INDEPENDENT MECHANISMS ARE UTILIZED FOR THE COORDINATE AND TRANSIENT ACCUMULATION OF 2 DIFFERENTIATION-SPECIFIC MESSENGER-RNAS DURING DIFFERENTIATION OF NAEGLERIA-GRUBERI AMEBAS INTO FLAGELLATES

During the differentiation of Naegleria gruberi amoebae into flagellat es, four differentiation-specific (DS) mRNAs are transiently and coord inately accumulated. Three of the four DS mRNAs, Class II, III, and IV , encode alpha-tubulin, beta-tubulin, and flagellar calmodulin, respec tively. The protein product of the Class I mRNA has not been identifie d. We examined the effects of inhibition of protein synthesis on trans cription and accumulation of beta-tubulin mRNA and Class I mRNA to und erstand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked trans cription of the beta-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription o f the beta-tubulin gene in less than 10 min as judged by nuclear run-o n experiments. However, once differentiation had proceeded for more th an 50 min, inhibition of protein synthesis did not inactivate transcri ption of the beta-tubulin gene. Rather, transcription of beta-tubulin mRNA was more active in cycloheximide-treated cells than in control ce lls. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of t he drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-liv es of beta-tubulin and Class I mRNA drastically. These data suggest th at: (1) the transient accumulation of the two DS mRNAs during differen tiation are regulated by changing both the rate of transcription and t he stability of the mRNAs; (2) protein synthesis is required for the t ranscriptional and post-transcriptional regulations; (3) the transcrip tional regulation mechanism of the beta-tubulin gene and that of the C lass I gene are distinct; and (4) the transcription of the beta-tubuli n gene is regulated by different mechanisms during differentiation. (C ) 1995 Academic Press, Inc.