F.T. De Castro

Characterization of messenger RNA populations of Crithidia fasciculata

Abstract Cells of Crithidia fasciculata taken from exponentially growing cultures display a relatively high content of poly(A) RNA (roughly 45% of the total) in a nonpolysomal compartment. The proportion of this fraction is significantly higher than that of the free ribosomal units. This RNA is translationally competent and is indistinguishable from polysomal mRNA by various criteria. The significance of these findings is discussed with respect to the protein synthesis capability of the cell and the metabolic relationships between the cytoplasmic compartments of mRNA.

Nalidixic acid and macromolecular metabolism in Tetrahymena pyriformis: effects on protein synthesis.

A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis.

The effect of temperature on nucleotide pool formation in Tetrahymena pyriformis.

Warming of exponentially growing T. pyriformis to 34°C results in severe inhibition of nucleotide pool formation. The utilization of the pool for stable RNA synthesis is poorly affected at the high temperature. It thus appears that the synthesis and processing of ribosomal RNA precursors are not primarily impaired at 34°C.

Poly(A)+ RNA metabolism during change of physiological state of Tetrahymena pyriformis cells

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.

Dynamic aspects of cytoplasmic poly(A)+ RNA of Tetrahymena pyriformis.

In the present work a study was made of the compartmentalization of the poly(A)+ RNA populations during the cultural development of cells of T. pyriformis that were pre-starved or derived from stationary cultures. It was found that the poly(A)+ RNA content increases when the cells change from stationary to lag phase. The increase in RNA poly(A)+ is manifested exclusively in the polysome compartment. The level of poly(A)+ RNA in the cytoplasmic non-polysomal compartment does not change. The increase in poly(A)+ RNA is concomitant with an expansion of the polysomes. Pre-starved cells initiate polysome formation soon after being transferred to a growing medium. During this time the poly(A)+ RNA content of the non-polysomal compartment decreases and that of polysomes increases in close proportion. Not only in the starved but also in stationary cells and in those that are beginning to grow, the proportion of poly(A)+ RNA in mRNP is higher than in the polysomes. These data are interpreted as indicating that cells of T. pyriformis, derived from stationary cultures are dependent on RNA synthesis for polysome formation; on the other hand, pre-starved cells use preformed non-polysomal poly(A)+ RNA for the same purpose, in the beginning of the cultural development.

Effect of the injection of ribosomes and RNA from Crithidia fasciculata on the experimental infection of mice by Trypanosoma cruzi.

Immunization of mice with Crithidia fasciculata (live suspension, ribosomal fraction and purified RNA) induced a certain degree of protection (decrease of parasitaemia) against infection with Trypanosoma cruzi.

The dynamic state of Tetrahymena pyriformis cytosol proteins during culture development

The dynamic state of cellular proteins has been demonstrated extensively in recent years [l-4] . The renewal of protein populations occurs apparently under strict control and appears to be linked to changes in physiological conditions of the cells [5-71. Although the knowledge of the mechanisms of this regulation is far from being understood, it has become clear that the turnover of cellular proteins is a highly selective process [8]. In the past the belief has been that microorganisms proteins were stable during growth and became subject to a degradative metabolism only under resting conditions. This argument has been recently questioned. Experimental evidence is compatible with significant protein breakdown during growth [9,10]. The present paper deals with an experimental study of protein stability in cells of Tetrahymena pyrijiormis that initiate a new cultural growth-cycle. It is shown that under these conditions the proteins of higher molecular weight turnover at a relatively high rate, whereas those of small size accumulate.