RNA synthesis in starved deciliated Tetrahymena pyriformis.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.     

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.     

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.     

Induction of DNA polymerase in Tetrahymena pyriformis

A number of different treatments will induce DNA polymerase in Tetrahymena pyriformis. In the present paper we have studied the induction of DNA polymerase by the inhibitor of DNA synthesis methotrexate plus uridine (M+U) and UV irradiation (after incorporation of bromodeoxyuridine (BUdR) into DNA) in synchronized Tetrahymena populations. We have found that M+U must be present in the nuclear S-period in order to induce the polymerase, and that a dose of UV irradiation which is too low to induce DNA polymerase will do so if the damaging effect of the irradiation is enhanced by incorporation of BUdR into DNA.     

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.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

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.