DNA methylation and demethylation in sea urchin development with special reference to cell defferentiation.

在海胆胚胎中，DNA甲基化率（以暴露于[甲基3H]蛋氨酸的胚胎DNA分数估算）在受精和桑葚胚期之间增加，然后在间充质囊胚和原肠胚期之间下降，然后又增加。在[甲基^3H] s -腺苷蛋氨酸孵育后，从胚胎不同阶段分离的细胞核中也获得了发育过程中DNA甲基化率的相同变化。在桑葚胚期和游泳囊胚期DNA去甲基化率较高，而在初级间质期之后的阶段DNA去甲基化率较低。根据用几种限制性内切酶消化^3H甲基化DNA得到的DNA片段电泳时^3H放射性分布的差异，假定原肠动物DNA的甲基化区域与桑葚动物DNA的甲基化区域不相同。放线菌素D抑制了前、后期和原肠胚期甲基化率的升高，随后C激酶、A激酶、CaM激酶和ADP核糖素三磷酸酯酶活性的升高，3-氨基苯甲酰胺和ADP核糖素转移酶抑制剂对甲基化率的抑制作用较强，而H7、H8和W7对甲基化率的抑制作用较弱。在某种程度上，DNA甲基化似乎是由核蛋白的[adp -核糖基]化所支持的。阻断DNA甲基化的SIBA和3-氨基苯甲酰胺在培养间充质囊胚至分裂对应阶段时，浓度依赖性地抑制了鞘臂和胚壁的形成。高浓度SIBA对大肠杆菌的形成有微弱的抑制作用。在原胚分离的外胚层细胞中，DNA甲基化率明显高于分离的内胚层和中胚层细胞。在孵育前用Zn^<2+>或A23187处理的动物胚胎中，DNA甲基化率明显高于原胚。在8-64细胞龄时，用Li^+处理的植物胚胎DNA甲基化程度很低。这些形态学观察表明DNA甲基化参与了外胚层细胞分化的过程。STBA暴露于胚胎前孵化期，其浓度依赖性导致胚胎死亡，但未产生异常胚胎。这一时期细胞命运的决定似乎不受DNA甲基化的支持。少

In sea urchin embryos, the rate of DNA methylation, estimated in DNA fraction in embryos exposed to[methyl ^3H]methionine, increased in the period between fertilization and the morula stage, then decreased and again increased in the period between the mesenchyme blastula and the gastrula stage. The same profile of change in the rate of DNA nietliylatiozi during development was also obtained in nuclei isolated from embryos at various stages, following their incubation with[methyl ^3H]S-adenosylmethionine. The rate of DNA demetliylation was high at the morula stage and also at the swimming blastula stage and was quite low at the stages later than the primary mesenchyme stage. Methylated regions of DNA in gastrulae are assumed to be not the same to those in morulae, on the basis of difference in the distribution of ^3H-radioacLivity on electroplioresis of DNA fragments obtained by digestion of ^3H nietliylaled DNA with several restriction enzymes.The increase in methylation rate in pre-ha … More tching period andin gastrulation period, which was inhibited by actinoniycin D, was followed by the increase in the activities of C kinase, A kinase, CaM kinase and ADP ribosyltraiisferase, and was strongly blocked by 3-aminobenzamide, inhibitors of ADP ribosyltransferase, but was hardly blocked by H7, H8 and W7. DNA methylation seems to be supported, to some extent, by[ADP-ribosyl]ation of nuclear protein.SIBA which blocked DNA methylation, as wellas 3-aminobenzamide, cocentrationdependently inhibited forination of pluteus arms and thim embryo wall, when mesenchyme blastulae were cultured with this compound up to the plism correspondig stage. The formation of archenteron was weakly inhibited at high cocentration of SIBA. In ectoderm cells isolated from gastrulae, the rate of DNA methylation was markedly hgher than in isolated elidoderm and mesoderm cells. In aninializ embtyos, obtained by the treatment with Zn^<2+> or A23187 in pre-hatchig period, the rate of DNA methylation was markedly higher than in gastrulae. DNA methylation was quite poor in vegetalezed embryos, obtained by the treatment with Li^+ at the 8-64 cell sbage. These morphological observations suggest that DNA metliylation takes a part in the process of ectodermal cell differentiation. STBA, exposed to embryos in pre-hatching period, concentration-dependently caused embryo-death but did not produced abnormal embryos. Determination of cell fate in this period does not seem to be supported by DNA methylation. Less