Bok J, Jin Y, Lee J
Department of Biology, Yonsei University, Seoul, Korea.
Exp Cell Res. 1995 Jul;219(1):47-53. doi: 10.1006/excr.1995.1203.
During the differentiation of Naegleria gruberi amoebae into flagellates, four differentiation-specific (DS) mRNAs are transiently and coordinately accumulated. Three of the four DS mRNAs, Class II, III, and IV, encode alpha-tubulin, beta-tubulin, and flagellar calmodulin, respectively. The protein product of the Class I mRNA has not been identified. We examined the effects of inhibition of protein synthesis on transcription and accumulation of beta-tubulin mRNA and Class I mRNA to understand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked transcription of the beta-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription of the beta-tubulin gene in less than 10 min as judged by nuclear run-on experiments. However, once differentiation had proceeded for more than 50 min, inhibition of protein synthesis did not inactivate transcription of beta-tubulin mRNA was more active in cycloheximide-treated cells than in control cells. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of the drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-lives of beta-tubulin and Class I mRNA drastically. These data suggest that: (1) the transient accumulation of the two DS mRNAs during differentiation are regulated by changing both the rate of transcription and the stability of the mRNAs; (2) protein synthesis is required for the transcriptional and post-transcriptional regulations; (3) the transcriptional regulation mechanisms of the beta-tubulin gene and that of the Class I gene are distinct; and (4) the transcription of the beta-tubulin gene is regulated by different mechanisms during differentiation.
在格氏耐格里变形虫向鞭毛虫分化的过程中，四种分化特异性（DS）mRNA会短暂且协同积累。四种DS mRNA中的三种，即II类、III类和IV类，分别编码α-微管蛋白、β-微管蛋白和鞭毛钙调蛋白。I类mRNA的蛋白质产物尚未确定。我们研究了蛋白质合成抑制对β-微管蛋白mRNA和I类mRNA转录及积累的影响，以了解协同调控的机制。在分化开始时抑制蛋白质合成完全阻断了β-微管蛋白基因的转录。根据细胞核连续转录实验判断，在分化开始后30或40分钟添加环己酰亚胺，不到10分钟就能使β-微管蛋白基因的转录失活。然而，一旦分化进行超过50分钟，抑制蛋白质合成并不会使β-微管蛋白mRNA的转录失活，在环己酰亚胺处理的细胞中β-微管蛋白mRNA比对照细胞中更活跃。在分化开始时用环己酰亚胺处理也阻断了I类基因的转录。然而，30分钟后添加该药物对I类基因的转录没有显著影响。环己酰亚胺处理还大幅增加了β-微管蛋白和I类mRNA的半衰期。这些数据表明：（1）分化过程中两种DS mRNA的短暂积累是通过改变转录速率和mRNA稳定性来调控的；（2）转录和转录后调控都需要蛋白质合成；（3）β-微管蛋白基因和I类基因的转录调控机制不同；（4）β-微管蛋白基因在分化过程中受不同机制调控。
