. // Epigenetic DNA diagnoctics, vol.2019(1), DOI: 10.26213/SE.2019. 76.40116
Abstract
DNA methylation in human genome is important for the cells specialization and functioning. An abnormal methylation of the regulation regions of some genes may cause the genes silencing and this phenomenon is often detected in cancer cells. Determination of differences of the genome-wide methylation in normal and tumor cells is useful for understanding the carcinogenesis process and for development of new methods of epigenetic diagnostics. The positions of methylated RCGY sites in the genomes of Raji, U-937 and L68 human cell lines have been determined using the previously developed method of massive parallel sequencing of Glal fragments. A comparison of the obtained data has revealed significant differences in methylation of CpG islands, putative regulatory regions and some repetitive DNA families between genomes of malignant and non-malignant cells. GO enrichment analysis of genes with highly methylated regulatory regions has shown the possible metabolic processes, which may be affected epigenetically in carcinogenesis. The new method allows to determine positions of many modified cytosine bases in the genomes and may be a simple alternative to the existing methods of genome-wide methylation analysis.
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Comparative analysis of RCGY sites methylation in the genomes of Raji and U-937 malignant and normal lung fibroblast cell lines // Conference papers: Cancer Epigenetics and Biomarkers, Osaka, Japan, October 2017.
Abstract
An aberrant methylation of the genomic regulatory regions may disrupt normal functioning of cells and often accompanies the human diseases including cancer. Thus, DNA methylation markers have significant diagnostic value and attract attention of many biomedical researchers. Unfortunately, the existing approaches for genome-wide methylation study are rather expensive and laborious. We have developed and successfully tested a novel method of the methylated sites mapping in the genomes. The method is based on a property of methyl-dependent site-specific DNA endonuclease GlaI to cleave DNA only at R(5mC)GY sites forming blunt-ended fragments [1]. The following next generation sequencing of the fragments obtained after GlaI hydrolysis of DNA from Raji malignant cell line allows to reveal more than 2,617,000 positions of R(5mC)GY sites in genome of [2]. We have applied this approach to find differentially methylated RGCY sites in the genomes of malignant Raji and U-937, as well as non-malignant L-68 lung fibroblast cell lines. The positions of methylated RCGY sites in the genomes have been determined using 75 bp pair-ended sequencing of 140-400 bp GlaI fragments at Illumina Genome Analyzer IIx. A comparison of the obtained data has revealed significant differences in methylation of RGCY sites in CpG islands, putative regulatory regions and some repetitive DNA families between all three genomes of malignant and non-malignant cells. GO enrichment analysis of genes with highly methylated regulatory regions has shown the metabolic processes, which may be affected epigenetically in carcinogenesis. The new method allows to determine positions of many modified cytosine bases in the genomes and may be a simple alternative to the existing methods of genome-wide methylation analysis.
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GLAD-PCR assay of selected R(5mC)GY sites in URB1 and CEPBD genes in human genome // Res J Pharm Biol Chem Sci, 8(1): pp.465-475, 2017.
Abstract
The optimal conditions of GLAD-PCR assay of A(5mC)GT site in URB1 gene in human DNA have been determined as follows: a hybrid primer with 4 additional bases,0.33 units of SP-Taq DNA polymerase in 20 µl of reaction mixture, concentration of MgCl2 is 3 mM, concentration of DNA ligase is 10 U/μl. GLAD-PCR assay of tandem of GCGC sites in CEBPD gene was carried out in these optimal conditions and onfirmed a complete methylation of tandem of GCGC sites in DNA from alignant cell lines Raji, U-937 and Jurkat. We believe these conditions may be also used for GLAD-PCR assay of other R(5mC)GY sites in the mammalian genomes. Distinct and clear curves of the fluorescence accumulation in case of methylated sites and an absence of fluorescence signal in case of non-methylated sites show a big potential of GLAD-PCR assay in DNA methylation analysis.
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Use of site-specific DNA endonucleases in genome-wide studies of human DNA]. // CGenetika. 2017 Jan;53(1):3-11. Review. Russian.(2017)
Abstract
Over the past few decades, site-specific DNA endonucleases have served as one of the key tools for studying the primary structure of DNA and for genetic engineering. This review examines examples of the use of these enzymes in whole-genome analysis of human DNA, including the use of restriction endonucleases in sample preparation for sequencing on NGS platforms, as well as the visualization of DNA repeat cleavage by restriction enzymes. Of particular interest are the first studies on the application of DNA endonucleases in the rapidly developing field of epigenetic genome analysis, which has been facilitated by the recent discovery of a new class of enzymes—5-methylcytosine-dependent site-specific DNA endonucleases.
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Complete Genome Sequence and Methylome Analysis of Acinetobacter calcoaceticus 65. // Genome Announc. 2017 Mar 23;5(12). pii: e00060-17. doi: 10.1128/genomeA.00060-17.(2017).
Abstract
Acinetobacter calcoaceticus 65 is the original source strain for the restriction enzyme Acc65I. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.
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Dimethyl Sulfoxide Changes the Recognition Site Preference of Methyl- directed Site-specific DNA Endonuclease GlaI. // Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 7(1),p. 1733-1739 (2016).
Abstract
Methyl-directed site-specific DNA endonuclease GlaI recognizes and cleaves methylated DNA sequence RCGY. GlaI activity in hydrolysis of recognition sequence containing two 5-methylcytosines is noticeably lower than in the case of the site with four or three 5-methylcytosines. We have shown that addition of DMSO to the reaction mixture significantly increases GlaI activity in hydrolysis of sites with two 5-methylcytosines but doesn't change a substrate specificity of the enzyme. A reaction buffer with DMSO may be used in epigenetic studies for GlaI digestion of eukaryotic genomes containing methylated CG pairs.
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Real time GlaI-PCR assay of regulation regions of human genes HDAC4, RARB and URB1 // // Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol 7(2), p.p. 667-676 (2016).
Abstract
Real-time GlaI-PCR assay is developed to determine DNA methylation status of the regulation regions of HDAC4, URB1 and RARB genes in DNA preparations from human leukocytes. Real-time GlaI-PCR assay is DNA hydrolysis with methyl-directed site-specific DNA endonuclese GlaI followed by real-time PCR from the primers located upstream and downstream of the studied DNA region. The obtained data confirm a full methylation of the studied ACGT and GCGC sites in the regulatory regions of the HDAC4 and URB1 genes and a complete hydrolysis of these sites with GlaI. A first exon of RARB gene is slightly methylated in the leukocytes DNA preparations and according to the results of GlaI-PCR assay we don’t observe GlaI hydrolysis of ACGCG site in RARB gene. The data obtained correspond to the literature data. The proposed method of real-time GlaI-PCR assay may be used to determine the methylation status of any unique parts in human and mammalian genomes.
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Application of GLAD-PCR assay for determination of the methylation sites in the regulatory regions of tumor-supressors gene ELMO1 and ESR1 in colorectal cancer // Translated from Problems in oncology, #1, p.116-120 (2016). (In Russian)
Abstract
Abnormal methylation of regulatory regions of tumor suppressor genes has been demonstrated in many types of cancer. In this study, the GLAD-PCR analysis method we previously developed [3] was applied to identify R(5mC)GY sites arising from aberrant methylation of the regulatory regions of the ELMO1 and ESR1 genes in DNA preparations from the SW837 cell line and colorectal tumor tissues. Four sites were selected for each gene, and it was found that in SW837 DNA, the most highly methylated sites are the GCGC site in the first exon of the ELMO1 gene and the GCGT site in the ESR1 gene promoter. The GCGT site in the ESR1 gene promoter is weakly methylated in healthy tissues and more highly methylated in most tumor tissues. The GCGC site in the first exon of the ELMO1 gene is unmethylated in healthy tissue DNA and methylated in 60% of tumor DNA samples. The potential for using the GLAD-PCR method to analyze extracellular DNA from patients’ blood for the diagnosis of colorectal cancer is discussed.
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Comparative analysis of biochemical properties of site specific methyl-directed DNA endonucleases BisI, BlsI and EcoBLI recognizing 5`-G(5mC)NGC-3`/3`-CGN(5mC)G-5` // Research Journal of Pharmaceutical, Biological and Chemical Sciences, RJPBCS, vol 7(2), p.p. 388-394. (2016)
Abstract
A comparative analysis of biochemical properties of three similar site specific methyl-directed DNA endonucleases BisI, BlsI and EcoBLI recognizing 5'-G(5mC)NGC-3'/3'-CGN(5mC)G-5' has been done. Optimal concentrations of MgCl2, NaCl or KCl, pH of reaction buffers as well as the optimal reaction temperatures for these enzymes were determined. It was shown that BisI has the maximal activity in the buffer 10 mM Tris-HCl (pH 9.0 at 25°C), 15 mM MgCl2, 150 mM KCl, 1 mM DTT. An optimal buffer for MD-endonuclease BlsI is 10 mM Tris-HCl (pH 8.0 at 25°C), 15 mM MgCl2, 100 mM NaCl, 1 mM DTT. The enzyme EcoBLI displays a maximal activity in the buffer 10 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 25 mM NaCl, 1 mM DTT. Optimal temperature for all enzymes is 37°C.
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GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer // Biol Med (Aligarh) , 8:7(2016).
Abstract
Hypermethylation of the gene regulatory regions is documented for many cancer diseases. Such an aberrant DNA methylation in cancer cells is catalyzed by DNA methyltransferases Dnmt3a and Dnmt3b, which predominantly recognize and methylate RCGY sequences with formation of R(5mC)GY sites. Recently, based on a new methyl-directed DNA endonuclease GlaI, we developed a GLAD-PCR assay, which allows determining R(5mC)GY site in a defi ned position of the genomic DNA. In this work we applied GLAD-PCR assay for identifi cation of the methylated RCGY sites in the regulatory regions of some downregulated genes associated with colorectal cancer (CRC). This list includes ADHFE1, ALX4, CNRIP1, EID3, ELMO1, ESR1, FBN1, HLTF, LAMA1, NEUROG1, NGFR, RARB, RXRG, RYR2, SDC2, SEPT9, SFRP2, SOCS3, SOX17, THBD, TMEFF2, UCHL1, and VIM genes. GLAD-PCR analysis of selected RCGY sites within the regulatory regions of some of these genes demonstrates a good prognostic potential with relatively high sensitivity and specifi city of CRC detection in tumor DNA.
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New DNA methyltransferase M.AgsI produces TTSA(m6A) // BioTechnology : An Indian Journal. V. 12 (2). – P. 100-106 (2016).
