Digestion of human chromosomes by means of the isoschizomers MspI and HpaII.

Abstract

The isoschizomers MspI and HpaII are four base cutter (C decrease CGG) restriction endonucleases, HpaII being sensitive to methylation of the internal cytosine. Human chromosomes treated with MspI have produced inconsistent results between laboratories, while HpaII has always been described as a nonbanding enzyme when used on human chromosomes. These results have been explained on the basis of both rarity of the CpG doublet in vertebrate genomes and high rate of CpG methylation (5mCpG). We demonstrated consistent banding patterns subsequent to digestions with MspI and HpaII. On euchromatin, MspI (but not HpaII) digests the DNA of R regions and thus R-bands apparently contain many more CCGG sites (mostly methylated) than G-bands. In heterochromatin, extensive digestion of the 9q12 region not only by MspI but also by HpaII reveals a heterochromatic domain with a high frequency of unmethylated CCGG sites, most probably within the satellite 3 DNA fraction. In addition, enzymatic digestions of the Yq12 heterochromatin, when this region is undercondensed by 5-azacytidine, contribute to deepen the insight into the mechanism of action of this cytidine analog and at the same time reinforce the idea of the heterogeneity of this chromosome region where domains with unmethylated CCGG sites may also exist.