| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Research Abstract |
1. Inward rectifier K+ channel (IRK) is known as one of the quantitative markers in the early neural differentiation process of the ascidian development. We carried out the screening of the genomic IRK gene from Halocynthia roretzi genomic library by the plaque hybridization, and cloned the gene. (1) We determined the total sequence of the genomic IRK gene (TuIRKA) including its coding region, 4.8 kb 5'-proximal promoter region and 2.5 kb 3'-UTR. (2) We examined the promoter activity of TuIRKA on the cleavage-arrested cell pairs, aa, bb and aA, from the 8-cell embryo, using GFP reporter. It was found that the activity was more remarkably detected in the epidermally differentiated aa and bb pairs than in the neurally differentiated aA pair. (3) BLAST2 reference represented that the TuIRKA amino acid sequence was more homologous to that of human IRK than those of Drosophila and C. elegans. While, the multiple splice sites in TuIRKA genomic gene, which existed in its membrane-spanning and pore segments as well as cytoplasmic regions, were considerably homologous to those of C. elegans.
2. During early ascidian development, anterior neuroectoderm cells (a4.2) from the 8-cell embryo are destined to become larval anterior neural structures, while presumptive notochordal neural cells (A4.1) become larval posterior neural structures. An anterior quadrant cell (A3) from the 4-cell embryo is their parent cell. We prepared the new mutually interactive system of cleavage-arrested cell triplets, which consisted of A3 and a pair of a4.2 or A4.1 cells. In the A3-aa triplet, all three cells in the triplet differentiated into either epidermal or neuronal cells according to the cell contact timing. When isolated A3 cells were neuralized with subtilisin, a protease, they predominantly became the anterior neural type. While, keeping isolated A3 in medium containing a bFGF, posterior neuralization was facilitated.
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