When exponentially growing cells of Tetrahymenapyrforrnis are transferred to a non-nutrient medium the loss of whole cell RNA, 90 % of which is ribosomal RNA, exhibits biphasic kinetics, whereas whole cell protein is lost at a constant rate. The ratio RNA/protein declines during the first 5 h of starvation and then remains constant during the subsequent period of starvation. The synthesis of the majority of the ribosomal proteins is coordinately regulated during a nutritional shift-down. Exponentally growing cells devote 17 % of their capacity for protein synthesis to the production of ribosomal proteins. Upon starvation this proportion is rapidly reduced 3.5-fold. In long-time-starved cells the absolute rate of ribosomal protein synthesis is only about 4.5 o/, of that of exponentially growing cells. The synthesis of ribosomal RNA and ribosomal proteins appears tightly coupled during the transition from growth to starvation. In long-time-starved cells the syntheses of ribosomal RNA and ribosomal proteins are stoichiometrically balanced with no significant degradation of de nouo synthesized ribosomal proteins.The potential of members of the genus Tetrahymena for studies on the regulation of ribosome synthesis has been amply demonstrated [I -31. Extraordinarily large variations in the rate of ribosome production occur during a starvationrefeeding cycle of Tetrahymena [I]. In long-time-starved cells less than 3% of the synthesized proteins accumulate in ribosomes, while up to 40% of the newly synthesized proteins can be recovered in ribosomes in refed cells [I]. It was calculated that this represents an 80-fold variation in the rate of accumulation of ribosomal proteins (r-proteins) in ribosomes [I]. The rates of pre-rRNA transcription and processing are severely depressed in starved cells [4,5], but increase rapidly upon refeeding [6]. However, from the data of Hallberg and Bruns [l], it is impossible to tell whether the rate of r-protein synthesis is reduced in concert with the synthesis of rRNA in the starved cells, or whether r-proteins are synthesized in excess of rRNA and then degraded [ 7 ] .In yeast starvation for a required amino acid leads to decrease in the rate of pre-rRNA transcription which is accompanied by a coordinate decrease in the rate of synthesis of most r-proteins [8]. In contrast, inhibition of rRNA transcription during differentiation of L,E, rat myoblasts into myotubes [9] or during a methyl-cellulose-mediated down-shift of mouse 3T3 cells [lo] is not parallelled by a decrease in the relative rate of r-protein synthesis. In fact, evidence was presented indicating that in the case of myoblast differentiation, transcription of r-protein mRNAs persisted unabated for several days. Analogous to other systems, r-proteins synthesized in excess relative to rRNA were rapidly degraded [7,9].The aim of this work was to analyze the synthesis of rproteins in Tetrahymena during a nutritional shift-down, in particular with respect to the coordination of the synthesis of Abbreaiation. r-protein, ribosoma...

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We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two‐dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid‐selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979‐bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3′ splice junction. The structure of the intron and the fact that it is found together with the well described self‐splicing rRNA intron is discussed in relation to the evolution of RNA splicing.

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.

