Morioka Mizue, Shimada Hiraku
Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Dev Growth Differ. 1985;27(4):453-459. doi: 10.1111/j.1440-169X.1985.00453.x.
For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs. DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.
为了研究海胆胚胎发育早期DNA复制的调控机制，建立了一种对外源供应的核苷酸具有通透性的胚胎细胞系统。通过将胚胎置于含有0.3M葡萄糖的低渗缓冲液中孵育使其通透化。通透化的胚胎细胞保持其形态完整性，并在供应外源脱氧核苷酸三磷酸（dNTPs）时合成DNA。这些通透化胚胎细胞中的DNA合成除了需要标记的三磷酸脱氧胸苷（dTTP）外，还需要ATP和其他三种脱氧核苷三磷酸的存在。DNA合成几乎完全被N-乙基马来酰亚胺抑制，并且以不连续的方式进行。只有在S期通透化的细胞才能将核苷三磷酸掺入DNA中：在其他时期通透化的细胞不合成DNA。在60分钟的孵育期内，在所使用的实验条件下，超过10%的基因组DNA被复制。