Abstract
DNA from Agrococcus sp. 25 was cloned into the pUC19 plasmid in Escherichia coli. The cloned DNA fragment contained two open reading frames encoding proteins with the conserved amino acid motifs characteristic of amino DNA methyltransferases. One of these genes was predicted to encode the first subunit of the adenine (N6)-DNA methyltransferase M.AgsI.
The enzyme was purified from the recombinant E. coli strain by sequential chromatography on P-11 phosphocellulose, heparin-Sepharose, and hydroxyapatite. The specificity of M.AgsI was determined by analyzing the protection of λ DNA methylated by the enzyme against cleavage by a panel of restriction endonucleases. The sensitivity of restriction endonucleases to M.AgsI-mediated methylation was also investigated. -
Cloning and characterization of a new site specific methyl- directed DNA endonuclease EcoBLI recognizing 5`- G(5mC)NGC-3`/3`-CGN(5mC)G-5` // BioTechnology: An Indian Journal. Volume 12, Issue 4, Pages 175-181 (2016).
Abstract
The gene encoding BisI, a site-specific methyl-directed DNA endonuclease recognizing the sequence 5′-G(5mC)NGC-3′/3′-CGN(5mC)G-5′, was recently identified in the sequenced genome of its producer strain, Bacillus subtilis T30. In the present study, we searched for bisI homologs among the sequenced genomes of enterobacteria. Sequence analysis revealed a small group of highly homologous open reading frames (ORFs) of unknown function, including one ORF in the genome of the well-characterized strain Escherichia coli BL21(DE3).
The ORF WP_001276099.1 from E. coli BL21(DE3) was amplified and cloned. The resulting recombinant strain, E. coli pEcoBLI, produced a methyl-directed endonuclease designated EcoBLI. The new enzyme exhibited the same substrate specificity as the methyl-directed endonuclease BisI. Thus, ORF WP_001276099.1 from E. coli BL21(DE3) encodes the site-specific methyl-directed DNA endonuclease EcoBLI, which recognizes and cleaves the sequence 5′-G(5mC)^NGC-3′/3′-CGN^(5mC)G-5′. -
Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes. // Sci Rep. 2016 Jun 29;6:28579. doi: 10.1038/srep28579. (2016).
Abstract
The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four (m5)C in the two strands, or hemi-methylated sites containing two (m5)C in one strand; Group II enzymes only cut GCNGC sites containing three to four (m5)C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four (m5)C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.
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Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5`-G(5mC)^NG(5mC)-3`. // Acta Naturae. 2016 Jan-Mar;8(1):117-25. (2016).
Abstract
A search for amino acid sequences homologous to the BisI MD endonuclease sequence in the genomes of enterobacteria revealed putative open reading frames encoding MD endonucleases. The high conservation of the nucleotide sequences of these open reading frames in enterobacteria of different genera (Escherichia, Klebsiella, and Cronobacter) made it possible to design primers for PCR screening of enterobacterial DNA collected from natural sources. Using genomic DNA from E. coli LM N17 as a template, a DNA fragment approximately 440 bp in length was amplified and cloned into the pMTL22 vector. Endonuclease activity was detected in the E. coli ER2267 strain transformed with the resulting construct. A preparation of the new enzyme ElmI was obtained from the biomass of this strain using chromatographic methods. It was found that ElmI, like BisI, specifically cleaves the methylated sequence 5’-GCNGC-3’ upstream of the central nucleotide “N,” provided that it contains two 5-methylcytosine residues. However, unlike BisI, ElmI cleaves this sequence more efficiently when it contains more than two methylated cytosine residues.
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Complete Genome Sequence Analysis of Bacillus subtilis T30. // Genome Announc. 2015 May 7;3(3). pii: e00395-15. doi: 10.1128/genomeA.00395-15. PMID:25953183.
Abstract
The complete genome sequence of Bacillus subtilis T30 was determined by SMRT sequencing. The entire genome contains 4,138 predicted genes. The genome carries one intact prophage sequence (37.4 kb) similar to Bacillus phage SPBc2 and one incomplete prophage genome of 39.9 kb similar to Bacillus phage phi105.
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Cloning and Study of NewDNA Methyltransferase M.FatI Modifying Cytosine in a Recognition Site CATG. // Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol 6 (6), P. 1341-1348 (2015).
Abstract
A fragment of Flavobacterium aquatile NL3 DNA carrying the gene of DNA methyltransferase M.FatI
was cloned in pUC19 plasmid. DNA was sequenced and M.FatI gene was analyzed. A recombinant strain
Esherichia coli was grown up and the enzyme was purified. M.FatI specificity was determined by a blocking of
some restriction endonucleases and computer modeling. It’s well known that M.NlaIII produces 5’-C(m6A)TG3’, whereas FatI MTase modifies the cytosine residue with formation 5’-(m5C)ATG-3’. The sensitivity of
restriction endonucleases to FatI-methylation has been studied.
Keywords: gene cloning, enzyme isolation, bacterial DNA methyltransferase, enzyme specificity, restriction
endonuclease, methylation sensitivity. -
Mapping of R(5mC)GY Sites in the Genome of Human Malignant Cell Line Raji // Biol Med (Aligarh) Volume 7, Issue 4, BM-135-15. (2015).
Abstract
The purpose of this study was to develop a method for the wide-scale determination of methylated RCGY sites in a genome using the next-generation sequencing (NGS) approach. We hydrolyzed genomic DNA from human, malignant cell line Raji with methyl-directed site-specific endonuclease GlaI and isolated DNA fragments 140-400 bp in length. DNA structure of 75 bp was determined for both ends of each DNA fragment based on NGS technology with Illumina MiSeq. The mapping of the obtained sequences on the human reference genome allowed to define the positions of more than one million R(5mC)GY sites represented in Raji genome with relatively high frequency. These data were compared with the results of the methylation study of regulatory regions of tumor suppressor genes studied earlier with a new version of PCR analysis. We have concluded that a suggested method of R(5mC)GY sites determination in a whole genome may be used for epigenetic studies, particularly for determination of methylation of regulatory regions of genes, which may affect the gene expression.
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Development and evaluation of physicochemical methods in ecological studies // Water: chemistry and ecology , No 10 (76), p. 119-122 (2014). (In Russian)
Abstract
Разработан комплексный подход использования физико-химических методов при решении экологических задач. Представлены результаты поиска индикаторов антропогенного влияния. Выявлено, что в прибрежной части экосистемы оз. Байкал наблюдается качественное изменение микроорганизмов по наличию или отсутствию в них ферментов эндонуклеаз рестрикции (рестриктаз ЭР). Применение этого метода позволит значительно повысить надежность индикации антропогенного загрязнения водных экосистем на ранней стадии
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Method of determining nucleotide sequence Pu(5mC)GPy at predetermined position of long-distance DNA. // Russian Federation patent RU 2525710 C1 (2014). (In Russian)
Abstract
The invention relates to biotechnology. The proposed method involves obtaining samples of highly purified DNA, fragmenting the isolated DNA with a restriction endonuclease that lacks a recognition site in the region to be amplified, hydrolysis of the fragmented DNA with the methyl-dependent site-specific DNA endonuclease GlaI or its isozomere, ligation of a universal oligonucleotide adapter to the hydrolyzed DNA, followed by real-time amplification using a primer and probe complementary to the DNA of interest and a hybrid primer, whose 3' end is complementary to at least 3 nucleotides of the 3' end of the DNA at the GlaI hydrolysis site of interest, and the remaining portion is complementary to the adapter sequence, and drawing a conclusion based on the fluorescent signal regarding the presence of the Pu(5mC)GPy sequence.
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Recombinant bacterial strain Escherichia coli n41 (pbpun4/mr)- producer of Bpun4i site specific endonuclease. // Russian Federation patent RU 2529362 C1 (2014). (In Russian)
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Development of a simple method for obtaining highly purified blood cell-related DNA // Pediatric Hematology/Oncology and Immunopathology, v.13, No.4, p.50-55 (2014). (In Russian)
Abstract
Предложен новый метод очистки ДНК, связанной с клетками крови. Метод позволяет получить высокоочищенный препарат ДНК, пригодный для диагностики с помощью полимеразной цепной реакции в режиме реального времени, в течение одного дня. Метод не требует использования дорогостоящих реагентов, а количество ДНК в получаемых препаратах выше, чем при выделении другими методами.
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Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome // BMC Genomics.2013, 14:771.
Abstract
Background: Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats.
Results: Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed.
Conclusion: Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes. -
Substrate specificity and properties of methyl-directed site-specific DNA endonucleases // Molecular Biology, v.47, No.6, p.900-913 (2013). (In Russian)
Abstract
Methyl-directed site-specific DNA endonucleases (MD endonucleases) are a small group of enzymes that specifically cleave only methylated DNA. The group includes N6-methyladenine- and 5-methylcytosine-directed enzymes. Although poorly understood, MD endonucleases are of interest for both basic research and application in biotechnology and epigenomics. The review for the first time summarizes the properties of MD endonucleases and considers their role in the bacterial cell and their possible uses in biotechnology and epigenomics.
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MteI-PCR assay - a new method of DNA methylation study in GC rich regulation regions of human tumor supressor genes // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.9, No.3, p.15-23 (2013). (In Russian)
Abstract
A new method for MteI-PCR analysis of GC-rich DNA regions has been proposed, based on the unique methyl-dependent site-specific DNA endonuclease MteI, which has an extended, highly methylated recognition site. The method involves hydrolysis of DNA by the MteI enzyme, followed by PCR either in real time or with the products analyzed by gel electrophoresis. The methylation status of regulatory regions of several tumor suppressor genes was determined using the MteI-PCR method in comparison with the previously proposed BlsI- and GlaI-PCR methods. The applicability of the MteI-PCR method for analyzing the methylation status of regulatory regions of the CEBPD, HS3ST2, RASSF1A, SEPT9b, and TWIST1 genes was demonstrated. In the case of the RASSF1A gene, real-time MteI-PCR analysis, unlike real-time GlaI- and BlsI-PCR analysis, does not detect methylation of this DNA region in control healthy cells from a lung fibroblast line. Thus, MteI-PCR analysis allows for a more precise determination and differentiation of the methylation status of the regulatory region of the RASSF1A gene and, likely, other GC-rich regions of human DNA.