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The addition of nutrients to long-time-starved cells of Tetrahymenapyriformis leads to a 50 -60-fold increase in the rate of synthesis of ribosomal proteins (r-proteins). This is achieved by a 6-fold increase in the relative rate of r-protein synthesis and a 8 -10-fold increase in the rate of total protein synthesis. Synthesis of r-proteins constitutes one third of total cellular protein synthesis 2 -4 h after refeeding and the absolute rate of r-protein synthesis is approximately three-times greater than in exponentially growing cells. The synthesis of the individual r-proteins is coordinately regulated during a nutritional shift-up, and de n o w synthesized r-proteins are stable. Addition of actinomycin D prevents the increase in the rate of r-protein synthesis. The rates of synthesis of rRNA and r-protein increase in concert, implying coordinate regulation. Furthermore, a comparison of the observed accumulation of r-proteins with the predicted accumulation based on the accumulation of rRNA suggests that rRNA and r-protein are synthesized in a stoichiometrically balanced way during the entire refeeding period.Addition of nutrients to long-time-starved Tetvahymena cells leads to an ordered sequential reactivation of macromolecular synthesis culminating in the initiation of a rather synchronous cell division about 4 h after refeeding [ 1 -51. An important and integral part of the events leading to the resumption of growth is the restoration of the cellular level of ribosomes [6]. The level of ribosomes in long-time-starved Tetrahymena cells is approximately halved in comparison to exponentially growing cells, and the rate of ribosome production is only about 4.5 % of the rate in exponentially growing cells [6,7]. Following refeeding, the rate of pre-rRNA processing increases rapidly [S]. During the initial period of refeeding the rate of pre-rRNA transcription increases slowly, but around 1.5 h after refeeding, concomitant with the initiation of rDNA replication, the rate of pre-rRNA transcription increases abruptly [8]. From studies using hydroxyurea to inhibit DNA replication, it was suggested that pre-rRNA transcription was gene-dose-dependent [8,9]. By measuring accumulation of total r-protein in mature ribosomes, Hallberg and Bruns estimated that the rate of r-protein synthesis increases about 80-fold during refeeding. Furthermore, it was inferred that r-proteins and rRNA were non-coordinately synthesized during the early period of refeeding [6,10].In this work we examined the rate of synthesis of individual r-proteins during a nutritional shift-up and attempted to measure the stoichiometry of r-protein and rRNA synthesis. We show that the rates of synthesis of individual r-proteins increase coordinately during refeeding. This increase in the rate of synthesis was prevented by the addition of actinomycin D. In addition we show that there is no degradation of de novo synthesized r-protein in refed cells, and finally, we present evidence that, contrary to the suggestion of Hallberg and coworkers [6,10], the s...

The codon usage of Tetrahymena thermophila and other ciliates deviates from the 'universal genetic code' in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

We have measured the levels of translatable total mRNA and ribosomal protein (r-protein) mRNAs in Tetrahymena pyriformis during nutritional shifts. After 15 min of starvation total mRNA is reduced 2-fold, and after 24 h 7.5-fold, relative to exponentially growing cells. Upon refeeding total mRNA increases rapidly reaching the level of exponentially growing cells after 2.5 h. The levels of the individual r-protein mRNAs are coordinately regulated throughout a starvation-refeeding cycle. The relative levels of r-protein mRNAs remain virtually unchanged during the first hour of starvation and then decrease gradually to 30% of the relative levels in exponentially growing cells. Following refeeding the relative levels of r-protein mRNAs increase 6-fold. Taking into account the changes in whole cell RNA, we have calculated that long-time-starved Tetrahymena cells contain only 4 %; whereas cells after 3 h of refeeding contain 200 % of the amount of r-protein mRNA in exponentially growing cells. The amount of r-protein mRNA thus increases 50-fold during the first 3 h of refeeding. A comparison between the relative levels of r-protein mRNAs and the relative rate of r-protein synthesis in vivo indicates that Tetrahymena employs a combination of control of translation and control of the level of r-protein mRNAs to ensure a rapid reduction in the rate of r-protein synthesis during the early period of starvation. In this period translation of r-protein mRNAs is preferentially inhibited. During refeeding the increase in the rate of r-protein synthesis parallels the increase in the abundance of r-protein mRNAs.Control of the synthesis of ribosomal proteins (r-proteins) in eukaryotic cells seems to encompass regulations at the levels of transcription, processing, degradation and translation of rprotein mRNAs. In the lower eukaryote yeast transcriptional controls seem to prevail during nutritional shifts [1,2] and during temperature shifts [3], whereas impaired splicing of r-protein mRNAs reduces r-protein synthesis at the nonpermissive temperature in at least 3 of the 11 non-allelic temperature-sensitive rna mutants [4,5]. Furthermore, the rate of turnover of r-protein mRNAs may vary [6,7] and, finally, Warner and co-workers have shown that yeast transformed with a multicopy plasmid harbouring the gene for r-protein L3 uses translational control to compensate for the increased abundance of mRNA for r-protein L3 in the transformed cells [7]. Translational controls also seem to be involved in the regulation of r-protein synthesis in Acanthamoeba castellanii during encystment [8] and in Dictyostelium discoideum during vegetative growth [9]. In higher eukaryotes evidence for regulation of r-protein synthesis at the level of transcription, processing or degradation of mRNAs [lo -121 and at the level of translation [12 -251 has been presented.We showed in the preceding papers [16,17] that in the ciliated protozoan Tetrahymena pyriformis the rate of r-protein synthesis changes 50 -60-fold during a starvationrefeeding cycle. In additio...