Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis.

We have characterized an embryonic antigen on the surface of chick erythrocytes using immunochemical electron microscopy. An indirect surface labeling technique (hemocyanin conjugated to goat antirabbit IgG and specific antisera prepared in rabbits) revealed that the antigenic sites, at hatching, nearly saturate the surface of erythrocytes with hemocyanin markers. The number of antigenic sites gradually decreases with age, and the antigen can no longer be detected at 7 months. Further, the antigen has been detected on the very earliest primitive erythrocytes which form in the extra-embryonic mesenchyme before circulation begins. The embryonic antigen appears to be firmly associated with the erythrocyte surface and cannot be removed by extensive washing either with phosphate-buffered saline or with EDTA. Labeling unfixed cells at 37 °C produces clustering of the surface markers, suggesting that the antigen is associated with a membrane component which is fairly free to move in the plane of the membrane. In addition, the erythrocytes from newly hatched chicks were found to agglutinate more readily with several different lectins, particularly Concanavalin A (ConA), than did the erythrocytes from adults. Three times more ConA is bound to chick erythrocytes than to adult erythrocytes, as estimated by electron microscopy. Although this difference in lectin binding suggests that the ConA-binding sites might be related to the embryonic antigen, the sugars known to block lectin-induced hemagglutination had no blocking effect on antiserum-induced agglutination or on antibody binding, as visualized by the electron microscope technique. Also, ConA binding was not inhibited by treatment of the chick erythrocytes with the specific antiserum.     

RNA synthesis and cell division in cold-synchronized cells of Tetrahymena pyriformis

Cells of Tetrahymena pyriformis have been cold-synchronized using a repetitive cycle of six, two-hour cold shocks (9.5°C) alternating with decreasing periods (60–30 minutes) at 28°C. This system gives a maximum division index of 70–80% occurring at 90 minutes from the end of the last synchronizing cold-treatment (EC). Examination of the division sensitivity of these cells to actinomycin D applied continuously at ten-minute intervals from EC reveals that division is essentially blocked until approximately 40 minutes past EC, after which a rapid decrease is sensitivity to the inhibitor occurs. Coinciding with this period of high sensitivity is the occurrence of a peak of C¹⁴ uridine incorporation at 40 minutes past EC. Inhibition of this peak is correlated with an inhibition of division, whereas strong inhibition of RNA synthesis beyond 60 minutes past EC has little effect on division activity. The similarity of these findings with those of the heat-synchronized system is discussed with the suggestion that both heat- and cold-synchronizing treatments result in the synchronous resynthesis of a division-associated fraction of RNA.     

Ribosomal proteins in growing and starved Tetrahymena pyriformis. Starvation-induced phosphorylation of ribosomal proteins.

The complements of ribosomal proteins in growing and starved cells of Tetrahymena pyriformis strain GL were examined by two-dimensional gel electrophoresis. In growing cells, the 40-S ribosomal subunit contained 30 proteins, 4 of which migrated toward the anode at pH 8.6, while the 60-S ribosomal subunit contained 46 proteins, 9 of which migrated toward the anode at pH 8.6. When exponentially growing cells were transferred into a non-nutrient medium pronounced phosphorylation of a single 40-S ribosomal subunit protein, S6, was induced. The phosphorylation was very specific; more than 99.5% of the [32P]phosphate incorporated into ribosomal proteins was associated with S6. Phosphate was incorporated into S6 as O-phosphoserine and O-phosphothreonine. Two-dimensional gel electrophoresis indicated that the complement of proteins associated with the ribosomes isolated from starved cells differed from that of growing cells. Careful examination, however, suggested that except for the phosphorylation of certain ribosomal proteins in starved cells, the observed differences did not reflect starvation-induced changes in vivo, but most probably different levels of artifactual modifications (limited proteolysis) during the preparation of the ribosomes.     

Site of tubulin synthesis in Tetrahymena pyriformis

Free and membrane-bound polysomes were isolated from the protozoa Tetrahymena pyriformis, and the contribution of these two types of polysomes to tubulin synthesis were studied using immunoprecipitation of the 35S-translational products in a rabbit reticulocyte lysate. One-dimensional electrophoretic analysis shows that tubulin is synthesized by polyadenylated RNA isolated from free and membrane-bound polysomes. Non-polyadenylated RNAs of free polysomes are also able to direct tubulin synthesis. Two-dimensional electrophoretic analysis using O'Farrell's system confirms these results and also reveals the existence of the alpha- and beta-tubulin subunits.     

The genes for 5 S ribosomal RNA and transfer RNA in Tetrahymena pyriformis.

In the present communication a characterization of the 5 S rRNA genes and the tRNA genes of Tetrahymena pyriformis has been performed. The number of 5 S rRNA and tRNA genes in the macromolecular DNA has been established. Furthermore no sequence homology is observed for these genes. The number of both types of genes does not change significantly under starvation conditions. The genomic organization of the 5 S rRNA and tRNA genes has been investigated. From in vivo replication studies it is concluded, that replication of both 5 S rRNA and tRNA genes takes place throughout the whole S-period.     

Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis.

Saturating concentrations of the initiation-specific inhibitors poly(I) and T-2 toxin inhibit protein synthesis by over 35% and cause ribosome 'run-off' from the polyribosomes. The elongation-specific inhibitor cycloheximide totally prevents protein synthesis and 'freezes' the ribosomes in the pattern of unincubated controls. These results prove that our Tetrahymena extracts are capable of protein-synthesis initiation, a conclusion which is confirmed by a 30% inhibition of synthesis by the mRNA cap analogue, 7-methylguanosine 5'-monophosphate.     

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.     

Phosphorylation of RNA polymerase I-associated polypeptides of Tetrahymena pyriformis

In the ciliate Tetrahymena pyriformis phosphorylation of RNA polymerase I [EC 2.7.7.6] and of polymerase-associated polypeptides was investigated in growing and growth-arrested cultures which differ widely in their rates of rRNA synthesis. Several putative subunits of RNA polymerase I (of 180, 21.5, and 19.5 kDa) and a polymerase-associated polypeptide of 27 kDa were found to be phosphorylated, independent of the growth conditions. However, an additional enzyme-associated polypeptide of 26 kDa was intensively labeled with 32P only after arrestment of growth by starvation. The molar quantities of both phosphorylated, enzyme-associated polypeptides thereby did not differ in growing and growth-arrested cultures, and the specific 32P-labeling of cellular ATP remained nearly unchanged under the different culture conditions. These findings indicate a selective, reversible phosphorylation of the RNA polymerase I-associated 26 kDa polypeptide correlated with conditions of repressed rRNA synthesis induced by the starvation procedure. In vitro phosphorylation in macronuclei isolated from growing and growth-arrested cultures using [gamma-32P]ATP revealed essentially the same pattern of labeling of the enzyme-associated polypeptides of 27 and 26 kDa as it was found in vivo.     

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.     

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.