|Budget Amount *help
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1990: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1989: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1988: ¥2,800,000 (Direct Cost: ¥2,800,000)
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
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