OVEREXPRESSION OF AN EPITOPE-TAGGED BETA-TUBULIN IN CHINESE-HAMSTER OVARY CELLS CAUSES AN INCREASE IN ENDOGENOUS ALPHA-TUBULIN SYNTHESIS
OVEREXPRESSION OF AN EPITOPE-TAGGED BETA-TUBULIN IN CHINESE-HAMSTER OVARY CELLS CAUSES AN INCREASE IN ENDOGENOUS ALPHA-TUBULIN SYNTHESIS
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DOI：
10.1002/cm.970310403
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发表时间：
1995-01-01
影响因子：
--
通讯作者：
CABRAL, F
中科院分区：
文献类型：
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作者：
GONZALEZGARAY, ML;CABRAL, F
将经工程改造以表达来自流感血凝素抗原（HA）的9个氨基酸表位的中国仓鼠β-微管蛋白cDNA转染到中国仓鼠卵巢（CHO）细胞中。根据以下标准，重组蛋白（HA β（1）-微管蛋白）似乎表现正常：免疫荧光表明HA β（1）-微管蛋白掺入所有类型的间期和纺锤体微管以及微管组织中心。表达HA β（1）-微管蛋白的细胞对秋水仙胺和紫杉醇的敏感性没有变化。一个210 kD微管相关蛋白（MAP）仍然与结合HA β（1）-微管蛋白的微管相关。内源性β-微管蛋白和HA β（1）-微管蛋白的合成均被秋水仙素抑制。HA β（1）-微管蛋白掺入微管的程度与内源性β-微管蛋白相同，转染细胞中微管组装的总体程度没有变化。最后，转染的细胞具有正常的生长速率和形态。当测量对内源性微管蛋白产生的影响时，发现HA β（1）-微管蛋白的表达减少了内源性野生型β-微管蛋白的合成，但增加了α-微管蛋白的合成。在稳定状态下，一个小的增加，总微管蛋白与增加的α-微管蛋白的合成相一致的found. The结果表明，过量的外源性β-微管蛋白的表达扰乱了内源性α-微管蛋白的合成的方式是不容易解释的微管蛋白调节的电流模型。微管蛋白合成的变化沿着过量微管蛋白亚基的降解可能反映了确保α-和β-微管蛋白组装的协调水平的机制。(C)1995 Wiley-Liss，Inc.
A Chinese hamster beta-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HA beta(1)-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HA beta(1)-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HA beta(1)-tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HA beta(1)-tubulin. The synthesis of both endogenous beta-tubulin and HA beta(1)-tubulin was repressed by colchicine. The HA beta(1)-tubulin incorporated into microtubules to the same extent as the endogenous beta-tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, transfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HA beta(1)-tubulin reduced the synthesis of endogenous wild-type beta-tubulin but increased the synthesis of alpha-tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of alpha-tubulin was found. The results indicate that expression of excess exogenous beta-tubulin perturbs the synthesis of endogenous alpha-tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of alpha- and beta-tubulin for assembly. (C) 1995 Wiley-Liss, Inc.