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Сloning of BpuN4I restriction-modification system from Bacillus pumilus N4 // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.9, No.3, p.40-47 (2013). (In Russian)
Abstract
The DNA of the Bacillus pumilus N4 bacterial strain, which carries the BpuN4I restriction-modification system (recognition site 5’-GGNNCC-3’), was hydrolyzed with the restriction endonucleases MfeI and EcoRI, and the resulting products were cloned into the pUC19 vector, which had been linearized with EcoRI. To select clones carrying the BpuN4I restriction endonuclease gene, a selection method was proposed based on the dilution of the resulting clone library, with direct determination of restriction endonuclease activity in total cell lysates. Using this method, a recombinant E. coli strain, N41(pBpuN4/MR), was identified as a producer of the BpuN4I enzyme. The resulting library was also screened using the traditional Venezianer method, yielding two distinct clones: one identical to E. coli N41(pBpuN4/MR), and the other, E. coli N7(pBpuN4/MR), which contains the plasmid insert in the opposite orientation and exhibits lower activity. The structure of the plasmid insert from E. coli strain N41(pBpuN4/M)R was determined, and it was shown to contain the genes for the DNA methyltransferase M.BpuN4I and the restriction endonuclease BpuN4I. A comparative analysis of the amino acid sequences of the enzymes encoded by these genes revealed homology between M.BpuN4I and other methyltransferases that recognize the 5’-GGNNCC-3’ site or a site similar to it. All domains characteristic of 5mC-DNA methyltransferases were identified in the amino acid sequence of M.BpuN4I.
Translated with DeepL.com (free version) -
A new site-specific DNA endonuclease BpuN4I cleaves nucleotides sequence 5`-G^GNNCC-3`. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.9, No.2, p.5-8 (2013). (In Russian)
Abstract
A new restriction endonuclease, BpuN4I, has been isolated from the bacterial strain Bacillus pumilus N4, and its substrate specificity and DNA cleavage site have been determined. The restriction endonuclease BpuN4I recognizes the DNA sequence 5’-G↓GNNCC-3’ and cleaves it as indicated by the arrow. This is the first time an enzyme that cleaves the 5’-GGNNCC-3’ site to form four-nucleotide 5’-sticky ends has been identified. The BpuN4I restriction enzyme is a neoschizomer of the NlaIV enzyme, which has the same recognition sequence but produces DNA fragments with blunt ends.
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Methyl-directed site-specific endonuclease BlsI recognizes and cleaves DNA sequence 5’-RYNRY-3’ carrying of at least two 5-methylcytosines // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.9, No.2, p.30-38 (2013). (In Russian)
Abstract
The substrate specificity of the methyl-dependent site-specific DNA endonuclease BlsI has been studied. It was shown that BlsI hydrolyzes the degenerate DNA sequence 5’-RYNRY-3’ (where R is A or G, and Y is T or C) when it contains two or more 5-methylcytosines. The efficiency of DNA hydrolysis by the enzyme depends on the number of 5-methylcytosines and their positions within the recognition sequence. Since the BlsI endonuclease cleaves only C5-methylated DNA, it can be used in molecular biology and epigenetic studies to assess the methylation of eukaryotic genomic DNA.
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A new methyl-directed site-specific DNA endonuclease MteI cleaves nine nucleotides sequence 5`-G(5mC)G(5mC)^NG(5mC)GC-3`/3`-CG(5mC)GN^(5mC)G(5mC)G-5`. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.8, No.1, p.16-26 (2012). (In Russian)
Abstract
A new methyl-dependent site-specific DNA endonuclease, MteI, has been isolated from the bacterial strain Microbacterium testaceum 17B. The enzyme recognizes a C5-methylated DNA sequence and does not hydrolyze unmethylated DNA. MteI is the first of the methyl-dependent site-specific DNA endonucleases to recognize an extended site, and its activity depends on the number of 5-methylcytosines and their positions within the recognized sequence. MteI cleaves the site 5’-G(5mC)G(5mC)↑NG(5mC)GC-3’/3’-CG(5mC)GN↓(5mC)G(5mC)G-5’ as indicated by the arrows, and this sequence represents the minimal recognition site. Enzyme activity increases significantly when the 5’-GC-3’ dinucleotides in the minimal recognition site are replaced by 5’-G(5mC)-3’ dinucleotides and additional 5’-G(5mC)-3’ dinucleotides appear at the 5’-ends of the nonanucleotide in both DNA strands. Due to its ability to cleave only long methylated DNA sequences, the MteI enzyme may find practical application in molecular biology and epigenetic research.
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A M.BssECI DNA Methyltransferase Forms 5`-m4CCNNGG-3`. Sensitivity of Restriction Endonucleases to the New Methylation. // Biotekhnologia (Moscow), No 2, p.32-42 (2012). (In Russian)
Abstract
[TRANSLATION] A DNA methyltransferase, M.BssECI, was isolated and purified from Bacillus stearothermophilus EC. The enzyme methylates the outer cytosine residue within the DNA sequence 5′-CCNNGG-3′, producing N4-methylcytosine (5′-m4CCNNGG-3′).
Procedures for enzyme isolation and purification, including gel filtration on Bio-Gel A-0.5m followed by chromatography on benzyl-DEAE-cellulose and heparin-Sepharose, as well as the method used to identify the methylated DNA sequence, are described. The optimal reaction conditions and methyltransferase activity were determined by measuring the protection of bacteriophage λ DNA from cleavage by the restriction endonuclease BssECI.
The methylated base within the recognition sequence was identified using a [^3H]-labeled oligonucleotide duplex. The specificity of M.BssECI was determined using "self-methylated" adenovirus type 2 DNA, data on the sensitivity of the restriction endonucleases BssECI, MvaI, and MspI to DNA methylation, and computer modeling.
The purified enzyme may serve as a useful tool for studies of DNA methylation. In particular, the approach described here allows discrimination between N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Bacteriophage λ and T7 DNA methylated by M.BssECI were also used to investigate the sensitivity of several restriction endonucleases to methylation within their recognition sites. -
Novel M.BstC8I Methyltransferase Forms 5`-Gm5CNNGC-3`.Investigation of Restriction Endonuclease Sensitivity to M.BstC8I Methylation. // Mol. Gen., Microbiol. and Virology, Vol. 27. No.1. P. 40-47 (2012). (In Russian)
Abstract
[TRANSLATION] A novel DNA methyltransferase, M.BstC8I, was identified in the cellular lysate of Bacillus stearothermophilus C8 grown on Luria agar at 37°C. Methylation of bacteriophage λ and T7 DNA at the sequence 5′-G(5mC)NNGC-3′ blocked cleavage by the restriction endonuclease BstC8I. The specificity of M.BstC8I was determined using λ DNA methylated by the enzyme, computer modeling, and data on the sensitivity of the restriction endonucleases BstC8I, BsuRI, AjnI, and PvuII to DNA methylation. Several restriction endonucleases were shown to be sensitive to methylation overlapping their recognition sites. These results may be useful for studies of DNA methylation.
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Epigenetic typing of human cancer cell lines by BlsI- and GlaI-PCR assays. // Ovchinnikov bulletin of biotechnology and physical and chemical biology V.7, No 3, pp 5-16, 2011.
Abstract
The BlsI- and GlaI-PCR analysis method was used to determine the methylation status of the regulatory regions of the tumor suppressor genes SEPT9b, IGFBP3, CEBPD, MGMT, and RASSF1A in the DNA of the malignant cell lines HeLa, Raji, U-937, and Jurkat, as well as in control DNA from the L-68 fibroblast cell line. BlsI- and GlaI-PCR analysis showed that the regulatory regions of the tumor suppressor genes RASSF1A, SEPT9b, IGFBP3, CEBPD, and MGMT may either contain the methylated 5’-R(5mC)GY-3’ sequence or lack these sites, depending on the malignant cell line from which the DNA was isolated. Various combinations of tumor suppressor genes with methylated sites were observed in the DNA of the studied cell lines. However, in healthy cells of the L-68 fibroblast line, no 5’-R(5mC)GY-3’ sites were detected in any of the aforementioned regulatory regions. Based on these results, we propose the use of BlsI- and GlaI-PCR analysis for the epigenetic typing of malignant cell lines.
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A new methyl-directed site-specific endonuclease KroI recognizes and cuts DNA sequence 5`-G^C(5mC)GGC-3`. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.7, No.1, p.14-20 (2011). (In Russian)
Abstract
A new 5-methylcytosine-dependent site-specific DNA endonuclease, KroI, has been isolated from Kocuria rosea 307. It recognizes and cleaves the DNA sequence 5'-G↑CCGGC-3'/3' -CGGCC↑G-5' in the presence of one or two 5-methylcytosines in the central dinucleotide and its complement, 5'-CG-3'/3'-GC-5'. KroI does not hydrolyze unmethylated DNA, nor does it hydrolyze the methylated sequences 5'-G(5mC)CGGC-3'/3'-CGGC(5mC)G-5' and 5'-G(5mC)CGG(5mC)-3'/3'-(5mC)GGC(5mC)G-5'. KroI is the first site-specific methyl-dependent endonuclease that recognizes a non-degenerate six-nucleotide sequence. Due to its ability to cleave only methylated DNA, the KroI enzyme can be used in molecular biology applications, particularly for determining the methylation status of eukaryotic DNA.
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A new site-specific DNA endonuclease FaiI recognizes and cuts tetranucleotide sequence 5`-YA^TR-3`. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.7, No.2, p.11-16 (2011). (In Russian)
Abstract
The substrate specificity of the new site-specific DNA endonuclease FaiI has been studied; it recognizes and cleaves the sequence 5’-YA↑TR-3’ and also hydrolyzes the 5’-YA↑TY site with lower activity. When one of the central AT nucleotides in the recognition sequence is substituted, the enzyme exhibits nuclease activity, cleaving the strand of the recognition site in which thymine is retained. FaiI cleaves DNA containing 5-methylcytosine, N4-methylcytosine, and N6-methyladenine at the recognition site. Keywords: site-specific endonuclease, degenerate recognition sequence, minor-cleaving endonuclease.
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A new site-specific methyl-directed DNA endonuclease PkrI recognizes and cuts methylated DNA sequence 5`-GCN^GC-3`/3`-CG^NCG-5` carrying at least three 5-methylcytosines. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.7, No.3, p.35-42 (2011). (In Russian)
Abstract
A new site-specific methyl-dependent
DNA endonuclease PkrI has been isolated from the Planomicrobium koreense 78k bacterial strain; this enzyme recognizes and cleaves, as indicated by the arrow, the methylated
DNA sequence 5'-GCN↑GC-3’/3’-CG↓NCG-5’ when it contains at least three 5-methylcytosines and
does not cleave unmethylated DNA. Due to its ability to cleave only
methylated DNA, the PkrI enzyme can find practical application in molecular biology
research, in particular, for determining the methylation status of eukaryotic DNA. -
Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI // Moscow University Biological Sciences Bulletin, Series 16: Biology, No.2, p.38-40 (2011). (In Russian)
Abstract
[TRANSLATION] The genes encoding the DNA methyltransferases of the BspACI restriction–modification system from Bacillus psychrodurans AC were cloned in Escherichia coli. Analysis of the amino acid sequences of the encoded proteins showed that both enzymes belong to the C5 DNA methyltransferase family. The M1.BspACI gene was subcloned into the expression vector pJW2.
