Lai E Y, Walsh C, Wardell D, Fulton C
Cell. 1979 Aug;17(4):867-78. doi: 10.1016/0092-8674(79)90327-1.
The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis 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 tublin. 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 from 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. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.
在格氏耐格里变形虫从阿米巴虫分化为鞭毛虫的长达一小时的过程中，鞭毛微管蛋白的程序性从头合成是我们研究细胞分化过程中基因表达的范例。本文报道了从小麦胚无细胞系统中由分化细胞提取的总RNA或聚腺苷酸化RNA指导下鞭毛微管蛋白mRNA的高效翻译。体外产物中的微管蛋白亚基分子量为55,000，在聚丙烯酰胺凝胶电泳的合适条件下可分离为α和β亚基，并与小牛脑微管蛋白共聚合。体外合成的微管蛋白至少有一半可被鞭毛微管蛋白特异性抗体沉淀，免疫沉淀的微管蛋白亚基产生的肽图与外双联体微管蛋白的肽图相似。鞭毛微管蛋白是无细胞系统中合成的主要蛋白质，约占由分化细胞总RNA指导合成的多肽的5%。相比之下，当无细胞系统由分化前的阿米巴虫提取的RNA指导时，几乎不合成或不合成鞭毛微管蛋白。翻译分析表明，至少92%的鞭毛微管蛋白mRNA在分化过程中出现。通过对无细胞产物进行定量免疫沉淀来测量该mRNA出现的时间进程。在分化开始60分钟后收集鞭毛细胞进行翻译的条件下，可翻译的鞭毛微管蛋白mRNA在20分钟时首次被检测到，在约60分钟时达到最大值，然后下降。在可翻译的鞭毛微管蛋白mRNA的量与先前在体内测量的鞭毛微管蛋白合成速率之间观察到了极好的相关性。这些结果表明，鞭毛微管蛋白的合成是其mRNA丰度的直接反映，并为剖析在分化过程中调节这种结构蛋白快速出现的因素提供了分子技术。
