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

海胆胚胎卵裂期S期调控的分子机制研究

基本信息

批准号：
62480015
负责人：
SHIMADA Hiraku
金额：
$ 4.16万
依托单位：
HIROSHIMA UNIVERSITY (1988)The University of Tokyo (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

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

在大多数卵多孔动物中，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胎细胞S期的特异性调控机制。(A)四磷酸腺苷（AP4A）对s相起始的调控作用。我们之前报道过，海胆胚胎卵裂期可溶性AP4A的水平在细胞周期中呈周期性波动，在S期开始时急剧下降，在S期再次上升。我们发现AP4A水平的下降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。海胆胚核基质的ap4a结合活性在S期开始时最为突出，在细胞周期的其他时期则最低。对核基质中ap4a结合蛋白进行凝胶电泳分析，发现存在两个不同的蛋白，分别为23 kda蛋白和70 kda蛋白。70 kda蛋白似乎与核层蛋白相对应。我们现在正试图纯化23kda的ap4a结合蛋白。(B)切割阶段DNA复制的调控。一般认为，海胆卵裂期胚胎极短的S期是由基因组DNA上许多额外复制起点的激活所支持的。在复制DNA相对缓慢的海胆细胞中，这些额外的起源将处于不活跃状态。为了证实这一假设，需要比较从不同发育阶段获得的已知长度的DNA片段和核苷酸序列上的活性起源的数量。为此，我们分离出了芳基磺化酶基因(约1。从该酶的纯化开始，通过传统的基因克隆程序从海胆胚胎中提取20 kbp)。少