| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We have isolated many RNAs localized in ascidian eggs, which are known as a mosaic egg, Among them, a number of RNAs are localized in the posterior region that are suggested to be essential to the axis specification and unequal cleavage patterns during embryogenesis, and are therefor named postplasmic RNAs. The aim of this study is to clarify the molecular mechanisms of the RNA sorting in asicdian eggs and embryos through identification of cis-elements controlling their localization and of trans-acting factors that bind to the elements. By analysis of the detailed RNA distribution patterns of six of the above-mentioned RNAs, we found that they are categorized into two groups by the localization pathways ; type I and type II. Injection of labeled synthetic RNAs of the postplasmic RNAs revealed that their 31 UTR sequences are necessary and sufficient for the correct localization of the RNAs in the embryos as th the both types of the RNAs. Furthermore, we have minimized the regions to about 50 nt. In order to identify and characterization of the trans-acting factors that bind to the regions, gel-shift experiments, East-Western analysis and UV cross-linking analysis using the cis-elements are on-going, by which we are establishing the assay systems to find sequence-specific RNA-binding proteins in the egg. Simultaneously biochemical purification of the RNA-binding proteins and isolation of the cDNAs encoding the proteins are also in progress. On the other hand, we have found that the two types of the postplasmic RNAs are localized in ultra-structurally distinct parts of the postplasm by the electron-microscopic in situ hybridization analysis. These will yield in the systematic knowledge about the RNA localization machanisms in ascidian embryogenesis.
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