PROGRAMMED APPEARANCE OF TRANSLATABLE FLAGELLAR TUBULIN MESSENGER-RNA DURING CELL-DIFFERENTIATION IN NAEGLERIA
PROGRAMMED APPEARANCE OF TRANSLATABLE FLAGELLAR TUBULIN MESSENGER-RNA DURING CELL-DIFFERENTIATION IN NAEGLERIA
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DOI：
10.1016/0092-8674(79)90327-1
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发表时间：
1979-01-01
期刊：
影响因子：
64.5
通讯作者：
FULTON, C
中科院分区：
文献类型：
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作者：
LAI, EY;WALSH, C;FULTON, C
鞭毛微管蛋白的程序性从头合成在N. gruberi从变形虫到鞭毛虫是研究细胞分化过程中基因表达的范例。本文报道了在小麦胚无细胞体系中，由从分化细胞中提取的总RNA或多聚腺苷酸化RNA指导鞭毛微管蛋白mRNA的高效翻译。体外产物中的微管蛋白具有55，000的亚基MW，分离成α-微管蛋白和α-微管蛋白。和β在聚丙烯酰胺凝胶电泳的合适条件下，将其亚基与小牛脑微管蛋白共聚合。至少有一半的微管蛋白在体外合成的鞭毛微管蛋白的特异性抗体沉淀和免疫沉淀的微管蛋白亚基产生类似于那些外部的双峰微管蛋白的肽图。鞭毛微管蛋白是在无细胞系统中合成的主要蛋白质，并且占由来自分化细胞的总RNA指导合成的多肽的约5%。相反，很少或没有鞭毛微管蛋白的合成时，无细胞系统是由RNA提取阿米巴分化前。翻译分析表明，至少92%的鞭毛微管蛋白mRNA出现在分化过程中。该mRNA的出现的时间过程通过无细胞产物的定量免疫沉淀来测量。在细胞分化开始后60 min形成鞭毛的条件下，在20 min时首先检测到可翻译的鞭毛微管蛋白mRNA，在约60 min时达到最大值，然后下降。一个很好的相关性之间的翻译鞭毛微管蛋白mRNA的量和先前测量的鞭毛微管蛋白在体内的合成率。鞭毛微管蛋白的合成可能是其mRNA丰度的直接反映，并提供了分子技术解剖的因素，调节这种结构蛋白在分化过程中的快速出现。
The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of N. gruberi from amoebae to flagellates was the paradigm for the study of gene expression during cell differentiation. The efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells is reported. The tubulin in the in vitro product has a subunit MW of 55,000, separates into .alpha. and .beta. subunits under suitable conditions of polyacrylamide gel electrophoresis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tubulin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells form flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. Synthesis of flagellar tubulin is probably a direct reflection of the abundance of its mRNA, and provides the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.