BMC Genomics 2009, 10:322 http://www.biomedcentral.com/1471-2164/10/322 ylated DNA sequences 5'-Pu(5mC)GPy-3'/3'-PyG (5mC)Pu-5'and 5'-G(5mC)Pu(5mC)-3'/3'-(5mC)GPyG- 5' [5,6]. Previously, we studied a cleavage of total mouse DNA with restriction endonucleases [7]. We compared the experimental patterns of DNA hydrolysis with calculated diagrams of genomic DNA fragments distribution, which had been obtained by computer analysis, and found a good correspondence [7]. We showed that patterns of mouse DNA hydrolysis were formed mainly by products of LINE1 repeats and γ-satellite DNA cleavage. Because of high proportion of γ-satellite DNA in a total mouse DNA preparation the bands, which correspond to products of γ- satellite DNA cleavage, were seen much better in electro- phoresis gels [7]. We cut mouse DNA with site-specific methyl-directed DNA endonucleases and observed DNA hydrolysis with GlaI only [7]. In this work we have undertaken a detailed study of mouse DNA cleavage with GlaI and analyzed a primary structure of γ-satellite DNA and methylation status of ACGT sites within it. Results γ-Satellite DNA digestion with GlaI Earlier we carried out a hydrolysis of total mouse DNA by restriction endonucleases and site-specific methyl- directed DNA endonucleases with subsequent separation of the reaction products by gel-electrophoresis [7]. A the- oretical analysis of mouse genomic DNA digestion at rec- ognition sequences of corresponding restriction endonucleases with subsequent construction of DNA frag- ments distribution diagrams has been performed and has shown a presence of peaks, which correspond mainly to products of LINE1 repeats cleavage [7]. A comparison of observed experimental results and calculation data has demonstrated a good coincidence, except presence of additional bright bands in the experimental cleavage pat- terns. These bands correspond to monomer and dimer of γ-satellite DNA 234 bp and 468 bp in length, respectively [7]. A proportion of γ-satellite DNA in a total mouse DNA preparation is about 8% [8] and higher than in total DNA preparations from rat and human [7]. This peculiarity allows us to use a total mouse DNA preparation in the experiments on study of mouse γ-satellite DNA hydrolysis. To analyze products of mouse γ-satellite DNA cleavage we have used gel-electrophoresis in PAAG and low melting point agarose. Figure 1 shows patterns of total mouse DNA cleavage with restriction enzymes Sse9I, FatI, Bst2UI and methyl-directed site-specific DNA endonuclease GlaI. In accordance with earlier published data [9,10] DNA hydrolysis with restriction enzymes results in formation BFMsiogt2uuUsreeIg1enomic DNA cleavage with enzymes GlaI, Sse9I, FatI, Mouse genomic DNA cleavage with enzymes GlaI, Sse9I, FatI, Bst2UI. 1,8% low melting point agarose, Tris- acetate buffer. M – DNA marker pUC19/MspI (the lengths of the fragments are shown at right). of the bands, which correspond to DNA fragments 234 bp and 468 bp and represent monomer and dimer of mouse γ-satellite DNA [11]. GlaI digestion pattern also contains a monomeric γ-satellite DNA fragment. However, unlike other enzyme, GlaI produces a number of shorter frag- ments, clearly visible in the gel photograph, and this pic- ture is not changed after additional GlaI treatment (data not shown). Comparing to DNA ladder, the lengths of DNA fragments in pattern of GlaI cleavage have been esti- mated 60, 85, 150, 180 and 234 bp, where the last one is a length of monomeric γ-satellite DNA. In our previous work on mouse DNA hydrolysis with restriction endonu- cleases we have shown that DNA fragments less than 240 bp are mostly the products of γ-satellite DNA cleavage (except BstX2I and RsaI digestions) [7]. Data on Figure 1 show that an intensity of 234 bp fragment in GlaI pattern is weaker and 468 bp band is missing if compared to other runs. This result corresponds to formation of short frag- ments in pattern of GlaI digestion from γ-satellite DNA. Location of GlaI digestion fragments To confirm the origin of DNA fragments in pattern of GlaI digestion and to determine their primary structure all five Page 2 of 10 (page number not for citation purposes)