Absence of an unusual ''densely methylated island'' at the hamster dhfr ori-beta

作者：Rein T; Natale DA; Gartner U; Niggemann M; DePamphilis ML; Zorbas H

来源：Journal of Biological Chemistry, 1997, 272(15): 10021-10029.

An unusual ''densely methylated island'' (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells 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 methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification 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. These 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. However, the ori-beta DMI was not detected in any of these cell populations using any of these methods.

• 出版日期1997-4-11