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| GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation |
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| | 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 ?-satellite DNA. Only small portion of mouse ?-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 ?-satellite DNA.
We have constructed a physical map and produced experimental patterns of mouse ?-satellite DNA hydrolysis with unique site-specific methyl-directed endonuclease GlaI and several restriction endonucleases. Fifty two DNA fragments of ?-satellite DNA have been cloned and sequenced. We have not observed any mutations of CG dinucleotide in position 208 of monomeric ?-satellite DNA and confirmed 50% methylation of this CG dinucleoitide. A comparison of consensus sequences of arrayed ?-satellite DNA and small blocks of satellite DNA (140 monomers and less) has shown a higher level of mutations and an absence of conserved CG dinucleotide in last ones. A replacement of CG dinucleotide by CA-dinucleotide in positions 178 and 17 in chromosomes 9 and 3, respectively, has been observed in blocks of monomers.
Arrayed ?-satellite DNA from mouse has at least one conservative CG-dinucleotide. Consensus sequences of this DNA and ?-satellite DNA in small blocks of monomers are differing. The last one displays a higher level of CG dinucleotides mutations and an absence of conservative CG-dinucleotide. Presence of conservative and half-methylated CG-dinucleotide supports an idea of importance of this CG dinucleotide methylation/demethylation in arrayed ?-satellite DNA functioning. | |
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| Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico |
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| | Theoretical diagrams of mouse chromosomal DNA cleavage at 18 nucleotide sequences 4-6 bp in length, which are the recognition sites of restriction endonucleases, have been plotted based on earlier proposed method of mammalian genomes restriction analysis in silico [2]. A set of mouse LINE1 repeats from the data base has been analysed and diagrams of these repeats cleavage at the same nucleotide sequences have been constructed. In general, the diagrams of mouse chromosomal DNA digestion correspond to diagrams of LINE1 repeats cleavage. Mouse DNA hydrolysis with corresponding restriction endonucleases has been performed. A comparison of mouse DNA cleavage patterns and the computed diagrams has revealed a good correspondence between the experimental and theoretical data. Hydrolysis of mouse DNA preparation and subsequent gel-electrophoresis allows visualizing only LINE1 repeats and satellite DNA cleavage products. Additionally, experiments on mouse chromosomal DNA cleavage with novel methyl-dependent site-specific DNA endonucleases BlsI, GlaI and GluI have been conducted for the first time. | |
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| Comparative analysis of human chromosomal DNA digestion with restriction endonucleases in vitro and in silico. |
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| | Theoretical analysis of human chromosomal DNA cleavage at 15 nucleotide sequences, which are the recongnition sites of various restriction endonucleases, has been carried out. Distribution diagrams of calculated DNA fragments have been constructed based on earlier proposed method of mammalian genomes digestion in silico. A similar study of human Alu- and LINE1-repeats nucleotide sequences, which are present in informational databases, has been performed and corresponding diagrams of DNA fragments distribution have been plotted. Distribution diagrams of chromosomal DNA digestion, which results in formation of low molecular weight DNA fragments, correspond to those for Alu-repeats; whereas the digestion, which results in formation of large molecular weight DNA fragments - are similar to those for LINE-repeats. All theoretical data have been compared to experimental patterns of human genomic DNA cleavages with respective restriction endonucleases and a good correspondence for the most of DNA diagrams has been observed. | |
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| Comparative restriction enzymes analysis of rat chromosomal DNA in vitro and in silico |
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| | Theoretical diagrams of rat chromosomal DNA cleavage at more than 25 different nucleotide sequences have been plotted based on earlier proposed method of restriction enzymes analysis of mammalian genomes in silico. A map of the rat LINE1 repeats digestions at the same nucleotide sequences has been calculated and a correspondence between the diagrams data and LINE1 repeat cleavage positions has been shown. Restriction enzymes analysis in vitro has been performed in order to obtain patterns of rat chromosomal DNA cleavage by endonucleases with respective recognition sequences. A perfect coincidence has been shown between theoretical DNA cleavage diagrams and experimental patterns of DNA hydrolysis with restriction endonucleases. | |
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| Mammalian chromosomal DNA digestion with restriction endonucleases in silico |
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| | A theoretical method to produce digestion patterns of mammalian chromosomal DNA cleavage by restriction endonucleases was proposed. Based on recently published data on primary structures of genomes, a computer analysis was performed and diagrams of chromosomal DNA fragments distribution were plotted for DNA cleavages at 5’-GGCC-3', 5'-CCGG-3', 5'-GATC-3' and 5'-CC(A/T)GG-3' sequences. Experiments on chromosomal DNA digestion with HaeIII, MspI, Kzo9I and Bst2UI restriction endonucleases, which recognize these sites, were carried out. A correspondence between the computed diagrams and experimentally observed patterns of restriction enzymes cleavage was shown. | |
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| An improved ARDRA method of microorganism identification and its application in identifying thermolabile alkaline phosphatase strains-producers |
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| | Based on the analysis of amplified ribosomal DNA fragmentation by restriction endonucleases, an improved method for microorganism identification (called ARDRA) has been suggested. A set of 6 restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI and RsaI) was used to get corresponding patterns of DNA cleavage. DNA samples were isolated from four strain-producers of thermolabile alkaline phosphatase, obtained from sea water. Carrying out of ARDRA, followed by a comparison with the calculated cleavage patterns of DNA from several referenced microorganisms, allowed us to conclude that the new strains-producers belong to the Alteromonas genus. | |
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