Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.
Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.
复制标题
盘基网柄菌中两个发育调节的 G 蛋白 α 亚基基因 G α 7 和 G α 8 的克隆和靶向突变。
DOI：
10.1091/mbc.5.6.691
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发表时间：
1994
影响因子：
3.3
通讯作者：
Devreotes,PN
中科院分区：
文献类型：
--
作者：
Wu,L;Gaskins,C;Zhou,K;Firtel,RA;Devreotes,PN
GTP结合蛋白（G蛋白）介导的信号转导途径在网骨藻的聚集和分化过程中起重要作用。除了五个已知的G蛋白α亚基基因，我们最近确定了三个新的α亚基基因，G α 6，G α 7和G α 8，使用聚合酶链反应技术。我们在这里提出了一个更完整的分析G α 7和G α 8。克隆了这两个基因的cDNA，并测定了它们的全核苷酸序列。序列分析表明G α 8具有一些不寻常的特征。它缺乏“TCATDT”基序，这是G α亚基中高度保守的氨基酸序列，并且在其C-末端具有由长的天冬酰胺延伸组成的另外50个氨基酸。此外，G α 8对蛋白酶消化具有不寻常的抗性，这可能表明GTP水解速率较慢。这些α亚基的可能功能通过产生缺乏G α 7或G α 8的突变体来评估，所述突变体通过同源重组的基因靶向和通过过表达G α 7或G α 8蛋白来产生。G α 7的过度表达导致在蛞蝓阶段开始的异常形态发生，而其他菌株的分析未能揭示任何明显的生长或发育缺陷，无论是正常或压力条件下。这些结果的影响进行了讨论。
GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed.