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Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

Various observations cited at the beginning of this paper and in the Discussion suggested that phalloidine, a poisonous constituent of the green mushroom Amanita phalloides, would not be toxic by itself, but would be rendered toxic previously in the liver. Using a tritiated toxic derivative of phalloidine ([³H]desmethylphalloin) we found that: (a) in rats, 60% of the administered substance are excreted unchanged in the urine within the first day. A second radioactive substance of low concentration, moving more slowly in paper chromatography turned out as a product of the self-degradation of [³desmethylphalloin; (b) respectively 98% and 99.9% of the radioactivity were extracted with methanol from homogenates of livers of rats or mice which had received [³H]desmethylphalloin 1 to 2 hours before. Aqueous homogenates of liver of poisoned rats also contained radioactivity firmly bound to a high molecular weight substance as shown by gel chromatography. The radioactive substance was totally removed from the carrier presumably ribosomal material, by adding methanol, and was identified as [³]desmethylphalloin; (e) [³H]desmethylphalloin was not metabolized on incubation with oxygen and rat liver ribosomes in a NADPH-regenerating system. A radioactive substance extracted from liver microsomes of a poisoned rat also proved to be original toxin. As a result of these experiments it can be concluded that [³H]desmethylphalloin and most probably also phalloidine are not metabolized in the livers of rats and mice. [ABSTRACT FROM AUTHOR]

A technique which releases ribosomes from membranes without the use of detergent described in the preceding paper is shown to allow the separation of two distinct pools of poly(A)-containing RNA in microsomes. Thus, using preparations from mouse myeloma tissue-culture cells, essentially 100 % release of immunoglobulin mRNA occurs under conditions in which only 50-60 % of the total microsomal poly(A)-containing RNA is released. The RNA which is not released is principally mitochondrial ribosomal RNA and the method thus provides a good preparative procedure for this fraction. Messenger ribonucleoprotein particles involved in immunoglobulin biosynthesis have been prepared from the released material. The heavy-chain mRNA-containing particle sediments in a neutral sucrose gradient at about 22 S, and that containing the light-chain mRNA at about 18 S. The ribosome-release technique has been used for the rapid preparation of light-chain mRNA in good yield and at over 60 % purity after a single sucrose gradient centrifugation. Further purification has been achieved by polyacrylamide gel electrophoresis. Using [32P]RNA, the contaminants can be estimated directly at the chemical leveI using a fingerprint assay. [ABSTRACT FROM AUTHOR]

In this paper we demonstrate that two compounds which are known to enhance the permeability of membranes to potassium, valinomycin, a cyclic dodecapeptide and dicyclohexyl-18-crown-6, a macrocyclic polyether, inhibit protein synthesis in reticulocytes and that this inhibition is not primarily due to changes in the intracellular potassium concentration. No effect is obtained by adding the drugs to a cell free system and the inhibition observed is likely to be membrane mediated. Dicyclohexyl-18-crown-6 and low concentration of valinomycin (10 nM) act reversibly probably by interacting with the lipid phase of the membrane. This interaction in turn affects the protein synthesizing apparatus by affecting ribosomal functions. Ribosomes once isolated from treated cells are perfectly normal in structural and functional respects. High concentrations of valinomycin, which change the K+ gradient between the inside and the outside of the cell, affect the protein synthesizing apparatus of reticulocytes irreversibly by blocking incorporation of amino acids at the level of elongation or termination. Ribosomes isolated from cells treated with high valinomycin concentrations are mostly in the form of polysomes which are inactive in cell free protein synthesis. The high valinomycin block on the ribosomal fraction can be relieved either by washing ribosomes with high salt or with puromycin. It is believed that with high concentrations of valinomycin an inhibitor is released (probably from the membrane) and attached to the ribosomes. This high valinomycin effect is believed to by a supplementary phenomenon which when added to the effect of low valinomycin renders the inactivation of ribosomal functions irreversible.

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

As found previously, 70 to 75% of the RNA present in previtellogenic oocytes of Xenopus laevis (average diameter: 50 to 150 μm) is 5-S RNA and transfer RNA. In this paper, we show that most of the RNA of the small oocytes is stored in nucleoprotein particles sedimenting at 58 S and 42 S. These particles are no longer detectable in vitellogenic oocytes (average diameter: 375 to 1100 μm) which accumulate ribosomes very rapidly. The 58-S particles contain mainly high-molecular-weight RNA. This RNA has a base composition close to that of DNA. Its main component sediments at about 19 S. The 42-S particles contain more than half of the RNA and a quarter of the protein present in homogenates of small oocytes. The only RNA species found in the 42-S particles are 5-S RNA and tRNA. The molar ratio of tRNA to 5-S RNA in the 42-S particles is 3:1. 5-S RNA is distributed as follows in the small oocytes: less than 1% of the total cell content is bound to the ribosomes; about 50% is in the 42-S particles and the remainder is apparently free in the cell sap. By its base composition and its hybridization properties 5-S RNA from the 42-S particles cannot be distinguished from 5-S RNA of the cell sap and from 5-S RNA of somatic cells. About 900 of the total tRNA content of the oocyte is included in the 42-S particles. The remaining 10% is either free or bound to the ribosomes. No difference was noticed in the hybridization properties of tRNA which is inside the 42-S particles and tRNA which is outside these particles. The 42-S particles present in homogenates of previtellogenic oocytes are not artifacts. These particles are probably storage particles in which 5-S RNA and tRNA produced in excess during the initial period of oogenesis are kept for many months until the ribosomes begin to accumulate. The role of the 58-S particles remains unknown. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KCl concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase. Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers. [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [ABSTRACT FROM AUTHOR]

1. The distribution of ribosomal particles between the soluble and membrane fractions of exponential-phase cells of Bacillus licheniformis was studied. The cells were very gently lysed by a combined treatment with lysozyme and Brij 58 in 10 mM. Tris-buffer, pH 7.6, with 60 mM K+, 10 mM Mg2+, 0.5 mM spermine and 0.75 mM spermidine. The proportion of the ribosomal material present in the membrane fraction was estimated after solubilization of the membranes by the combined action of Brij 58, deoxyribonuclease and lipase. 2. Under these conditions of lysis 96% of the total ribosomal material, consisting of subunits (12%) monosomes (20%) and polyribosomes (68%) and most of the cellular DNA, is found to be membrane-bound. 3. In the soluble fraction, containing only 4% of the total cellular amount of the ribosomal material, only subunits and 70S particles and no polyribosomes are found. 4. DNA does not seem to be involved in the binding of the ribosomal material to the membranes. 5. The results will be discussed considering the question of whether the distribution described in this paper represents the true distribution in vivo of ribosomes. [ABSTRACT FROM AUTHOR]