The recombinant enzyme was purified to homogeneity by chromatography on several matrices. M1.BspACI was shown to methylate the first cytosine in the sequence 5′-CCGC-3′. The kinetic parameters of the DNA methylation reaction catalyzed by the enzyme were determined. The catalytic constant (kcat) was 0.095 ± 0.002 min⁻¹, the Km for λ DNA was 0.053 ± 0.007 μM, and the Km for S-adenosyl-L-methionine (SAM) was 5.1 ± 0.3 μM. -
Bacterial strain Microbacterium testaceum 17B - producer of MteI site specific endonuclease. // Russian Federation patent RU 2475533 C1 (2011).
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Bacterial strain Planomicrobium koreense 78K - producer of PkrI site specific endonuclease. // Russian Federation patent RU 2475534 C1 (2011).
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Analysis of substrate specificity and biochemical properties of DNA methyltransferase M3.BstF5I from BstF5I restriction-modification system // Biochemistry (Moscow), Vol. 75, No 1, p.78-87 (2010). (In Russian)
Abstract
[TRANSLATION] The optimal conditions for DNA methylation by M3.BstF5I from Bacillus stearothermophilus were determined, and the kinetic parameters of the modification reaction were established using bacteriophage λ DNA and a series of oligonucleotide substrates.
Comparison of the kinetic properties of M1.BstF5I and M3.BstF5I showed that, despite having similar temperature optima and DNA-binding affinity, M3.BstF5I exhibits an approximately fourfold lower turnover number (0.24 min⁻¹) under optimal conditions and methylates hemimethylated recognition sites less efficiently than unmethylated sites.
Unlike the other three DNA methyltransferases of the BstF5I restriction–modification system, M3.BstF5I is also capable of methylating the noncanonical DNA sequence 5′-GGATC-3′, although the rate of modification is more than an order of magnitude lower than that observed for the canonical recognition sequence 5′-GGATG-3′. -
Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: purification, properties and amino acids sequence analysis. // Molecular Biology(Moscow), Vol.44, No. 4, 688-698 (2010). (In Russian)
Abstract
The gene encoding the DNA methyltransferase M1.Bst19I of the Bst19I restriction–modification system (recognition sequence 5′-GCATC-3′) from Bacillus stearothermophilus 19 was cloned into the expression vector pJW, carrying tandem thermally inducible λ phage PR/PL promoters.
A highly purified preparation of the enzyme was obtained from Escherichia coli cells, in which the protein accumulated in soluble form, by sequential chromatography on several chromatographic media.
M1.Bst19I exhibits a temperature optimum of 50°C and maximal activity at pH 8.0. The enzyme methylates the adenine residue within the sequence 5′-GCATC-3′. The kinetic parameters of the DNA methylation reaction catalyzed by M1.Bst19I were determined. The Michaelis constant (Km) was 0.68 ± 0.07 μM for bacteriophage λ DNA and 2.02 ± 0.31 μM for S-adenosyl-L-methionine (SAM). The catalytic constant (kcat) was 1.8 ± 0.05 min⁻¹.
Comparative analysis of the amino acid sequence of the substrate recognition domain of M1.Bst19I and other N6-adenine DNA methyltransferases suggests the existence of two classes of enzymes recognizing DNA sequences containing either the ATG or ATC triplet. -
Abstract
A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. Gene bspACIM1 was cloned in pJW2 vector and expressed in E. coli cells. High purity enzyme M1.BspACI preparation was obtained by chromatography on different carriers. Study of the properties of M1.BspACI revealed that the enzyme has a temperature optimum of 30°C and maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the sequence 5′-CCGC-3′. Kinetic parameters of the M1.BspACI DNA methylation reaction were determined as follows: Km for lambda DNA is 0.053 μM, Km for S-adenosyl-L-methionine is 5.1 μM, and the catalytic constant (kcat) is 0.095 min⁻¹.
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BlsI- and GlaI-PCR assays – a new method of DNA methylation study // Ovchinnikov bulletin of biotechnology and physical and chemical biology , V.6, No 1, p. 5-12 (2010). (in Russian)
Abstract
Based on unique methyl-dependent site-specific DNA endonucleases, a new method for BlsI- and GlaI-PCR analysis of methylated DNA regions has been proposed. The method involves treating DNA with BlsI or GlaI enzymes, respectively, followed by PCR analysis of the resulting hydrolysates. Using the methylation of regulatory regions of several human tumor suppressor genes as an example, the effectiveness of the new method and the agreement of the results obtained using it with bisulfite DNA sequencing data are demonstrated. Analysis of methylation in the promoter region of the DAPK1 gene, the promoter region and first exon of the RARB gene, and the first exon region of the RASSF1A gene using the new method revealed distinct methylation patterns in these DNA regions across the HeLa, Raji, U-937, and Jurkat cell lines, as well as in the control L-68 cells. GlaI-PCR analysis detected methylation of the regulatory region of the RARB gene in the DNA of all malignant cell lines and the absence of this modification in the DNA of L-68 cells. BlsI- and GlaI-PCR analysis of the first exon region of the RASSF1A gene revealed the presence of methylated DNA regions only in Raji and Jurkat cells. BlsI-PCR analysis of the promoter region of the DAPK1 gene demonstrated additional methylation of this DNA region only in Raji cells. The potential use of BlsI- and GlaI-PCR analysis for detecting malignant cells and determining the type of oncological pathology is discussed.
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Purification of recombinant DNA methyltransferase M2.BstSEI from nickase-modification system NM.BstSEI and study of the enzyme properties. // Molecular Biology(Moscow), Vol.43, No. 1, 10-18 (2009). (In Russian)
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GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation // BMC Genomics 2009, 10:322.
Abstract
Patterns of mouse DNA hydrolysis with restriction enzymes are coincided with calculated diagrams of genomic DNA digestion in silico, except presence of additional bright bands, which correspond to monomer and dimer of γ-satellite DNA. Only small portion of mouse γ-satellite DNA sequences are presented in databases. Methyl-directed endonuclease GlaI cleaves mouse DNA and may be useful for a detailed study of primary structure and CG dinucleotides methylation in γ-satellite DNA.
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New site-specific endonuclease BspACI from BACILLUS PSYCHRODURANS AC recognizes 5`-C^CGC-3`/3`-GGC^G-5`. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.5, No.1, p.16-24 (2009). (In Russian)
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Defining the Specificity of DNA Methyltransferase M.Bsc4I in Cellular Lysate by Blocking Restriction Endonucleases and Computer Modeling // Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya (Moscow), No 3, p.114-120 (2009).
Abstract
The specificity of DNA methyltransferase M. Bsc4I was determined in cellular lysate of Bacillus schlegelii 4. The methylation sensitivity of restriction endonucleases and methylation modeling were used for this purpose. Modeling consisted of editing DNA sequences using replacements of methylated bases and their complementary bases. Substrate DNA treated with M. Bsc4I were used to study the sensitivity of some restrictases to methylation. It was shown that M. Bsc4I methylated 5′-Cm4CNNNNNNNGG-3′ and overlapping dcm-methylation blocked its activity. The suggested approach would be universal and simple for determining the specificity of DNA methyltransferases
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Analysis of Specificity of DNA-Methyltransferase M.AspS9I in Cell Lysate by means of Restriction Endonucleases Blocking. // Biotekhnologia (Moscow), No 4, p.30-39 (2009). (In Russian)
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Bacterial strain Kocuria rosea - producer of KroI site specific endonuclease. // Russian Federation patent RU 2394099 C1 (2009).
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A method for identifying hypermethylated CpG islands in the regions of tumour-suppressor genes in human DNA. // Patent RU 2413773 C1 (2009). (In Russian)
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Bacterial strain Arthrobacter oxydans - producer of AoxI site specific endonuclease. // Russian Federation patent RU 2399663 C1 (2009). (In Russian)
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Substrate specificity of new methyl-directed DNA endonuclease GlaI . // BMC Molecular Biology 2008, 9:7.
Abstract
Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI.
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A physical map of human Alu repeats cleavage by restriction endonucleases // BMC Genomics 2008, 9:305.
Abstract
Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico.
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New restriction endonuclease GsaI, neoschizomer of BseY I, recognizes 5’-CCCAGC(-1/-5)-3’. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.4, No.1, p.19-24 (2008). (In Russian)
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Bacterial strain Paracoccus carotinifaciens 3K- producer of PcsI site specific endonuclease. // Russian Federation patent RU 2377294 C1 (2008). (In Russian)
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New Restriction Endonuclease AluBI from Arthrobacter luteus B - AluI Isoshizomer, nonsensitive to Presence of 5-methylcytosine in the Recognition Sequence AGCT. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.3, No.1, p.21-27 (2007). (In Russian)
Abstract
The properties of a new restriction endonuclease, AluBI, which recognizes and cleaves the AGCT sequence and is an isozomere of the well-known AluI restriction endonuclease, are described. Unlike AluI, AluBI cleaves DNA when the cytosine bases at the recognition site are C5-methylated. AluBI also cleaves the recognition sequence when one cytosine is C5-methylated and the other is N4-methylated; however, it does not cleave DNA when both cytosines are N4-methylated.
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Site-specific endonuclease BlsI recognizes DNA sequence 5’-G(5mC)N^GC-3’; and cleaves it producing 3’ sticky ends. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.3, No.1, p.28-33 (2007). (In Russian)
Abstract
A bacterial strain, Bacillus simplex 23, which produces the site-specific endonuclease BlsI, has been isolated from natural sources. This enzyme recognizes the methylated DNA sequence 5’-G(5mC)N↑GC-3’, just like the previously discovered site-specific endonuclease BisI (recognition site 5’-G (5mC)↑NGC-3’), but, unlike the latter, it cleaves DNA to form single-nucleotide 3’-overhanging ends. Due to its ability to cleave only methylated DNA, the BlsI enzyme can find practical application in genetic engineering, as well as for determining the methylation status of eukaryotic DNA.
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A novel site-specific endonuclease GluI recognizes methylated DNA sequence 5’-G(5mC)^NG(5mC)-3’/3’-(5mC)GN^(5mC)G. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.3, No.2, p.13-17 (2007). (In Russian)
Abstract
A new site-specific endonuclease, GluI, was isolated and characterized from the bacterial strain Glacial ice bacterium GL24. This enzyme recognizes the methylated DNA sequence 5’-G(5mC)^NG(5mC)-3’/3’-(5mC)GN^(5mC)G-5’ and cleaves it as indicated by the arrow. Due to its ability to cleave only highly methylated DNA, the GluI enzyme may find practical applications in genetic engineering, as well as for determining the methylation status of eukaryotic DNA.
