ABSENCE OF AN UNUSUAL DENSELY METHYLATED ISLAND AT THE HAMSTER DHFR ORI-BETA

An unusual ''densely methylated island'' (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has be en reported at mammalian origins of DNA replication. This report had f ar-reaching implications in understanding of DNA methylation and DNA r eplication. For example, since this DMI appeared in about 90% of proli ferating cells, but not in stationary cells, it may regulate origin ac tivation. In an effort to confirm and extend these observations, the D MI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was check ed for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cell s and in cell populations enriched in the G(1), S, or G(2) + M phases of their cell division cycle. DNA was subjected to 1) cleavage by meth ylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate mod ification of 5-methylcytosines (C-m) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of C- m residues that complements the more commonly used hydrazine method. T hese methods were capable of detecting C-m in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. Ho wever, the ori-beta DMI was not detected in any of these cell populati ons using any of these methods.