The use of prokaryotic DNA methyltransferases as experimental and analytical tools in modern biology

Buryanov, Yа. I.; Шевчук, Т. В.

doi:10.1016/j.ab.2004.02.048

Cited by 22 publications

(23 citation statements)

References 85 publications

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“…We and others have shown that amplicons can be characterized by performing PCR followed by methyltransferase and restriction digestion reactions (45,50). This genotyping was performed using …”

Section: Discussionmentioning

confidence: 99%

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Mundo

Gilardoni

Hoffman

et al. 2013

Appl Environ Microbiol

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Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

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Section: Discussionmentioning

confidence: 99%

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Mundo

Gilardoni

Hoffman

et al. 2013

Appl Environ Microbiol

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“…Three different DNA preparations were used for macrophage activation studies. For further control experiments, DNA was either digested with DNase or cytosine residues (C5) were methylated by the Sss I methyltransferase [26] .…”

Section: Dna Isolation Digestion and Methylationmentioning

confidence: 99%

Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

et al. 2008

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Alveolar macrophages are the first line of host defence against mycobacteria, but an insufficient host response allows survival of bacteria within macrophages. We aimed to investigate the role of Toll-like receptor 9 (TLR9) activation in macrophage defence against mycobacteria. Human in vitrodifferentiated macrophages as well as human and mouse alveolar macrophages showed TLR9 mRNA and protein expression. The cells were markedly activated by DNA isolated from attenuated mycobacterial strains (H37Ra and Mycobacterium bovis BCG) as assessed by measuring cytokine expression by real-time PCR, whereas synthetic phosphorothioate-modified oligonucleotides had a much lower potency to activate human macrophages. Intracellular replication of H37Ra was higher in macrophages isolated from TLR9-deficient mice than in macrophages from wild-type mice, whereas H37Rv showed equal survival in cells from wild-type or mutant mice. Increased bacterial survival in mouse macrophages was accompanied by altered cytokine production as determined by Luminex bead assays. In vivo infection experiments also showed differential cytokine production in TLR9-deficient mice compared to wild-type animals. Both human monocyte-derived macrophages as well as human alveolar macrophages showed reduced activation upon treatment with DNA isolated from bacteria from virulent (M. bovis and H37Rv) compared to attenuated mycobacteria. We suggest attenuated TLR9 activation contributes to the insufficient host response against virulent mycobacteria.

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“…Expression of the bacterial Dam enzyme, that methylates adenine residues in GATC sequences, apparently produced no side effects in Saccharomyces cerevisiae and Drosophila melanogaster (Buryanov and Shevchuk 2005;Hoekstra and Malone 1985;Wines et al 1996). Moreover, natural methylation of adenine was not detected in mammals (Kudriashova et al 1976), thus making Dam methylase suitable for analysis of chromatin structure.…”

Section: Resultsmentioning

confidence: 97%

“…Recently, there appeared methods based on the expression of modifying enzyme genes, in particular DNase I, in eukaryotic cells (Wang and Simpson 2001). Among them, an in vivo approach that employs bacterial Dam methylase which methylates adenine residues in GATC sequences was proposed (Singh and Klar 1992), reviewed in (Buryanov and Shevchuk 2005). Obviously, this method can be applied only to the organisms lacking endogenous adenine methylation in the GATC sequence, such as Saccharomyces cerevisiae, Drosophila melanogaster and Homo sapiens (Hattman et al 1978;Kudriashova et al 1976).…”

Section: Introductionmentioning

confidence: 99%

Identification and mapping of open chromatin regions within a 140 kb polygenic locus of human chromosome 19 using E. coli Dam methylase

2006

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Using transient expression of the E. coli Dam methylase gene and analysis of the distribution of methylated GATC sites, we studied the distribution of open chromatin regions within a 140 kb long human genome segment in HEK-293 cells. Dam methylated sites were found in gene introns, exons, and intergenic regions, and their distribution along DNA was uneven. There were regions of high and low density of Dam methylated GATC sites, presumably corresponding to "open" and "closed" chromatin regions, respectively, and to the functional profile of the genomic locus under study. The Dam methylation profile was also generally in agreement with transcriptional activity of genes in the locus. Moreover, DNA regions accessible to Dam methylase apparently coincided with those hypersensitive to DNase I.

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The use of prokaryotic DNA methyltransferases as experimental and analytical tools in modern biology

Cited by 22 publications

References 85 publications

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

Identification and mapping of open chromatin regions within a 140 kb polygenic locus of human chromosome 19 using E. coli Dam methylase

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