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The Sse9I restriction-modification system: organization of genes and comparative analysis of proteins structures. // Molecular Biology, V.41, No 3, p. 491-498 (2007). (In Russian)
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Gene cloning, comparative analysis of the protein structures from Fsp4HI restriction-modification system and biochemical characterization of the recombinant DNA methyltransferase M.Fsp4HI. // Molecular Biology, V.41, No 1, p. 43-50 (2007). (In Russian)
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Comparative analysis of human chromosomal DNA digestion with restriction endonucleases in vitro and in silico. // Medical genetics, V.6, No 8, p. 29-36 (2007). (In Russian)
Abstract
The potential for using the method of cleaving human chromosomal DNA with restriction endonucleases in medical genetics research has been investigated. Based on previously published work on the in silico analysis of mammalian genomes, distribution diagrams of human chromosomal DNA fragments were calculated following digestion with 15 nucleotide sequences that serve as restriction enzyme recognition sites. An analysis of the primary structure of Alu and LINE1 DNA repeats was conducted, and distribution diagrams of DNA repeat fragments were constructed upon digestion with these 15 nucleotide sequences. It was shown that the distribution diagrams of the resulting low-molecular-weight fragments of chromosomal DNA correspond to the distribution diagrams of Alu repeat fragments, whereas the distribution patterns of high-molecular-weight fragments are analogous to the distribution diagrams of LINE1 repeats. Profiles of human DNA hydrolysis by the corresponding restriction endonucleases were obtained, and a comparison was made between theoretical fragment distribution profiles and the experimentally obtained results. The prospects for the use of site-specific endonucleases in DNA diagnostics are discussed.
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Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // Ovchinnikov bulletin of biotechnology and physical and chemical biology V.3, No 4, p. 19-27.(2007) (In Russian)
Abstract
Based on a previously proposed method for in silico restriction analysis of mammalian genomes, distribution diagrams of mouse chromosomal DNA fragments were calculated following its digestion with 18 nucleotide sequences that serve as recognition sites for restriction endonucleases. An analysis was conducted of the mouse LINE1 repeat nucleotide sequences available in the database and the DNA fragments formed upon digestion of these repeats. It was found that the vast majority of peaks in the chromosomal DNA fragmentation patterns correspond to similar peaks formed upon digestion of numerous mouse LINE1 DNA repeats at the same nucleotide sequences. Experimental patterns of mouse DNA hydrolysis by the corresponding restriction endonucleases were obtained. A comparison was made between theoretically calculated fragment distribution diagrams and the obtained patterns of DNA hydrolysis by restriction endonucleases. It was shown that, upon analysis of DNA preparations by gel electrophoresis, only the cleavage products of LINE1 repeats and mouse γ-satellite DNA are visualized. Experiments were conducted on the hydrolysis of mouse chromosomal DNA using the 5-methylcytosine-dependent site-specific endonucleases BlsI, GlaI, and GluI.
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Bacterial strain Arthrobacter luteus B - producer of AluBI site specific endonuclease. // Russian Federation patent RU 2340670 C1 (2007). (In Russian)
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Substrate specificity of new restriction endonuclease SetI // Biotechnology and Bioengineering: Conference Proceedings (2006)
Abstract
Only a few site-specific endonucleases are currently known to recognize and cleave short degenerate DNA sequences of three or four nucleotides. Among them are the restriction endonuclease CviJI and several of its isoschizomers isolated from the eukaryotic Chlorella virus, which recognize and cleave the sequence 5′-RGCY-3′ [1]. In the present study, we describe the substrate specificity of a new site-specific endonuclease, SetI, which recognizes and cleaves the DNA sequence 5′-ASST^-3′ (the four principal target sequences are ACGT^, AGCT^, ACCT^, and AGGT^). SetI was isolated from an Escherichia coli strain carrying the cloned setI gene from Streptomyces werraensis 37.
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BstKTI, a New dam-Sensitive Neoschizomer of Restriction Endonuclease MboI, which is Able to Cleave Hemimethylated Substrate. // Biotekhnologia (Moscow), No.2, 5-10 (2006). (In Russian)
Abstract
A strain of Bacillus stearothermophilus KT was isolated that produces a new restriction endonuclease, BstKTI, which is a neoschizomer of the MboI restriction enzyme that recognizes the sequence 5’-↑GATC-3’. An enzyme preparation was obtained, and its properties were studied, including substrate specificity and the site of DNA cleavage. It was shown that DNA hydrolysis occurs at the 5’-GAT↑C-3’ position. Unlike other isoschizomers of the MboI restrictase, BstKTI is sensitive to dam methylation but is capable of cleaving a substrate that is half-methylated at adenine. Furthermore, BstKTI hydrolyzes the 5’-GATC-3’ site, in which cytosine is modified at the C5 position.
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A novel restriction endonuclease GlaI recognizes methylated sequence 5′-G(5mC)^GC-3′. // Biotechnologia V 4. P. 31-35(2006). (In Russian)
Abstract
A new restriction endonuclease, GlaI, was isolated and characterized from bacterial strain GL29. This enzyme recognizes the methylated DNA sequence 5’-G(5mC)↓GC-3’ and cleaves it as indicated by the arrow. Due to its ability to cleave only methylated DNA, GlaI may find practical applications in genetic engineering, as well as for determining the methylation status of eukaryotic DNA.
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New Eight Bases Cutter AbsI from Arthrobacter species Recognizes Palindromic DNA Sequence 5’-CC^TCGAGG-3’. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), V.2, No.2, p.29-34 (2006). (In Russian)
Abstract
A bacterial strain, Arthrobacter species 7M06, has been identified as the producer of a new site-specific endonuclease, AbsI. The AbsI enzyme recognizes the palindromic octanucleotide DNA sequence 5’-CC↑TCGAGG-3’ and hydrolyzes it after the second cytosine, thereby forming 5’-protruding “sticky” ends that are compatible with the ends formed by the hydrolysis of DNA by the restriction endonucleases XhoI (5’-C^TCGAG-3’), PspXI (5’-VC↑TCGAGB-3’), and SalI (5’-G↑TCGAC-3’). Among all known infrequent-cleavage restriction endonucleases, AbsI is the only enzyme that lacks recognition sites on standard substrates, such as lambda and T7 phage DNA, as well as type 2 adenovirus DNA.
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Bacterial strain Bacillus simplex - producer of BlsI site specific endonuclease. // Russian Federation patent RU 2322494 C1 (2006). (In Russian)
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Bacterial strain Glacial ice bacterium - producer of GluI site specific endonuclease. // Russian Federation patent RU 2322492 C1 (2006). (In Russian)
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Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5`-GCGC-3`. // Bulletin of biotechnology and physico-chemical biology V.2, No 1, p. 30-39 (2006). (In Russian)
Abstract
We investigated the dependence of the activity of the site-specific endonuclease GlaI—which recognizes and hydrolyzes the methylated DNA sequence 5’-GCGC-3’/3’-CGCG-5’—on the number and position of cytosine residues methylated at the 5th position in the recognition sequence. Significant DNA hydrolysis occurs only when two to four bases in the recognition site are modified, and in the former case, both methylcytosines must be internal. Substrate cleavage efficiency increases with the number of methylated bases and is maximal when all four cytosines in the recognition site are modified. GlaI cleaves sites containing 5-methylcytosines but not N4-methylcytosines.
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Mammalian chromosomal DNA digestion with restriction endonucleases in silico // Ovchinnikov bulletin of biotechnology and physical and chemical biology , V.2, No 3, p. 29-38 (2006). (In Russian)
Abstract
A method is proposed for generating theoretical patterns of mammalian chromosomal DNA cleavage by restriction endonucleases. Based on recently published data on the primary structure of genomes, calculations were performed and distribution diagrams were constructed for mouse, rat, and human chromosomal DNA fragments obtained by digestion at the sequences 5’-GGCC-3', 5'-CCGG-3', 5'-GATC-3', and 5'-CC(A/T)GG-3'. Experiments were conducted on the hydrolysis of chromosomal DNA using the restriction endonucleases HaeIII, MspI, Kzo9I, and Bst2UI, which have corresponding recognition sites, and a correspondence was demonstrated between the calculated diagrams and the experimentally obtained DNA restriction patterns.
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Comparative restriction enzymes analysis of rat chromosomal DNA in vitro and in silico // Ovchinnikov bulletin of biotechnology and physical and chemical biology , V.2, No 3, p. 39-46 (2006). (In Russian)
Abstract
Based on a previously proposed method for in silico restriction analysis of mammalian genomes, distribution diagrams of rat chromosomal DNA fragments were calculated following its digestion with more than 25 nucleotide sequences that serve as recognition sites for restriction endonucleases. During in vitro restriction analysis, electrophoresis patterns were obtained in agarose or polyacrylamide gels of rat DNA hydrolysis products using all of these enzymes. A comparison was made between the theoretically calculated fragment distribution diagrams and the experimentally obtained data on DNA hydrolysis by the corresponding restriction endonucleases.
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The Use of Restriction Analysis of Amplified 16S rRNA Genes for the Identification of Microorganisms: A Case Study of Bacterial Producers of Thermolabile Alkaline Phosphatase // Biotechnology, No. 6. – pp. 3–11 (2005). (In Russian)
Abstract
A method for identifying microorganisms has been proposed, based on the analysis of restriction fragment length polymorphism (RFLP) of the PCR product of the 1,500-nucleotide 16S rRNA gene. A set of six restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI, and RsaI) was selected, enabling the identification of a wide range of microorganisms using RFLP.
Four strains producing thermolabile alkaline phosphatase were isolated from natural seawater isolates. PCR analysis of these strains, when compared with calculated results obtained for the 16S rRNA genes of various microorganisms, established that the identified producers belong to the genus Alteromonas. -
Heat-Labile Alkaline Phophatase from Psychrophilic Bacteria Alteromonas undina P2. // Biotekhnologia (Moscow), No.2, 38-43 (2005). (In Russian)
Abstract
From natural isolates, we isolated a producer of thermolabile alkaline phosphatase, identified as the bacterial species Alteromonas undina P2. A method for purifying the enzyme is described. The optimal conditions for alkaline phosphatase activity were determined: 0.1 M glycine-NaOH at pH 9.5, 10 mM MgCl₂, and 0.1 mM ZnCl₂. The enzyme is most active at 30°C. At 6°C, the activity level of alkaline phosphatase exceeds 50% of the maximum. Heating the enzyme preparation for 20 minutes at 65°C leads to complete and irreversible inactivation of the phosphatase.
