A novel approach for expression cloning of small GTPases: Identification, tissue distribution and chromosome mapping of the human homolog of rheb
A novel approach for expression cloning of small GTPases: Identification, tissue distribution and chromosome mapping of the human homolog of rheb
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DOI：
10.1016/0014-5793(95)01349-0
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发表时间：
1995-12-18
期刊：
影响因子：
3.5
通讯作者：
Celis, JE
中科院分区：
文献类型：
--
作者：
Gromov, PS;Madsen, P;Celis, JE
我们报告了一种新的方法，用于确定单体GTP结合蛋白，这是基于探测cDNA表达文库与[α-P-32]GTP。简而言之，用2%SDS处理来自平板cDNA表达文库的硝酸纤维素复制品，以阻断由E.用这种方法，我从人角质形成细胞中克隆了几种小的GTP结合蛋白，包括rheb的人同源物，rheb是ras相关GTP结合蛋白的新成员，人rheb cDNA与大鼠对应物具有90%的同一性，并且它在各种来源的转化的人细胞中高度上调，北方分析表明，人I hcb是普遍表达的，在骨骼肌和心肌中观察到最高水平，而不是在大脑中，因为它是在大鼠rheb的情况下。将人RHEB基因定位于染色体10 q11。
We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with [alpha-P-32]GTP. In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E. coli, thus allowing the direct identification of positive clones, Using this procedure me have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins, Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin, Northern analysis showed that human I hcb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb. The human RHEB gene was mapped to chromosome 10q11.