Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro

We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 10⁵ and 24 × 10⁵. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 10³ and 320 × 10³. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Synthesis of messenger and ribosomal RNA precursors in isolated nuclei of the cellular slime mold Dictyostelium discoideum

When incubated with all four ribonucleoside triphosphates, isolated nuclei of the cellular slime mold, Dictyostelium discoideum, will synthesize RNA linearly for 10 to 50 minutes, depending on the salt concentration of the reaction. A fraction (10 to 30%) of the RNA labeled in isolated nuclei binds to immobilized polyuridylic acid. By the following criteria this RNA species is identical to the messenger RNA precursor characterized in whole cells: (a) both contain polyadenylic acid sequences of identical size; (b) they have the same base composition; (c) they have the same mean size as determined by dimethylsulfoxide—sucrose centrifugation; (d) they renature to excess nuclear DNA with similar kinetics; and (e) synthesis of both RNAs is resistant to 2 to 3 μg of actinomycin D/ml. Two independent RNA polymerase activities appear to synthesize poly(A)-containing RNA in isolated nuclei. One is equally active at 0.01 m-KCl and 0.25 m-KCl and is resistant to α-amanitin; the other is considerably more active at the higher salt concentration and is sensitive to α-amanitin. By the criteria of sedimentation coefficients, base composition and sensitivity of synthesis to actinomycin D, the remainder (70 to 90%) of the RNA synthesized by isolated nuclei was identical to cellular ribosomal RNA or its precursors.     

The nature of the changes in the pattern of RNA synthesis by the juvenile hormone analogue altosid

The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-¹⁴C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

Effects of actinomycin D on ribosomal RNA and protein synthesis as revealed by high resolution autoradiography.

Incorporation of tritiated amino acids and uridine was studied in untreated and actinomycin D treated HeLa cells by high resolution autoradiography. Results showed a non-selective inhibition of protein synthesis by actinomycin, as measured by the decrease in radioactive amino acid uptake. When cells pretreated with actinomycin D were incubated with radioactive amino acids and uridine, amino acid uptake in the nucleolus still occurred, while uridine uptake was almost completely eliminated. These findings suggest that in the absence of ribosomal RNA precursor synthesis, nucleolar protein synthesis continues to some extent, and that this protein is transported to the nucleolus.     

Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins in Escherichia coli.

Summary The rates of functional decay of messenger RNA coding for total soluble, total ribosomal and individual ribosomal proteins were measured in Escherichia coli strain AS-19, at 30^o. This was accomplished by blocking RNA synthesis with the inhibitor thiolutin and measuring residual protein synthesis at various times thereafter. The data obtained expressed as a decay constant (Hartwell and Magasanik, 1963) show that both total soluble and total ribosomal protein decay with similar rates (K ₂=0.64 and 0.61 respectively) which are slightly faster than the decay rate of -galactosidse (k ₂=0.43) under these conditions. All the individual ribosomal proteins appear to comprise a population of cistrons whose individual mRNA's decay with very similar rates with the possible exception of protein L3, whose mRNA appears consistently to decay very rapidly.Additional data on the stability of the total soluble and total ribosomal proteins during thiolutin treatment (that is, proteins synthesized in the absence of concommitant ribosomal RNA synthesis) fail to demonstrate any marked difference between these two protein populations. Examination of the stability of the individual ribosomal proteins however, reveals that some are degraded up to 35% in 15 min of thiolutin exposure, some to about 15% and some appear to be completely stable. In general, a degree of correlation exists between the stability of a given protein and the observed decay rate of its messenger RNA. This observation may explain in part the spread among the rates of mRNA decay. Nevertheless, we conclude that although degradation is occurring, it is not sufficient to alter the main conclusion that the rates of functional decay of mRNA cistrons coding for the ribosomal proteins are very similar.