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Analysis of Morphogenesis and Differentiation of Dictyostellium by restriction enzyme mediated insertion (REMI)

通过限制性内切酶介导的插入 (REMI) 分析盘基网柄菌的形态发生和分化

基本信息

批准号：
07308052
负责人：
TAKEUCHI Ikuo
金额：
$ 2.3万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Takeuchi, I.organized Dictyostelium cDNA project with several members of this research group and started in the last autumn. He also found that prestalk and prespore cells sucked into a capillary tube do not differentiate at a constant propotion, as in ordinary slugs.Molecular mechanism of morphogenesis Suto, K.analyzed a REMI mutant which dose not develop after aggregation, and found that the mutated genes is a homologue of SSN6 gene of yeast. Ochiai, H.prepared rabbit polyclonal antibody raised against the membrance fraction isolated from Polysphondylium cells transformed with an antisence RNA-expressing plasmid to reduce cell-adhesion protein. As the antibody efficiently inhibits Polysphondylium cell-adhesion, it will be a good tool to analyze the mechanism in the formation of multeicellular structure of the organism. Molecular machanism of differentiation To investigate a switch mechanism from growth to differentiation, Maeda, Y.cloned two genes (Quit2,3) which are expressed around the critical point (PS point) of the cell cycle. He found that Quit2 encodes a new calcium-binding protein and Quit3 has a sequence coding for the complementary strand of annexin Vll gene. Oohata, A.isolated a factor induction of prespore differentiation in vitro. Maeda, M.revealed that the gene is essential for aggregation and that hetero-trimeric G proteins are not required for this regulation. Tanaka, Y.established the techniques for an insertional mutagenesis with Polysphondylium cells by the use of REMI method. Ishida, S.isolated 'sporogenous mutant', in which even single cells differentiate into spores and tag mutants which stop at the 'tight aggregate' stage. Urushihara, H.analyzed two sorts of REMI mutants (TMC1 and MCF1). TMC1 was normal for sexual cell fusion, but was unusual for chemotaxis. MCF1 had a defect in sexual cell fusion.

Takeuchi, i .与本研究组的几名成员组织了盘齿骨菌cDNA项目，并于去年秋天启动。他还发现，吸进毛细管的前柄细胞和前孢子细胞不能像普通鼻涕虫那样以恒定的比例分化。Suto, k .分析了一个聚集后不发育的REMI突变体，发现该突变基因与酵母的SSN6基因同源。Ochiai, h .制备了兔多克隆抗体，该抗体针对从Polysphondylium细胞分离的膜部分，该膜部分经反肽rna表达质粒转化以减少细胞粘附蛋白。由于该抗体能有效抑制Polysphondylium细胞粘附，为分析机体多细胞结构形成机制提供了良好的工具。为了研究细胞从生长到分化的开关机制，Maeda, y .克隆了两个在细胞周期临界点（PS点）附近表达的基因（Quit2,3）。他发现Quit2编码一个新的钙结合蛋白，而Quit3有一个序列编码膜联蛋白Vll基因的互补链。Oohata, a .分离出一种体外诱导孢子前分化的因子。Maeda, m .揭示了该基因是聚集所必需的，而异三聚体G蛋白不需要这种调节。Tanaka, y .建立了利用REMI方法对多孢子虫细胞进行插入诱变的技术。石田，s.s分离出“孢子性突变体”，在这种突变体中，即使是单细胞也分化成孢子和标签突变体，后者在“紧密聚集”阶段停止。Urushihara, h .分析了两种REMI突变体（TMC1和MCF1）。TMC1在性细胞融合方面是正常的，但在趋化性方面是异常的。MCF1在性细胞融合方面存在缺陷。