BMC Genomics 2009, 10:322 http://www.biomedcentral.com/1471-2164/10/322 DNA bands have been eluted from gel and cloned into pUC19. DNA insertions of 50 plasmids have been sequenced. Primary structures of 39 insertions correspond to DNA sequence of consensus monomeric γ-satellite DNA [11], which is given in Figure 2. According to this consensus sequence, the recognition sites of restriction enzymes Bst2UI (CCWGG), FatI (CATG) and Sse9I (AATT) are located in positions 4, 150 and 98 of γ-satellite DNA, respectively. In a course of γ-sat- ellite DNA hydrolysis with these enzymes monomer and multimers are formed (Figure 1). Analysis of consensus DNA sequence (Figure 2) with methylated CG dinucle- otides has shown that only three GlaI recognition sites with nucleotide sequence ACGT are present in the main monomeric γ-satellite DNA in positions 61, 121 and 207. In Figure 3 we have presented a diagram with location of all 39 sequenced fragments in monomeric γ-satellite DNA based on their primary structure and positions of GlaI rec- ognition sites. Thus, we have determined DNA sequence of six insertions from lower 60 bp band (positions on γ- satellite DNA 61–121), fourteen insertions of 85 bp band (including four 86 bp fragments 121–207 and ten 88 bp fragments 207-61), six 150 bp insertions (including three 146 bp fragments 61–207 and three 148 bp fragments 207-121), seven 174 bp insertions (121-61) from 180 bp band and six 234 bp fragments. This set includes all pos- sible DNA fragments with a length of 234 bp and less, which are produced in the course of limited GlaI hydroly- sis of γ-satellite DNA at positions 61, 121 and 207, except a monomer 207–207. A primary structure of all 39 sequenced fragments and their alignment are presented in Additional file 1 (top 39 sequences of the multiple align- ment). Data in Additional file 1 show that in all cases GlaI cleaves only ACGT sites in position 61, 121 and 207. So, we haven't observed any non-specific hydrolysis of γ-sat- ellite DNA with GlaI. Incomplete hydrolysis of ACGT sites in γ-satellite DNA As can be seen from a map in Figure 3, a complete hydrol- ysis of fully methylated γ-satellite DNA should produce only three DNA fragments: 60 bp, 86 bp and 88 bp. These FCiognusreens2us sequence of mouse γ-satellite DNA monomer Consensus sequence of mouse γ-satellite DNA mono- mer. CG sites are underlined, ACGT sites (GlaI recognition sites) are shown by inverted color. fragments correspond to the bottom bands 60 bp and 85 bp in Figure 1, taking into account a double intensity of the last one. However, experimental pattern of GlaI hydrolysis (Figure 1) includes three additional bands 150 bp, 180 bp and 234 bp, which are a result of partial hydrolysis of γ-satel- lite DNA because of mutations [7,11,12] and undermeth- ylation [11,12] of ACGT sites. A ratio of molar intensities of DNA bands obtained in GlaI cleavage pattern (234 bp, 180 bp, 150 bp, 85 bp and 60 bp) has been determined as 0.2 : 0.3 : 0.3 : 1.5 : 1. According to sequencing data of the cloned insertions, 174 bp DNA fragment corresponds to 180 bp band, 146 bp and 148 bp DNA fragments are presented in 150 bp band and 234 bp is a monomeric γ-satellite DNA. In Table 1 we have summarized data on DNA fragments, which contain unmethylated and mutated GlaI recogni- tion sequence(s). There are approximately 24%, 30% and 2% mutated sites in positions 61, 121 and 207, respec- tively. Additionally, 3%, 7% and 50% of unmethylated sites have been found in the same positions. The last data correspond to earlier published results on 50% methyla- tion of CG dinucleotide in position 208, whereas other CG dinucleotides (underlined on Figure 2) are extensively methylated [11,12]. In total, 27%, 37% and 52% of tetra- nucleotides at positions 61, 121 and 207, respectively, are resistant against GlaI hydrolysis and corresponding prob- abilities of these positions cleavage are 73%, 63% and 48%. Based on the probability data we have calculated a ratio of molar intensities of DNA bands obtained in GlaI cleavage (234 bp, 174 bp, 146 bp + 148 bp, 86 bp + 88 bp and 60 bp) as 0.37 : 0.52 : 0.45 : 1.41 : 1. A comparison of these calculations and molar intensities of DNA bands in experiment has shown a similarity of both ratios. Figure 4 demonstrates patterns of total mouse DNA hydrolysis with restriction endonucleases HpyCH4IV, which recognizes unmethylated site ACGT, and SspI (rec- ognition sequence 5'-AATATT-3'). We can see a weak hydrolysis of genomic DNA and similar patterns of incomplete cleavage of γ-satellite DNA with HpyCH4IV and SspI. A double digestion of mouse γ-satellite DNA with GlaI and restriction enzymes We have carried out the experiments on double digestion of mouse DNA with GlaI and some restriction endonucle- ases, which have a unique recognition site in γ-satellite DNA. These restriction endonucleases are Bst2UI (recog- nition site CCWGG at position 4), AcsI (RAATTY, 97), FatI (CATG, 150) and BstF5I (GGATG, 166). The photos of double digestions experiments are provided in Figure 5A. Page 3 of 10 (page number not for citation purposes)