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A Novel Restriction Endonuclease BisI from Bacillus subtilis T30, Recognizes a Methylated DNA Sequence 5′-G(m5C)^NGC-3′. // Biotekhnologia (Moscow), No.3, 22-26 (2005). (In Russian)
Abstract
A bacterial strain, Bacillus subtilis T30, which produces a new restriction endonuclease, Bis I, has been isolated from natural sources. This enzyme recognizes the methylated DNA sequence 5’-G(5mC)↓NGC-3’ and cleaves it as indicated by the arrow. Because it cleaves only modified DNA, the Bis I enzyme has broad potential for identifying methylated regions in eukaryotic DNA.
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PspXI, a novel restriction endonuclease that recognizes the unusual DNA sequence 5′-VC^TCGAGB-3′. // Bulletin of biotechnology and physico-chemical biology named by Yu.A.Ovchinnikov (Moscow), No.1, p.18-23 (2005). (In Russian)
Abstract
We have identified a bacterial strain of the Pseudomonas species, X11, which produces a novel restriction endonuclease named PspXI. This enzyme recognizes an eight-nucleotide DNA sequence with an unusual, V-B degeneracy type: 5’-VCTCGAGB-3’, where V stands for A, C, or G, and B stands for T, C, or G. A PspXI restriction endonuclease preparation with a concentration of 10,000 units/mL was obtained through purification using a four-step chromatographic process. It has been shown that PspXI hydrolyzes the recognition site between C and T, forming 5’-protruding “sticky” ends compatible with the ends formed upon hydrolysis of DNA by the restriction endonucleases XhoI (5’-C^TCGAG-3’) and SalI (5’-G^TCGAC-3’) restriction endonucleases.
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Bacterial strain Glacial ice bacterium I - producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2005). (In Russian)
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Determination and Analysis of the Primary Structure of NM.BstSEI Operone from Bacillus starothermophilus SE-589 which Produces N.BstSEI Site-Specific Nickase. // Molecular Biology (Moscow), Vol. 39, No.6, 990-994 (2005). (In Russian)
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Isolation and exploration of biochemical propeties of DNA Methyltransferase FauIA modifying Non-palindromic sequence 5'-CCCGC-3'. // Biochemistry (Moscow), Vol. 70, No 6, (2005). (In Russian)
Abstract
The FauIA DNA methyltransferase gene from the FauI restriction-modification system (recognition site 5'-CCCGC-3') was cloned into the pJW expression vector, which was used to transform E. coli RR1 cells, followed by thermal induction and accumulation of bacterial cell biomass. A highly purified preparation of the DNA methyltransferase—M.FauIA—was obtained using chromatography on various media. The isolated enzyme has a molecular weight of ~39 kDa, which corresponds to the theoretically calculated molecular weight of the corresponding translation frame. A study of the properties of the obtained enzyme showed that M.FauIA has an optimal temperature of 33° and exhibits maximum activity at pH 7.5. Methylation of a synthetic oligonucleotide by the M.FauIA enzyme, followed by digestion with various restriction enzymes and analysis of the resulting products, established that M.FauIA modifies the second cytosine in the sequence 5'-CCCGC3-'. The kinetic parameters of the methylation reaction of a synthetic oligonucleotide duplex by the M. FauIA enzyme were determined, and it was shown that the Michaelis constant (Km) for this substrate is 0.16 μM, Km for S-adenosyl-L-methionine (SAM) is 0.78 μM, and the catalytic constant (kcat) is 0.05 min–1.
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An improved ARDRA method of microorganism identification and its application in identifying thermolabile alkaline phosphatase strains-producers // Biotechnologia , No 6, p. 3-11 (2005). (In Russian)
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Restriction Endonuclease AjnI from Acinetobacter johnsonii R2, an Isoschizomer of EcoRII, recognizes 5′- ↓CCWGG-3′ and cleaves dcm-methylated sites. // Biotekhnologia (Moscow), No.3, 19-24 (2004). (In Russian)
Abstract
A new producer of a restriction endonuclease (restriction enzyme) was isolated from natural isolates and identified as Acinetobacter johnsonii R2; its restriction enzyme was named AjnI. A method for purifying and assessing the enzyme’s activity is described. It has been shown that the AjnI restrictase generates DNA fragments with 5’-CCWGG overhanging ends, similar to EcoRII, but is not inhibited by dcm methylation. The AjnI restrictase may find wide application in genetic engineering.
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Bacterial strain Bacillus subtilis - producer of BisI restriction endonuclease. // Russian Federation patent RU 2270859 C1 (2004). (In Russian)
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Cloning of the Genes for DNA Methyltransferases of the SfaNI and Bst19I Restriction--Modification Systems and Primary Structure Analysis of Protein Products // Molecular Biology (Moscow), Vol. 38, No.6, 997-1004 (2004). (In Russian)
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Determining the G + C Content in Bacterial DNA using Restriction Endonucleases. // Biotekhnologia (Moscow), No.4, 77-82 (2004). (In Russian)
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Restriction endonuclease BmtI from Bacillus megaterium S2 cleaves DNA in 5′-GCTAG^C-3′. // Biotekhnologia (Moscow), No.1, P. 11-15 (2003). (In Russian)
Abstract
A new strain producing the restriction endonuclease (restriction enzyme) BmtI has been discovered and identified as the bacterial species Bacillus megaterium S2. The enzyme purification procedure and some of its properties are described. It has been shown that the BmtI restrictase is a heterochromer of the well-known NheI enzyme (which cleaves 5'-G^CTAGC-3') and cleaves DNA to form fragments with protruding CTAG-3'-ends. The BmtI restrictase can be used in genetic engineering.
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Restriction endonucleases FalI and FalII from Flavobacterium aquatile Ob10 Recognize 5′-(8/13)AAGN5CTT(13/8)-3′, Respectively. // Biotekhnologia (Moscow), No.6, P. 24-29 (2003). (In Russian)
Abstract
From aquatic isolates, we have isolated the Flavobacterium aquatile strain Ob10, which produces two restriction endonucleases, FalI and FalII. FalI is a new prototype and recognizes the DNA sequence 5'-(8/13)AAG(N)5CTT(13/8)-3'. The FalI enzyme is activated by S-adenosylmethionine and cleaves DNA on both sides of the recognition sequence. Thus, FalI belongs to the BcgI subtype of restriction endonucleases. FalII cleaves DNA at the 5'-CG↓CG-3' site, making it an isozomere of FnuDII.
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The unique FauI restriction-modification system: cloning and comparative analysis of protein structure // Molecular Biology (Moscow), Vol. 37, No.4, 619-624 (2003). (In Russian)
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M.BstF5I-2 and M.BstF5I-4 DNA Methyltransferases from BstF5I restriction-modification system of Bacillus stearothermophilus F5. // Biochemistry (Moscow), Vol. 68, No 9, (2003). (In Russian)
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Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP. // Biochemistry (Moscow), Vol. 68, No 12, (2003). (In Russian)
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Preparation and characterization of endonuclease I from Proteus vulgaris 84. // Biotekhnologia (Moscow), No.1, 15-20 (2002). (In Russian)
Abstract
An enzyme with a molecular weight of 33 kDa was isolated and purified more than 200-fold. The optimal conditions for enzyme activity were determined: pH 9.5, 25 mM MgCl₂, 100 mM NaCl. The enzyme is most active at 37°C. It has been shown that the isolated enzyme is an endonuclease that hydrolyzes native and heat-denatured DNA and RNA into oligonucleotides 3–5 nucleotides in length with a 5'-phosphate group at the terminus.
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Restriction endonuclease FatI from Flavobacterium aquatile NL3 that cleave DNA in site 5′-^CATG-3′. // Biotekhnologia (Moscow), No.5, 3-7 (2002). (In Russian)
Abstract
A new producer of a restriction endonuclease was obtained from natural isolates and identified as the bacterial species Flavobacterium aquatile NL3, and the corresponding restriction enzyme was named FatI. Methods for purifying the enzyme and determining its activity are described. It has been shown that the FatI restriction enzyme produces DNA fragments with 5'-CATG overhanging ends and may find wide application in genetic engineering.
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Restriction endonuclease Bst19I from strain Bacillus stearothermophilus, that recognize DNA sequence 5′-GCATC-3′. // Biotekhnologia (Moscow), No.6, 17-20 (2002). (In Russian)
Abstract
A strain of Bacillus stearothermophilus 19, which produces a new restriction endonuclease, Bst19I, has been isolated. The enzyme preparation was obtained, and its properties—including its substrate specificity and DNA cleavage site—were studied. The restriction endonuclease Bst19I has been identified as a heteroisomer of the SfaNI restriction enzyme, which recognizes the sequence 5’-GCATC-3’ and cleaves DNA at distances of 4 and 6 nucleotides from the recognition site on the upper and lower strands, respectively.
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The restriction-modification system from Bacillus stearothermophilius F5: Comparison study of the DNA methyltransferases M.BstF5I-1 and M.BstF5I-3. // Molecular Biology(Moscow), vol.36, p.1-8 (2002). (In Russian)
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An express method for testing the tctivity of a repair enzyme, uracil-DNA-glycosylase. // Bioorg. chemistry (Moscow), Vol.26, No.6, 442-447 (2000). (In Russian)
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M.BstF5I-1, the forth DNA-methyltransferase of the BstF5I restriction-modification system from Bacillus stearothermophilius F5. // Biorg. Chemistry(Moscow), vol.28, p.84-86 (2002). (In Russian)
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Novel site-specific DNA-nickase N.Bst9I from Bacillus stearothermophilus 9. // Biotekhnologia (Moscow), No.4, 3-8 (2001). (In Russian)
Abstract
During the screening of bacterial strains isolated from natural sources, we identified a strain of Bacillus stearothermophilus 9 that produces a novel site-specific DNA nuclease named N.Bst9I. N.Bst9I recognizes and cleaves the DNA sequence 5'-GAGTCNNNN^-3', thus being an isozomere of the enzyme N.BstSEI, which we had previously identified. However, the enzymatic properties of these two enzymes differ significantly, and N.Bst9I is preferable for use in genetic engineering and biotechnology applications, as it exhibits virtually no additional activity characteristic of N.BstSEI.
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Restriction endonuclease ZraI from Zoogloea ramigera 11 recognize 5′-GAC^GTC-3′. // Biotekhnologia (Moscow), No.6, 3-7 (2001). (In Russian)
Abstract
From natural isolates, we have isolated a producer of a restriction endonuclease (restriction enzyme), identified as the bacterial species Zoogloea ramigera 11, and its restriction enzyme has been named ZraI. A method for purifying the enzyme and determining its activity is described. It has been shown that the ZraI enzyme cleaves DNA to form blunt ends and may find wide application in genetic engineering.
