The Amount of Ribosomal RNA Genes in Tetrahymena pyriformis in Different Physiological States

Abstract

DNA-RNA hybridization studies have been performed to determine the amount of rRNA genes present in purified nuclear DNA of Tetrahymena pyriformis cultured under various conditions of growth. It was found that 0.32O/, of the nuclear DNA isolated from exponentially growing cells hybridizes with an equimolar mixture of 17-S and 25-S rRNA. Nuclear DNA isolated from starved cells and from cells in the stationary growth phase hybridizes 30-40°/, less with rRNA than the nuclear DNA of the exponentially growing cells. This variation of the amount of rRNA genes to the total amount of nuclear DNA suggests that the replication of the rRNA genes and the rest of the nuclear DNA are under separate controls. With the possible exception of a minor homology between 17-5 and 25-S rRNA, the saturation and competition experiments indicate that there are separate genes for the two classes of rRNA.Many studies on nucleolar morphology in Tetrahymena pyriformis have shown the nucleoli to be structures capable of reflecting nutritional changes by undergoing fusion and disaggregation. Thus, starvation, heat synchronization, treatment with pfluorophenylalanine or actinomycin D and decellerating growth prior to stationary phase conditions are known to induce the multiple, peripherally located, small nucleoli in the macronuclei of exponentially growing Tetrahymena to aggregate to a few large fusion bodies [ 1 -51, When stationary phase cells or starved cells are inoculated into fresh growth medium the process of fusion body formation is reversed. It is not apparent from cytological observations whether changes in nucleolar organization and total surface area are associated with changes in total nucleolar mass or nucleolar DNA. Nucleolar DNA in Tetrahymena has recently been shown to include the rRNA genes (unpublished results) in agreement with observations in other organisms [6]. Furthermore, biochemical analyses have demonstrated that net RNA accumulation declines in parallel with the formation of fusion bodies and that net RNA accumulation begins when the fusion bodies dissociate into small nucleoli [1,7].I n the study reported here we have used the technique of DNA-RNA hybridization to test if the change in net RNA synthesis (850/, of which is ribosomal) could be related to a change in the amount Abbreviations. rRNA, ribosomal ribonucleic acid; sarkosyl, sodium lauryl-saroosinate.of rRNA genes in the cell. I n addition the hybridization technique was used to evaluate sequence homologies between the two classes of rRNA. MATERIALS AND METHODS Culture MethodsTetrahymenu pyriformis, amicronucleate strain GL, was grown axenically in complex medium containing 2 O / , proteose peptone (Difco) with 0.lo/, liver extract fraction L (Nutritional Biochemical Co.). Cultures were grown a t 22 "C in 1-3-1 batches in 6-1 Erlenmeyer flasks with continuous magnetic stirring under vigorous aeration produced by bubbling filtered air through the medium. Cell densities were measured electronically using a Coulter counter. The cultures were in the midexponenti...

Nucleocytoplasmic translocation of RNA in Tetrahymena pyriformis and its inhibition by actinomycin D and cycloheximide

“…the average duration time of potential cytoplasmic RN A molecules in the nucleus) of nuclear RNA molecules of about 3.7 min which is in accord with the calculations of turnover and processing times for macronuclear prerRNA reported by other authors [15,31,36,57]. From the amount of ribosomal units synthesized and translocated into the cytoplasm per min (about 5x 10 5 ; see also [15]), the number of rRN A cistrons in the macronucleus (170-200; refs [53,54]), the average number of simultaneously growing chains per cistron (ca 100; assuming a situation similar to what has been shown in various other organisms, 55; for refs, see [38]), the chain growth rate of rRNA can be estimated to be at least 50 nucleotides per second (for similar data in other eukaryotes see [56]). This means that the apparent rRNA precursor molecule (2.2x 10 6 D) needs max.…”

Studies on the Amount and Location of the tRNA and 5-S rRNA Genes in Tetrahymena pyriformis GL

“…Centrifugation was performed with a type-40 fixedangle rotor in a Beckman L2-65 ultracentrifuge at 22 "C and 37000 rev./min for 18 h. Fractions were collected from the bottom of the centrifuge tube. The DNA-containing fractions were pooled and passed through a Sephadex G-50 column [11]. The absorbance ratio at 260/280nm of the nuclear DNA was 1.83-1.91 and the hyperchromicity, when DNA was denatured with alkali was about 25 %.…”

Replication and functions of macronuclear DNA in synchronously growing populations of Tetrahymena pyriformis

“…However, macro-and micronuclei differ in their amounts of ribosomal genes. The number of genes coding for 17S and 25S ribosomal RNA is amplified in the macronucleus, and the relative amplification per genome is around 200 fold (38,115). These genes are localized in the numerous (500-1000) nucleoli which are positioned in close contact with the macronuclear membrane.…”