Study on Molecular mechanisms regulating S phase in sea urchin embryo at cleavage stage.

在大多数有卵动物中，卵裂期的S期非常短，在这个发育阶段的细胞周期中几乎没有间隙期。本项目旨在阐明海胆卵裂胚细胞S期的特异性调控机制。(A)腺苷四磷酸（AP 4A）在S期起始调控中的作用。我们以前曾报道过，在卵裂期的海胆胚胎中可溶性AP 4A的水平在细胞周期中周期性波动，在S期结束时急剧下降，并在S期再次增加。我们发现AP4A水平的降低不是由于该物质的酶水解。在S期开始时，一部分游离的AP4A与核基质蛋白结合。海胆胚胎核基质的AP4A结合活性在S期开始时最显著，在其它时期最低。 关于我们 细胞周期凝胶电泳分析显示，核基质中的AP 4A结合蛋白存在两种不同的蛋白，23 kDa蛋白和70 kDa蛋白。70 kDa蛋白质可能是核纤层蛋白。我们现在试图纯化23 kDa的AP 4A结合蛋白。(B)DNA复制在切割阶段的调节。一般认为，海胆卵裂期胚胎中极短的S期是由基因组DNA上许多额外的复制起点的激活支持的。在繁殖海胆的细胞中，它们复制DNA的速度相对较慢，这些额外的起源将处于不活跃状态。为了证实这一假说，需要比较从不同发育阶段获得的已知长度和核苷酸序列的DNA片段上的活性起点的数量。为此，我们分离了芳基硫酸酯酶基因（约。20 kbp）。少

In most oviporous animals, the S phases during cleavage stage are extremely short, and little, if any, gap phases are detectable in the cell cycles at this stage of development. This project has been planned to elucidate the mechanism specifically regulating the S phases in the cells of cleavage embryos of sea urchin.(A) Role of deadenosine tetraphosphate (AP4A) on the regulation of s phase initiation. We have previously reported that the level of the soluble AP4A in sea urchin embryos at cleavage stage fluctuates cyclically during the cell cycle, drastically decreasing at the beginnning of S phase and increasing again during the S phase. We found that the decrease in the AP4A level is not due to enzymatic hydrolysis of this substance. A part of the free AP4A becomes bound to the nuclear matrix proteins at the beginning of S phase. The AP4A-binding activity of nuclear matrix of sea urchin embryos is most prominent at the beginning of S phase and become lowest during the other period of … More cell cycle. Gel electrophoretic analysis of the AP4A-binding proteins from nuclear matrix revealed the presence of two distince proteins, 23 kda protein and 70 kda protein. It seems that the 70 kda protein corresponds to nuclear lamins. We are now trying to purify the AP4A-binding protein of 23 kda.(B) Regulation of DNA replication at cleavage stage. It is generally believed that the extremely short S phases in cleavage-stage embryos of sea urchin are supported byactivation of many extra replication origins on genomic DNA. In the cells of abult sea urchin which replicate their DNA relatively slowly, these extra origins would be in an inactive state. To confirm this hypothesis, it is needed to compare the numbers of active origins on the DNA fragmenst with known length and nucleotide sequnce which are obtained from various stages of development. For this purpose, we have isolated the arylsulfatase gene (approx. 20 kbp) from sea urchin embryos by the conventional gene-cloning procedure starting from purification of this enzyme. Less