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

在大多数卵巢动物中，裂解阶段的S相非常短，在此发育阶段，在细胞周期中几乎无法检测到间隙相。该项目已计划阐明在海胆裂解胚胎细胞中专门调查S相的机制。（a）deadenenosine tetraphate（AP4A）对S相启动的调节作用。我们先前已经报道说，在裂解阶段，海胆胚胎中的固体AP4A水平在细胞周期中周期性波动，在S相开始时大大降低，并在S相中再次增加。我们发现，AP4A水平的降低不是由于该物质的酶水解。自由AP4A的一部分在S相开始时与核基质蛋白结合。海胆胚胎的核基质的AP4A结合活性在S期开始时最为突出，并且在……更多的细胞周期的另一个时期中变得最低。核基质中AP4A结合蛋白的凝胶电泳分析表明，存在两种不可思议的蛋白，23 kDa蛋白和70 kDa蛋白。似乎70 kDa蛋白对应于核层粘连。现在，我们正在尝试纯化23 kDa的AP4A结合蛋白。（b）在裂解阶段调节DNA复制。人们普遍认为，海胆裂解阶段的极短S相可以支持基因组DNA上许多额外复制起源的吸收。在默认的海胆细胞中，复制其DNA相对较慢，这些额外的起源将处于不活跃状态。为了确认这一假设，需要比较从多个发育阶段获得的已知长度和核苷酸sequnce的DNA Fragmenst上的活性起源数量。为此，我们通过从纯化该酶开始的常规基因粘结程序将芳基硫酸蛋白酶基因（约20 kbp）从海胆胚胎中分离出来。较少的