The identification of Pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum
The identification of Pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum
复制标题
DOI：
10.1091/mbc.e02-06-0335
复制
发表时间：
2003-01-01
影响因子：
3.3
通讯作者：
Larochelle, DA
中科院分区：
文献类型：
--
作者：
Abysalh, JC;Kuchnicki, LL;Larochelle, DA
在这里，我们描述了细胞质分裂缺陷突变细胞系17HG5的鉴定和特性，该突变细胞系是通过限制性内切酶介导的整合突变筛选而产生的，该突变筛选旨在分离盘基网柄菌胞质分裂所需的基因。17HG5细胞系的表型特征表明，除了在悬浮培养中观察到的胞质分裂缺陷外，肌动球蛋白细胞骨架的整体功能没有明显缺陷。用质粒救援法鉴定导致胞质分裂缺陷的基因座(pats1；与信号转导相关的蛋白)。在几个独立的细胞系中，通过同源重组重建了pats1基因座的破坏，每个细胞系都重现了胞质分裂缺陷的表型，从而证实了该基因座对于正常的胞质分裂是重要的。通过对插入的限制性内切酶介导的整合质粒两侧基因组区域的序列分析，发现了一个8892bp的基因组开放阅读框，编码2964个氨基酸的蛋白质。Pats1蛋白包含3个调控结构域(RI-磷酸酶、RII-GTP结合、R-III蛋白激酶)、13个富含亮氨酸的重复序列和8个WD-40重复序列。这些调控结构域与蛋白质-蛋白质相互作用结构域相结合，表明Pats1参与了网柄基菌胞质分裂过程中的信号转导。
Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1) that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.