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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：
10680689
负责人：
YOKOYAMA Takahiko
金额：
$ 1.6万
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：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680689/
关键词：
left right asymmetry inv situs inversus ankyrin intracellular localization 遺伝子 Xenopus

项目摘要

Almost all internal organs of vertebrate body have distinctive left/right asymmetry such as heart and spleen on the left. The inv mutant shows a constant reversal of such left/right polarity. We have identified the gene that is responsible for the inv mutation. The gene encodes 1062 amino acids and 15 repeats of ankyrin motif and two bipartite nuclear localization signals. The gene has not previously reported and its cellular function is unknown. To study the intracellular function of the inv gene, we determined the localization of the inv gene product. GFP (green fluorescent protein) fusion constructs linked to the full length inv gene are generated and are transfected in cos 7 cells. Curiously, signals are observed in the cytoplasm when GFP is connected in front of the inv gene whereas in nucleus when GFP is connectd behind the inv gene. When flag tag or myc tag is connected in front of or behind the inv gene, signals are observed in the cytoplasm. Therefore we concluded that the inv protein is normally localized in the cytoplasm. We next deleted the inv gene and connected behind GFP gene. These constructs are also transfected in cos 7 cells and their localization was examined. When GFP constructs have bipartite nuclear localization signal without ankyrin repeats, signal are observed in the nucleus. Therefore, nuclear localization signals in the inv gene are functional. If the constructs retained ankyrin repeats, signals were observed in the cytoplasm but not in the nucleus. Although it is still unclear where the inv gene product works, these results suggested that the inv protein may be function by transporting between the nucleus and the cytoplasm.

脊椎动物体内几乎所有的内脏器官都具有明显的左右不对称性，如心脏和脾脏在左侧。inv突变体显示这种左/右极性的恒定逆转。我们已经确定了导致inv突变的基因。该基因编码1062个氨基酸、15个锚蛋白重复序列和两个二分核定位信号。该基因以前没有报道，其细胞功能是未知的。为了研究inv基因的细胞内功能，我们确定了inv基因产物的定位。产生与全长inv基因连接的GFP（绿色荧光蛋白）融合构建体，并转染到cos 7细胞中。奇怪的是，当GFP连接在inv基因之前时，在细胞质中观察到信号，而当GFP连接在inv基因之后时，在细胞核中观察到信号。当flag标签或myc标签连接在inv基因的前面或后面时，在细胞质中观察到信号。因此，我们得出结论，inv蛋白通常定位于细胞质中。我们接下来删除inv基因并连接到GFP基因后面。还将这些构建体转染到cos 7细胞中并检查它们的定位。当GFP构建体具有不含锚蛋白重复序列的二分核定位信号时，在核中观察到信号。因此，inv基因中的核定位信号是功能性的。如果构建体保留锚蛋白重复序列，则在细胞质中观察到信号，但不在细胞核中观察到信号。虽然inv基因产物在哪里起作用还不清楚，但这些结果表明inv蛋白可能通过在细胞核和细胞质之间转运来发挥作用。