Tetrahymena cells secrete a low-molecular weight factor which inhibits RNA synthesis in vivo and in vitro.

Medium which has been dialyzed against a dense population of Tetrahymena cells contains a factor which reduces the rate of RNA synthesis in other Tetrahymena cells inoculated on that medium. Furthermore, this extracellular factor inhibits the ribosomal RNA synthesis in vitro when added to an RNA synthesizing r-chromatin preparation.     

A peptide from Tetrahymena disrupts subunit organization of E. coli RNA polymerase.

Incubation of the E. coli RNA polymerase with a polypeptide factor from the protozoan Tetrahymena reduces the affinity of the holoenzyme for DNA. SDS-polyacrylamide gel electrophoresis of the peptide-treated RNA polymerase showed that the band pattern of the polymerase subunits was strongly altered. The three large subunits, beta', beta and sigma, disappear and a high number of rapidly migrating bands appeared. However, a brief heat treatment of the samples almost restored the original RNA polymerase subunit composition, and in addition a high molecular weight protein band approximately 240 kDa appeared. It is suggested that the Tetrahymena peptide specifically binds to the RNA polymerase and changes the structures of the large subunits.     

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.     

Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.     

Chemosensory Responses in Tetrahymena: The Involvement of Peptides and other Signal Substances1

Starved Tetrahymena thermophila (or cells growing in Holz's defined medium) are attracted by a chemosensory response to complex peptide mixtures as proteose peptone, yeast extract, and extracts of blood platelets containing platelet-derived growth factor. Starved cells are also significantly attracted by mixtures of amino acids and of nucleosides of Holz's defined medium; however, no individual amino acid or nucleoside could be identified as the major chemo-attractant. The positive chemosensory response can be abolished by cycloheximide (CHX) but is insensitive to actinomycin D and puromycin, possibly indicating that de novo RNA and protein synthesis are not necessary for a change in chemosensory behavior. Three mutants resistant to CHX show normal response in the presence of the drug. The possible role of peptides as naturally occurring food signals of Tetrahymena is discussed.     

A cytophotometric study of the influence exerted by epidermal growth factor on RNA and protein synthesis in the ciliate Tetrahymena pyriformis

The influence of EGF (10(-8) M) on RNA and protein synthesis in the ciliate Tetrahymena pyriformis was studied. It was found that EGF stimulated RNA and proteins synthesis in T. pyriformis within 3 h after addition of EGF.     

The Effect of 2-Mercapto-1-(Beta-4-Pyridethyl) Benzimidazole (MPB) on Cell Differentiation and RNA Synthesis in the Protozoon Tetrahymena vorax1

SYNOPSIS. Differentiation of small-mouthed cells (microstomes) into large-mouthed, potentially carnivorous cells (macrostomes) in Tetrahymena vorax is prevented by 2-mercapto-1-(β-4-pyridethyl) benzimidazole (MPB). This differentiation, induced by the transforming principle, stomatin, isolated from the potential prey, Tetrahymena pyriformis, is a synchronous process in which 70–95% of the population of T. vorax microstomes transform into macrostomes within 450 min. MPB also inhibits RNA synthesis in transforming microstomes while having little effect on protein synthesis. Finally, the effect of MPB on both transformation and RNA synthesis is reversible.     

Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro.

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.     

Meiosis-Reinitiation-Inducing Factor of Tetrahymena Functions Upstream of M-Phase-Promoting Factor

Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.     

Meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor.

Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.     

In vivo facilitation of Tetrahymena group I intron splicing in Escherichia coli pre-ribosomal RNA.

The observation that the large ribosomal RNA intron of Tetrahymena is spliced 20-50-fold more rapidly in vivo than in vitro (Brehm SL, Cech TR, 1983, Biochemistry 22:2390-2397; Bass BL, Cech TR, 1984, Nature 308:820-826) suggests facilitation of RNA folding in vivo. To determine whether a specific group I splicing factor is required in Tetrahymena, the intron was inserted into the analogous position of the Escherichia coli 23S rRNA. We report that the intron is rapidly excised from pre-rRNA in bacteria and that the magnitude of the in vivo rate enhancement is similar to that in Tetrahymena. These results demonstrate that a species-specific protein is not required. Instead, a common mechanism of assisting RNA folding is sufficient to accelerate the removal of self-splicing introns from ribosomal RNA.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vito

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred with a 2.4 X 10(6) Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template-primers in other systems for the purpose of isolating replication proteins.     

Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro.

Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

Ciliate telomerase RNA structural features.

Telomerase RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. The RNA moiety contains a templating domain that directs the synthesis of a species-specific telomeric repeat and may also be important for enzyme structure and/or catalysis. Phylogenetic comparisons of telomerase RNA sequences from various Tetrahymena spp. and hypotrich ciliates have revealed two conserved secondary structure models that share many features. We have cloned and sequenced the telomerase RNA genes from an additional six Tetrahymena spp. (T. vorax, T. borealis, T. australis, T. silvana, T. capricornis and T. paravorax). Inclusion of these sequences, most notably that from T. paravorax, in a phylogenetic comparative analysis allowed us to more narrowly define structural elements that may be necessary for a minimal telomerase RNA. A primary sequence element, positioned 5' of the template and conserved between all previously known ciliate telomerase RNAs, has been reduced from 5'-(C)UGUCA-3' to the 4 nt sequence 5'-GUCA-3'. Conserved secondary structural features and the impact they have on the general organization of ciliate telomerase RNAs is discussed.     

The breakdown of Tetrahymena ribosomes in vivo. The effects of inhibitors.

1. When Tetrahymena were deprived of nutrients 50% of the polysomes disaggregated within 20 min and 20% of the total RNA broke down in 2 h. Ribosomal RNA accounted for 75% of the RNA breakdown. 2. RNA labelled by a long incubation with [14C]uridine was stable in growing cells and in the presence of actinomycin D, but broke down at the same rate as bulk RNA in starved cells. 3. The following substances inhibited the loss of RNA during starvation: cycloheximide (which inhibited both polysome disaggregation and protein synthesis), inhibitors of energy metabolism and puromycin (all of which caused polysome disaggregation and inhibited protein synthesis), and chloroquine and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK') (neither of which affected polysomes or protein synthesis). 4. Starvation appears to activate a ribosome degradation mechanism that may involve lysosomal and non-lysosomal enzymes.     

Murine splenocytes release an inhibitory peptide of lymphocyte DNA synthesis into serum-free culture medium

Murine splenocytes release a novel repressing activity of Concanavalin A-induced lymphocyte DNA synthesis. This activity was purified by gel filtration, ion exchange and thin layer chromatography. It was characterized to be a heat-stable peptide whose molecular weight was estimated to be about 1 000 daltons. The strong inhibition by this peptide was observed when added at early periods of the cell culture. This factor does not affect early RNA synthesis in lymphocytes, but nucleotide (UTP) incorporation at early periods was considerably decreased. In addition, although the factor inhibits drastically lymphocyte DNA synthesis, the cell viability is not changed significantly. These results suggest that the peptide suppresses the cell membrane function at early periods and specifically blocks the concomitant DNA synthesis.