BMC Genomics 2009, 10:322 http://www.biomedcentral.com/1471-2164/10/322 cleotide, whereas γ-satellite DNA from blocks of mono- mers doesn't have a conservative CG dinucleotide in any positions. Absence of mutations [this work] and 50% methylation [[11,12] and this work] of CG dinucleotide in position 208 allow to say about important role of this CG dinucle- otide methylation/demethylation in function of arrayed γ-satellite DNA from mouse liver. Conclusion Hydrolysis of total DNA from mouse liver with new site- specific methyl-directed endonuclease GlaI provides a clear digestion pattern, which corresponds to a partial cleavage of γ-satellite DNA at methylated ACGT sites in positions 61, 121 and 207. Determination of primary structure of 39 obtained DNA fragment has confirmed data on extensive modification of CG dinucleoitide in positions 62 and 122 but only 50% modification in posi- tion 208. Study of 52 sequenced fragments of γ-satellite DNA has shown that CG dinucleotide in position 208 is highly conservative and its methylation/demethylation is, likely, important for function of γ-satellite DNA arrays. Consensus sequence of arrayed γ-satellite DNA is coin- cided with earlier published one and differs from a con- sensus sequence of γ-satellite DNA in small (140 and less) monomers blocks. Methods DNA fragments cloning and sequencing Male A/He mice aged 5–6 months (Breeding Laboratory of Experimental Animals, Institute of Cytology and Genet- ics, Novosibirsk) have been used in the experiments. Genomic DNA from the animal liver was isolated as described previously [15]. DNA was digested with endo- nucleases according to manufacturer's recommendations. DNA fragments, which formed visible bands, were extracted from PAAG as described earlier [16]. The obtained DNA fragments were ligated with pUC19 linear- ized with SmaI, and E. coli XL10-Gold competent cells were transformed with the obtained ligation mixture according to [16]. The obtained plasmids were isolated with NucleoSpin Plasmid Kit (Macherey-Nagel, Ger- many). The sequences of insertions were determined using ABI Prism 310 Genetic Analyzer ("Applied Biosys- tems"). Hydrolysis of chromosomal DNA and electrophresis The following restriction endonucleases manufactured by SibEnzyme Ltd. were used in the work (the recognition site of respective restriction enzyme is given in the brack- ets): GlaI (5'-R(5mC)GY-3'), Bst2UI (5'-CCWGG-3'), FatI (5'-CATG-3'), Sse9I (5'-AATT-3'), AcsI (5'-RAATTY-3'), BstF5I (5'-GGATG-3' and 5'-CATCC-3'), SspI (5'-AATATT- 3'). HpyCH4IV restriction enzyme (recognition site 5'- ACGT-3') was purchased from New England Biolabs. Hydrolysis reactions were performed in 40 μl of the reac- tion mixture containing 6 μg of DNA, SE-buffers recom- mended by the manufacturer and 3 μl of restriction enzyme at optimal temperatures for 3 h. Electrophoresis in 8%–10% polyacrylamide gel was used to separate DNA fragments from 40 to 500 bp (6 μg of hydrolyzed DNA was applied on gel in each run). Electro- phoresis in 1.8% of low melting point agarose ("Sigma", USA) was used to separate DNA fragments in the range of 200–2000 bp. Electrophoresis in 1% agarose "Type I-A, Low EEO" ("Sigma", USA) was used to separate DNA frag- ments in the range of 500–20000 bp. 3 μg of hydrolyzed DNA was applied on agarose gel in each run. Tris-acetate buffer was used for electrophoresis in all the cases. After electrophoresis DNA bands were stained with ethidium bromide and photographed in UV light. The following DNA preparations were used as DNA frag- ment length markers: 1 kbp DNA ladder and pUC19/MspI DNA ladder (SibEnzyme Ltd., Russia). Sequence analysis BioEdit 7.09 software was used to obtain and refine mul- tiple alignments of all mouse γ-satellite DNA fragments [17]. The blocks of γ-satellite DNA in the sequenced mouse genome were derived from Entrez Genomes View site by searching for "GSAT_MM" keyword [14]. Authors' contributions VAC and DAG carried out DNA hydrolysis, cloning and sequencing of DNA fragments. MAA carried out the data analysis and drafted the manuscript. SKD coordinated the project and prepared the final manuscript. All authors have read and approved the final manuscript. Additional material Additional file 1 Alignment of the sequenced GlaI, Sse9I and AspS9I fragments. Num- bering is given according to consensus sequence [11]. ACGT sites are given with yellow background. Nucleotides which differ from those in con- sensus are given with blue background. Sse9I fragments (names start with S) and AspS9I fragments (names start with A) are indicated in green. Some pieces of fragments (shown in magenta) were moved in the align- ment to fit to a total length of 234 bp. CG dinucleotides are shown in blue. Yellow background indicates ACGT sites (cleaved or not cleaved). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-322-S1.pdf] Page 9 of 10 (page number not for citation purposes)