MESSENGER-RNAS FOR ALPHA-TUBULIN AND BETA-TUBULIN AND FLAGELLAR CALMODULIN ARE AMONG THOSE COORDINATELY REGULATED WHEN NAEGLERIA-GRUBERI AMEBAE DIFFERENTIATE INTO FLAGELLATES
MESSENGER-RNAS FOR ALPHA-TUBULIN AND BETA-TUBULIN AND FLAGELLAR CALMODULIN ARE AMONG THOSE COORDINATELY REGULATED WHEN NAEGLERIA-GRUBERI AMEBAE DIFFERENTIATE INTO FLAGELLATES
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DOI：
10.1083/jcb.105.3.1303
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发表时间：
1987-09-01
影响因子：
7.8
通讯作者：
WALSH, CJ
中科院分区：
文献类型：
--
作者：
SHEA, DK;WALSH, CJ
四种mRNA中的三种特异性地将格鲁伯耐格里阿米巴分化为鞭毛虫（Mar，J.，J. H. Lee，D. Shea和C. J.沃尔什，1986，细胞生物学杂志，102：353-361）已被鉴定为编码鞭毛蛋白。这些mRNA的产物，这是协调调节分化过程中，通过在体外翻译的混合选择的RNA，然后通过二维凝胶电泳和抗体结合进行了鉴定。6个与1.7-kB RNA（II类）互补的交叉杂交克隆，所有选择的mRNA被翻译成两个α-RNA。微管蛋白主要的体外产物，α- 1，与细胞质α-微管蛋白，而具有更酸性pI的次要产物α-微管蛋白，2，与鞭毛α-微管蛋白而Naegleria鞭毛α-基于微管蛋白与对该形式特异的单克隆抗体的反应，发现微管蛋白被乙酰化，我们建议α-微管蛋白是乙酰化的。2不太可能由于体外乙酰化而产生，但可能代表第二个α-微管蛋白基因III类克隆也与1.7-kb RNA互补，选择β-微管蛋白mRNA。在这项工作的过程中，发现使用针对α-和β-微管蛋白的亚基，即耐格里属α-微管蛋白比β-微管蛋白迁移更快。微管蛋白的SDS-PAGE分析。IV类克隆与0.5 kb RNA杂交，选择了翻译成热稳定钙结合蛋白鞭毛钙调蛋白的mRNA。
Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kB RNA (class II) all selected mRNA that was translated into two .alpha.-tubulins. The principal in vitro product, .alpha.-1, comigrated with a cytoplasmic .alpha.-tubulin, while the minor product with a more acidic pI, .alpha.-2, comigrated with flagellar .alpha.-tubulin. While Naegleria flagellar .alpha.-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that .alpha.-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second .alpha.-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected .beta.-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the .alpha.- and .beta.-subunits of tubulin, that Naegleria .alpha.-tubulin migrated faster than .beta.-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.