权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Functional analysis of left-right determining (inv) gene'-''

左右决定(inv)基因-的功能分析

基本信息

批准号：
12680723
负责人：
YOKOYAMA Takahiko
金额：
$ 2.24万
依托单位：
Tokyo Women's Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680723/
关键词：
left right asymmetry inv (3) situs inversus ankyrin intracellular localization xenopus

项目摘要

Establishment of left-right asymmetry in internal organs is essential for normal development of vertebrates. The inυ mutant in mice shows a constant reversal of left-right asymmetry. We have cloned the inv gene. The gene was novel. Therefore, its biochemical and cell biological functions were undefined. Using yeast two hybrid system, we discovered that calmodulin binds with inv protein at two sites (IQ1 and IQ2). The binding was confirmed by a Gel-overlay assay. The binding of calmodulin to the IQ2 site occurred in the absence of Ca2+, whereas it was inhibited in the presence of Ca2+. We also found that injection of mouse inυ mRNA into the right blastomere of Xenopus embryos at two-cell stage randomized the left-right asymmetry and altered patterns of Xnr-1 and PitX2 expression. Importantly, inυ RNA lacking the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inυ to randomize the left-right asymmetry in Xenopus. These results suggest a possiblity that calmodulin-binding regulates inυ function.To see whether inυ gene has any effect on cultured cells, we introduced inυ gene into cos cells. Long-term cultured cos cells that carry inυ gene tends to show hyperploidity, suggesting a possiblity that the gene may affect cytokinesis.

内脏器官左右不对称的建立对脊椎动物的正常发育至关重要。在小鼠中的inclusion突变体显示出左右不对称的持续逆转。我们克隆了inv基因。这个基因很新奇。因此，其生物化学和细胞生物学功能尚不明确。利用酵母双杂交系统，我们发现钙调素与inv蛋白在两个位点（IQ 1和IQ 2）结合。通过凝胶覆盖测定证实结合。钙调素与IQ 2位点的结合发生在没有Ca 2+的情况下，而在Ca 2+的存在下被抑制。我们还发现，将小鼠inυ mRNA注射到两细胞期爪蟾胚胎的右侧卵裂球中，会随机化Xnr-1和PitX 2表达的左右不对称性并改变其表达模式。更重要的是，缺乏IQ 2位点的RNA不能随机化非洲爪蟾胚胎的左右不对称，这意味着IQ 2位点对于随机化非洲爪蟾的左右不对称是必不可少的。为了观察in-resistin基因对培养细胞是否有影响，我们将in-resistin基因导入cos细胞中。长期培养的COS细胞中携带有Cos基因，易出现超倍性，提示该基因可能影响胞质分裂。