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Btr I, a novel restriction endonuclease, recognizes the non-palindromic sequence 5′-CACGTC-3′ (-3/-3). // Nucleic Acids Research, Vol. 28, No 11.(2000).
Abstract
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.
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Novel restriction endonuclease SmiI from Streptococcus milleri recognize 5′-ATTT^AAAT-3′. // Molecular Biology and Virusology, No.1, 23-27 (2000). (In Russian)
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The second DNA methyltransferase from BstF5I restriction-modification system is homologous to C-domains of methylases FokI and StsI. // Molecular Biology (Moscow), vol.33, p.87-94 (2000). (In Russian)
Abstract
The nucleotide sequence of the M.BstF5I-2 DNA methyltransferase gene, which is part of the restriction-modification system of the Bacillus stearothermophilus F5 strain, has been determined. On the bacterial chromosome, the bstF5IM-2 gene is located downstream of the M.BstF5I-1 methyltransferase gene and has the same orientation. The protein encoded by the bstF5IM-2 gene contains conserved regions specific to adenine-binding DNA methyltransferases of class D12. The M.BstF5I-2 DNA methyltransferase is homologous to the C-terminal domains of the FokI and StsI DNA methyltransferases, which share the same recognition sequence. Thus, the BstF5I restriction-modification system comprises a unique enzyme, M.BstF5I-1, which modifies the upper strand of the recognition sequence, and M.BstF5I-2, which is homologous to the C-terminal regions of the isozyms M.FokI and M.StsI, which modify the second DNA strand within the recognition site.
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Multiplicity of site-specific DNA-methyltransferases of the BstF5I restriction modification system from Bacillus stearothermophilus F5. // Molecular Biology(Moscow), Vol.34, No.3, 443-447 (2000). (In Russian)
Abstract
The nucleotide sequence of a Bacillus stearothermophilus F5 DNA fragment adjacent to the 3'-end of the region containing the M.BstF5I-1 and M.BstF5I-2 DNA methyltransferase genes from the BstF5I restriction-modification system has been determined. A gene encoding the M.BstF5I-3 protein, which is structurally and functionally homologous to the N-terminal domain of the M.FokI methylase, has been identified. Thus, unlike all known restriction-modification systems, the BstF5I system comprises three genes encoding DNA methyltransferases, two of which are homologous to two different domains of M.FokI.
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Multiplicity of site-specific DNA-methyltransferases of the BstF5I restriction modification system from Bacillus stearothermophilus F5. // Molecular Biology(Moscow), Vol.34, No.3, 443-447 (2000). (In Russian)
Abstract
A fragment of Streptococcus faecalis SE72 plasmid DNA containing the genes of the SfeI restriction-modification system was cloned. Analysis of the nucleotide sequence of the fragment revealed high homology between the operons of the SfeI and LlaBI systems from Lactococcus lactis subsp. cremoris W56 (99.2%) with the same recognition site 5-CTRYAG-3'. Significant differences between the SfeI system and the LlaBI system include an additional 198-nucleotide fragment in the DNA methyltransferase gene and a longer gene for the putative regulatory protein. The lack of homology between the nucleotide sequences adjacent to the operons of the two systems indicates horizontal transfer of operons between two closely related bacterial genera.
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Thermostable DNA polymerase from Thermus thermophilus B35: Influence of divalent metal ions on the interaction with deoxynucleoside triphosphates. // Biochemistry (Moscow), Vol. 65, No 5 (2000). (In Russian)
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Psi I, a novel restriction endonuclease recognizing the DNA sequence 5′-TTA^TAA-3′. // Biochemistry (Moscow), Vol. 64, No 4, 481 - 482 (1999). (In Russian)
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Thermostable DNA Polymerase from Thermus thermophilus B35: Cloning, Sequence Analysis, and Gene Expression // Biochemistry (Moscow), Vol. 64, No 11, (1999). (In Russian)
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Actinobacillus and Streptococcus: producers of isoschizomers of the restriction endonucleases R.Hph I, R.Sau I, R.Nhe I, R.Mbo I and R.Swa I. // Biol. Chem., Vol. 379, 573-574, (1998).
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Sse9I, a Restriction Endonuclease from Sporosarcina sp. 9D, Recognizing the DNA Sequence 5′-AATT-3′. // Apllied Biochemistry and microbiology, Vol.34, No.2, 139-141 (1998). (In Russian)
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Thermostable DNA polymerase from Thermus thermophilus B35: purification and characterization. // Biochemistry (Moscow), vol.63, no.11, p.1490-1495 (1998). (In Russian)
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Cloning and characterization of the gene encoding M.Fau I DNA methyltransferase. // Biol. Chem., Vol. 379, 567-568 (1998).
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Photoaffinity labeling of DNA polymerase from Thermus thermophilus and DNA template by photoreactive analogs of dCTP. // Biochemistry (Moscow), Vol. 63, No. 8, 1090-1096 (1998). (In Russian)
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BstAP I, an ApaB I isoschizomer, cleaves DNA at 5′-GCANNNN^NTGC-3′. // Nucleic Acids Research, Vol. 25, No 12., 2301-2302(1997).
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AccBS I: a new restriction endonuclease from Acinetobacter calcoaceticus BS. // Apllied Biochemistry and microbiology, Vol. 33, No 5, 496-498 (1997). (In Russian)
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Primary structure and strand specificity of BstF5 I — 1 DNA methyltransferase which recognizes 5′-GGATG-3′ // Gene, 187, 217-219 (1997).
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Cloning and characterization of Sse9I DNA Methyltransferase recognizing 5′-AATT-3′. // Nucleic Acids Research, Vol. 24, No 14., 2790-2792 (1996).
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Bacillus badius - a strain producer of restriction endonuclease, that recognize and cleave nucleotide sequence 5′-(A/T)CCGG(A/T)-3′ // Russian Federation patent RU2053299 (1996). (In Russian)
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N.BstSE, site-specific nickase from Bacillus stearothermophilus SE-589. // Molecular biology, Vol.30, No.6, 754-758 (1996). (In Russian)
Abstract
A site-specific nicasase capable of recognizing and hydrolyzing the DNA sequence indicated by the arrow has been isolated from the Bacillus stearothermophilus SE-589 strain:
5'-G-A-G-T-C-N-N-N-N↑N-N-3'
3'-C-T-C-A-G-N-N-N-N-N-N-5'
A study of the properties of this enzyme, named N.BstSE, suggests that it is likely related to type II restriction endonucleases. -
BstF5 I, an unusual isoschizomer of Fok I. // Gene, 172, 4951 (1996).
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Acinetobacter calcoaceticus 65 - a strain producer of restriction endonuclease, that recognize and cleave sequence 5′-GGTACC-3′. // Russian Federation patent RU2034922 (1995) (In Russian).
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CciNI, an isoschisomer of NotI from Curtobacterium citreum recognizes 5′-GC^GGCCGC-3′. // Gene, No. 157, 99-100 (1995).
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Sim I, a new restriction endonuclease that recognizes non-palindromic sequence 5′-GGGTC-3′ (-3/0). // Nucleic Acids Research, Vol. 23, No 14., 2571-2572 (1995).
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Biochemical characterization of Vsp I methyltransferase. // Gene, 157, 65-66 (1995).
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Bsp24I, a new unusual restriction endonuclease. // Gene, No.131, 93-95 (1993).
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Interaction of Vsp I and Tru9 I restriction endonuclease with synthetic oligonucleotides. // Biochimica et Biophysica Acta, 1172, 89-94 (1993).
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Vsp I methylase belongs to m6A-gamma class of adenine methylases. // Nucleic Acids Research, Vol. 21, No 8., 2015 (1993).
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Bacillus sphaericus - a strain producer of restriction endonuclease BsiI. // Soviet Union patent SU 1784642 (1992). (In Russian)
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Acs I, a new restriction endonuclease from Arthrobacter citreus 310 recognizing 5′-Pu^AATTPy-3′. // Nucleic Acids Research, Vol. 20, No.14 (1992).
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Acl I, a new restriction endonuclease from Acinetobacter calcoaceticus recognizing 5′-AA^CGTT -3′. // Nucleic Acids Research, Vol. 20, No. 14, 37-87 (1992).
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The resctriction endonucleases detection in colonies of microorganisms Streptomyces and Nocardia. // Applied Biochemistry and Microbiology (Moscow), Vol 28, No. 2, 309- 313 (1992). (In Russian)
Abstract
A method is proposed for testing various strains of the actinomycetes Streptomyces and Nocardia for the presence of restriction endonucleases. The testing was performed directly on cells isolated from colonies in Petri dishes. The harvested cells were treated with lysozyme and Triton X-100, after which the cell lysate was analyzed for specific endonuclease activity. The method allows for the detection of the enzymes NcoI, NotI, NruI, SfiI, and Sfr303I in lysates from the corresponding producer strains.
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Comparison of express-methods used for the detection of resctriction endonucleases in microorganisms. // Applied Biochemistry and Microbiology (Moscow), Vol 28, No. 1, 152-155 (1992). (In Russian)
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Proteus vulgaris - a strain producer of restriction endonuclease PvuII. // Soviet Union patent SU 1632974 (1991). (In Russian)
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Streptococcus faecalis - a strain producer of restriction endonuclease SfaNI. // Soviet Union patent SU 1645293 (1991). (In Russian)
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Streptomyces fradiae - a strain producer of restriction endonuclease Sfr I. // Soviet Union patent SU 1645300 (1991). (In Russian)
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Bacillus licheniformis - a strain producer of restriction endonuclease Bli13I. // Soviet Union patent SU 1650697 (1991). (In Russian)
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Flavobacterium acuatile - a strain producer of restriction endonuclease Fau I. // Soviet Union patent SU1661213 (1991). (In Russian)
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Substrate specificity determination of restriction endonuclease Tru9I. // Sibirian Biological Journal, No 1, 57-59 (1991). (In Russian)
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Substrate specificity determination of restriction endonuclease Acc65I. // Sibirian Biological Journal, No 1, 59-60 (1991). (In Russian)
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Substrate specificity determination of restriction endonuclease Vha464I. // Sibirian Biological Journal, No 1, 60-61 (1991). (In Russian)
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Substrate specificity determination of restriction endonuclease Mlu113I. // Sibirian Biological Journal, No 2, 66-67 (1991). (In Russian)
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Substrate specificity determination of restriction endonuclease Msp17I. // Sibirian Biological Journal, No 6, 19-20 (1991). (In Russian)
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Bsp1720 I, an isoschizomer of Esp I from Bacillus species 1720 recognizing 5′-GC^TNAGC-3′. // Nucleic Acids Research, Vol. 19, No. 9, 2504 (1991).
