Shiraishi M, Oates A J, Li X, Hosoda F, Ohki M, Alitalo T, Lerman L S, Sekiya T
Oncogene Division and Radiobiology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku 104-0045, Tokyo, Japan.
Nucleic Acids Res. 1998 Dec 15;26(24):5544-50. doi: 10.1093/nar/26.24.5544.
We isolated fragments containing parts of CpG islands from human chromosomal regions chosen for expected differences in gene density by segregation of partly melted molecules. Restriction fragments of P1 bacteriophage clones covering a region of 11q13 and those of cosmid clones derived from Xp22 were recovered from bands in denaturing gradient gels that were retained following prolonged exposure to electric field. Forty-five independent fragments derived from 11q13 and five from Xp22 were isolated. Nucleotide sequence analysis revealed that 11 of the 45 fragments from 11q13 contained CpG islands including four derived from known genes in 11q13. None of the five fragments derived from Xp22 resembled CpG islands. The number of CpG island fragments obtained was consistent with the expectation based on the number of Not I restriction endonuclease sites present at these regions. Adjustment of parameters in our quasi-theoretical approach to the rate of fragment dissociation improves the discrimination between retention and non-retention. The results support probable identification of CpG island fragments by their reduced rate of strand dissociation when retarded in a denaturing gradient gel.
我们通过部分解链分子的分离，从因基因密度预期存在差异而选择的人类染色体区域中分离出包含部分CpG岛的片段。从覆盖11q13区域的P1噬菌体克隆的限制性片段以及源自Xp22的粘粒克隆的片段中，从变性梯度凝胶的条带中回收，这些条带在长时间暴露于电场后仍保留。分离出了45个源自11q13的独立片段和5个源自Xp22的片段。核苷酸序列分析表明，45个源自11q13的片段中有11个包含CpG岛，其中4个源自11q13中的已知基因。源自Xp22的5个片段中没有一个类似于CpG岛。获得的CpG岛片段数量与基于这些区域中Not I限制性内切酶位点数量的预期一致。在我们对片段解离速率的准理论方法中调整参数，可以提高对保留和非保留的区分。结果支持通过变性梯度凝胶中滞留时链解离速率降低来可能鉴定CpG岛片段。
