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

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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