Ohgane J, Aikawa J, Ogura A, Hattori N, Ogawa T, Shiota K
Laboratory of Cellular Biochemistry, Animal Resource Science/Veterinary Medical Science, University of Tokyo, Japan.
Dev Genet. 1998;22(2):132-40. doi: 10.1002/(SICI)1520-6408(1998)22:2<132::AID-DVG3>3.0.CO;2-7.
Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells.
大鼠滋养层巨细胞每个细胞核所含的基因组DNA比二倍体细胞多至少100倍。这种不寻常的现象似乎与胎盘细胞分化和基因表达的分子机制有关。在本研究中，我们使用甲基化敏感的地标酶Not I和Bss HII，通过限制性地标基因组扫描（RLGS）分析了滋养层巨细胞的CpG岛。在胎盘交界区，分别使用Not I和Bss HII通过RLGS检测到1000多个和1900多个斑点，其中超过90%的基因组DNA存在于DNA含量较高的细胞中。在这些斑点中，在交界区发现的斑点中有97%（1009个斑点）和99%（1911个斑点）与二倍体细胞对照呈现相同的模式和相同的强度，二倍体细胞对照的基因组DNA是从迷路区和母肾中提取的。因此，巨细胞基本上是多倍体。更重要的是，使用Not I通过RLGS检测到24个组织特异性斑点。随后对基因组DNA的四个典型斑点进行克隆和测序证实，这些DNA片段含有丰富的CpG二核苷酸，并显示出CpG岛的特征。在这24个斑点中，有10个是胎盘特异性的，其中3个是交界区特异性的，这表明胎盘组织中CpG岛的甲基化状态在交界区和迷路区之间存在差异。这些结果表明，在巨细胞分化过程中发生了多轮核内复制和通过胞嘧啶甲基化对CpG岛的修饰。