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Isolation of restriction endonuclease RsaI from Rhodopseudomonas sphaeroides and study of its properties. // Applied biochemistry and microbiology(Moscow), Vol.27, No.3, 330-337 (1991). (In Russian)
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Preparation method of Streptomyces biomass, that enriched by restriction endonuclease SfiI. // Soviet Union patent SU 1701745 (1991). (In Russian)
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Effect of some cultivation parameters on the yield of resctriction endonuclease SfaNI. // Applied Biochemistry and Microbiology (Moscow), Vol 27, No. 4, 486-490 (1991). (In Russian)
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II-Q restriction endonucleases - new class of type II enzymes. // Nucleic Acids Research, Vol.18, No. 19, 5807-5810 (1990).
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Streptomyces fradiae - a strain producer of restriction endonuclease Sfr274I. // Izvestia SB SA USSR, Biological sciences series, Vol.1, 32-34 (1990). (In Russian)
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BsiI - novel unusual restriction endonuclease. // Molecular Biology(Moscow), Vol.24, No. 1, 244-247 (1990). (In Russian)
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Specificity determination of restriction endonuclease Bse21I. // Izvestia SB SA USSR, Biological sciences series, Vol.1, 138-139 (1990). (In Russian)
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Type II restriction endonucleases: scheme of possible evolution relations between enzymes with palindrome tetra nucleotide recognition sequences. // Molecular Biology(Moscow), Vol.24, No. 5, 1393-1398 (1990). (In Russian)
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Preparation method of restriction endonuclease Vsp I // Soviet Union patent SU1641024 (1990). (In Russian)
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Discovery of strains producers of restriction endonucleases among water microorganisms from the lake Baikal. // Izvestia SB SA USSR, Biological sciences series, No1, 35-37 (1990). (In Russian)
Abstract
Restriction endonucleases (restriction enzymes) are widely used in genetic engineering, which highlights the importance of identifying new strains that produce these enzymes and are more suitable for industrial applications than existing ones. The search for new strains capable of producing restrictases is conducted among microorganisms isolated from various natural sources. Aquatic microorganisms have been relatively poorly studied as potential producers of restriction enzymes, although enzymes with novel specificity have recently been identified among them. The aim of this study was to discover and identify new restriction enzyme-producing strains among microorganisms inhabiting the unique ecological niche of Lake Baikal. The isolation of the Lake Baikal ecosystem, as currently established, provides favorable conditions for the formation of new species of organisms. Since it is well known that, all other things being equal, the rate of species evolution is inversely proportional to generation time, it is clearly advisable to search for endemic species and, consequently, producers of new enzymes among Baikal microorganisms—that is, strains isolated from the pelagic zone and bottom sediments.
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Staphilococcus saprophyticus - a strain producer of restriction endonuclease SsrI. // Soviet Union patent SU 1486512 (1989). (In Russian)
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Moraxella species -a strain producer of restriction endonuclease MspAI. // Soviet Union patent SU 1514774 (1989). (In Russian)
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Bacillus species 21 - a strain producer of restriction endonuclease Bse21I. // Soviet Union patent SU 1518372 (1989). (In Russian)
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The cultivation of Bacillus species 21 - Bse21I restriction endonuclease producer. // Izvestia SB SA USSR, Biology sciences seria, Molecular byology, vol.1, 108-111 (1989). (In Russian)
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Paracoccus denitrificans - a strain producer of restriction endonuclease Pde12I. // Soviet Union patent SU 1532583 (1989). (In Russian)
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Brevibacterium immotum - a strain producer of restriction endonuclease BimI. // Soviet Union patent SU 1532584 (1989). (In Russian)
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Preparation method of Vibrio species biomass, that enriched by restriction endonuclease Vsp I. // Soviet Union patent SU1559704 (1989). (In Russian)
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Substrate specificity determination of restriction endonuclease Bsu15I. // Izvestia SB SA USSR, Biological sciences series, No.2, 108-109 (1989). (In Russian)
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Discovery and substrate specificity determination of restriction endonucleases Bme18I and Kzo9I. // Izvestia SB SA USSR, Biological sciences series, No.3, 25-26 (1989). (In Russian)
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Substrate specificity study of DNA-methylase from Flavobacterium okeanokoites. // Bioorg. chem (Moscow), Vol.15, No 3, 417-418 (1989). (In Russian)
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Aerobic spore formative bacteria as restriction endonucleases producers. // Biological Journal of Armenia, Vol.42, No 11, 969-972 (1989). (In Russian)
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Substrate specificity determination of restriction endonuclease Bpu10I with unusual recognition sequence. // Molecular Biology(Moscow), Vol.23, No 4, 1051-1056 (1989). (In Russian)
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Ssr I - second class restriction endonuclease from Staphylococcus saphrophyticus. // Molecular Genetic, Microbiology and Virusology, No 11, 24-25 (1989). (In Russian)
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Determination of substrate specificity of restriction endonuclease FauI. // Bioorg. chem (Moscow), Vol.15, No 1, 130-132 (1989). (In Russian)
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Immobilized oligonucleotides as affine sorbent for restriction endonucleases. // Bioorg. chem (Moscow), Vol.15, No. 3, 358-362 (1989). (In Russian)
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Preparation method of restriction endonuclease Fok I from Flavobacterium okeanokoites // Soviet Union patent SU1436160 (1988). (In Russian)
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Kurthia zopfii - a strain producer of restriction endonuclease Kzo9I // Soviet Union patent SU 1440919 (1988). (In Russian)
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Preparation method of restriction endonuclease SfaNI, that recognizes and cleaves DNA sequence 5'-GCATC(N)5^, 3'-CGTAG(N)9^. // Soviet Union patent SU 1442546 (1988). (In Russian)
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Substrate specificity determination of restriction endonuclease SfeI. // Bioorg. chem (Moscow), Vol.14, No 6, 848-849 (1988). (In Russian)
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New restriction endonuclease from soil strain Bacillus megaterium 12 // Izvestia SB SA USSR, Biological sciences series, No 14, Vol. 2, 105-107 (1988). (In Russian)
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Providencia stuartii - a strain producer of restriction endonuclease PstI. // Soviet Union patent SU 1472493 (1989). (In Russian)
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Search of strains producers of restriction endonucleases among soil microorganisms. // Izvestia SB SA USSR, Biological sciences series, No.2, 109-112 (1988). (In Russian)
Abstract
We investigated the dependence of the activity of the site-specific endonuclease GlaI—which recognizes and hydrolyzes the methylated DNA sequence 5’-GCGC-3’/3’-CGCG-5’—on the number and position of cytosine residues methylated at the 5th position in the recognition sequence. Significant DNA hydrolysis occurs only when two to four bases in the recognition site are modified, and in the former case, both methylcytosines must be internal. Substrate cleavage efficiency increases with the number of methylated bases and is maximal when all four cytosines in the recognition site are modified. GlaI cleaves sites containing 5-methylcytosines but not N4-methylcytosines.
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A simple technique for detection of restriction endonucleases in bacterial colonies. // Applied Biochemistry and Microbiology (Moscow), Vol. 24, No. 1, 121 - 124 (1988). (In Russian)
Abstract
A method is proposed for testing bacterial strains for the presence of restriction endonucleases. The analysis is performed directly on cells from colonies in a Petri dish. The harvested cells are treated with lysozyme and Triton X-100, and after centrifugation, the supernatant is analyzed for specific endonuclease activity. This method allows for the analysis of up to 100 colonies in 3–4 hours.
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Modified method for determination of restriction endonucleases cleavage position on DNA. // Biotekhnologia (Moscow), Vol.5, No5, 618-620 (1988). (In Russian)
Abstract
Advances in molecular biology and genetic engineering are largely determined by the availability of a wide range of restriction endonucleases (restriction enzymes), to which new entries are added every year. The enzymes identified must be characterized both by the sequence they recognize and by the site at which they cleave the DNA strand. Identifying the recognition site is currently not a problem. For this purpose, researchers typically use co-hydrolysis of DNA by the enzymes under study and enzymes of known specificity, or they compare experimental data with predicted patterns of marker DNA cleavage at recognition sites.
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Determination of restriction endonuclease preparations purity. // Izvestia SB SA USSR, Biological sciences series, No. 14, Vol. 2, 102-105 (1988). (In Russian)
Abstract
In the early 1970s, the first restriction endonucleases (restriction enzymes) were discovered, and today a wide range of these enzymes is being produced for use in genetic engineering. One of the most important characteristics of commercially produced enzyme preparations is their purity with respect to off-activities, particularly impurities such as nucleases and phosphatases.
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Substrate specificity detrmination of restriction endonuclease VspI. // Bioorg. chem (Moscow), Vol.13, No 3, 420-421 (1987). (In Russian)
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Substrate specificity determination of restriction endonuclease VneI. // Bioorg. Chem (Moscow), Vol.13, No 3, 422-423 (1987). (In Russian)
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Vibrio nereis - strain producer of restriction endonuclease VneI, that recognize and cleave DNA sequence 5'-GTGCAC-3'. // Soviet Union patent SU 1413954 (1986). (In Russian)
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Cleavage of DNA that adsorbed on surface of phospholipids membranes by type II restriction endonucleases. // Biochemistry (Moscow), Vol. 51, No. 9, 1496 – 1498 (1986). (In Russian)
Abstract
The hydrolysis of λ-phage DNA by type II restriction enzymes in the presence of model membranes made from egg phosphatidylcholine was studied. It was shown that the extent of DNA hydrolysis by the enzymes Bsp I, Pst I, and Bam HI under these conditions is sharply reduced. This is not due to irreversible inactivation of the enzymes or their interaction with the membrane. The most likely explanation for the inhibition of DNA hydrolysis in the presence of phospholipid vesicles is a change in the substrate properties of DNA resulting from its adsorption onto the surface of the phospholipid membrane with the participation of Mg²⁺ ions.
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Nucleic acid fragment and recombinant plasmid DNA pDKR 85 for testing of rare cutters restriction endonucleases SfiI and NotI. // Soviet Union patent SU 1433020 (1986). (In Russian)
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Preparation method of restriction endonuclease Rsa I from Rhodopseudomonas sphaeroides // Soviet Union patent SU1431328 (1986). (In Russian)
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Preparation method of restriction endonuclease Hga I // Soviet Union patent SU1389292 (1985). (In Russian)
Nothing found.