Rapidly-labeled RNA Research Articles

Effects of postmicrosomal rat-liver fractions on the activity of cell-free amino acid incorporating systems

The X fraction, obtained by centrifuging rat-liver cell sap for 12–15 h at 105 000 × g, markedly stimulated the endogenous and poly (U)-directed amino acid incorporation by a cell-free rat-liver system containing preincubated or non-preincubated ribosomes. Density gradient centrifugation resolved the X fraction into two small, ribonucleoprotein-containing subfractions, with sedimentation coefficients of approx. 55 S and 40 S, and one larger, light subfraction, containing protein and some tRNA. Although rapidly-labelled RNA, probably associated with some messenger activity, accumulated in the 40 S subfraction, the stimulatory effect was concentrated in the light subfraction. Removal of the tRNA did not appreciably reduce this effect, provided that the tRNA content of the test sytem was satisfactory. Aminoacyl-tRNA synthetases did not selectively accumulate in the X fraction. On the other hand, this fraction contained most of the aminoacyl transferase activity of the cell sap. The experimental evidence suggests that this activity contributes to its stimulatory effect in the commonly used test systems.

Attachment of rapidly labelled RNA to polysomes in the absence of ribosomal RNA synthesis during normal cell differentiation.

Anucleotate mutant tadpoles of Xenopus laevis synthesize rapidly-labelled RNA but no detectable ribosomal RNA or ribosomes. Experiments demonstrate that high molecular weight RNA synthesized by these mutants attaches to polysomes active in protein synthesis.

Base composition of rapidly-labelled RNA in [formula omitted] undergoing thymineless death

Base composition of rapidly-labelled RNA in [formula omitted] undergoing thymineless death

RNA synthesis in liver and heart of growing and adult mice

Cells of liver and heart of 12-day-old mice actively synthesize DNA, whereas in 5-month-old mice, the rate of DNA synthesis in these two organs is reduced to a negligible level. Livers and hearts of 12-day- and 5-month-old mice were therefore compared in order to determine differences in some aspects of RNA synthesis. RNA synthesis was studied by determining nucleic acid concentration, incorporation in vivo of radioactive cytidine into whole cells and into subcellular fractions, the size of the endogenous cytidine pool, RNA turnover over a period of 30 days, the effect of actinomycin D and sucrose gradient analysis of phenol-extracted RNA. The rate of synthesis of rapidly-labeled RNA was higher in 12-day-old than in 5-month-old tissues (50 % higher in liver and 2.4 times higher in heart). The specific activity of the cytidine precursor pool was doubled in the adult tissues. In addition, the turnover of RNA was decreased in the adult heart, and the concentration of RNA per cell in the adult liver was almost twice the amount found in growing liver. Other parameters of RNA synthesis were substantially similar in growing and adult livers an hearts.

Intracellular distribution and base composition of rapidly-labelled RNA fractions in Chlorella

Intracellular distribution and base composition of rapidly-labelled RNA fractions in Chlorella

RNA Synthesis in Normal Leucocytes, Leukaemia and Macroglobulinaemia

DIFFERENCES in the sedimentation characteristics and incorporation of radioactive precursors in the RNA of HeLa cells, ascites tumour cells and rat liver tissue have been reported1–3. Marks et al.4, in the course of extensive studies on RNA synthesis in reticulocytes, reported that rapidly-labelled RNA was absent from reticulocytes while it could be detected in leucocytes from normal subjects and patients with chronic lymphocytic leukaemia5. The present report describes patterns of RNA synthesis in the leucocytes from patients with leukaemia or macro-globulinaemia and from normal subjects.

Studies on rapidly labelled ribonucleic acid in HeLa cells

A study of the sedimentation pattern in sucrose gradients of rapifly-labelled RNA (5–30 min labelling with [ 14C]adenine) was done on whole extracts of HeLa cells. Two regions of radioactivity have been found: a lighter region sedimenting in the range of 4 to 20-S particles and a heavier heterogeneous region sedimenting from about 30 S to the bottom of the gradients even when 30 % sucrose was used. The nature of the various types of RNA's likely to sediment in these regions is discussed. The heavier heterogeneous region contained very small amounts of DNA, but no definite evidence for the existence of DNA-RNA hybrids was obtained. On the other hand, the heavy heterogeneous region is active in amino acid incorporation and consisted of a variety of polysomes; their characterization by electron microscopy is described in a further paper. These polysomes, after a short labelling period, consist of unlabelled 74-S ribosomes to which is attached rapidly-labelled RNA; it was shown that it takes roughly 5 min for [ 14C]adenine to become integrated into RNA sedimenting with the polysomes. Comparison of autoradiographic and sedimentation analysis shows that a large proportion of the polysomes might be formed in the cell nucleus.

Effect of puromycin on RNA synthesis in HeLa cells

Sedimentation analyses of RNA from HeLa cells have revealed that rapidly-labeled RNA sediments faster in a sucrose gradient than ribosomal RNA ( Scherrer and Darnell, 1962). This RNA fraction is confined to the nucleus and its synthesis is prevented by a low level (0.01 μg/ml) of actinomycin D ( Tamaoki and Mueller, 1962). Recent papers by Perry (1963) and Scherrer et al. (1963) indicate that at least two kinds of RNA are present in the rapidly sedimenting fraction: (1) a precursor RNA which converts into ribosomal RNA in the presence of actinomycin D, and (2) DNA-like RNA which is capable of forming a hybrid with HeLa cell DNA and stimulates amino acid incorporation in a cell-free E. coli system. In this paper, the effect of puromycin on RNA metabolism in HeLa cells will be presented. The data illustrate that the synthesis and turnover of a fraction of the heavy RNA continued in the presence of puromycin, whereas the introduction of guanine-8-C 14 into other classes of RNA (28 and 16s) was blocked by puromycin.