Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.     

Microsomal components in relation to amino acid incorporation by preparations from the developing rat brain

1. After incorporation of [(14)C]valine in vitro, cerebral microsomes were separated into membrane-bound and free ribosomes by sucrose-density-gradient centrifugation. 2. In preparations from both 4-day-old and adult rats, free and bound ribosomes incorporated [(14)C]valine. Free ribosomes could be found as polysomes, which were highly active. 3. Microsomes labelled with [(14)C]valine in vitro were fractionated after deoxycholate treatment into a preliminary sediment, sedimented at 105000g (5min.), and ribonucleoprotein particles, sedimented at 150000g (70min.), to determine the role of membrane-bound ribosomes. In the adult the ribonucleoprotein particles retained most of the radioactivity, whereas in the young the preliminary sediment was as highly labelled as the ribonucleoprotein particles. 4. The labelled preliminary sediment from young preparations was both ribonuclease- and deoxycholate-resistant, and the nature of this material is discussed in terms of a possible structural component of microsomal membranes.     

Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.     

Inhibition of bacterial growth by metal salts. The accumulation of ribonucleic acid during inhibition of Escherichia coli by cobalt chloride

During inhibition of the growth of Escherichia coli by cobalt chloride protein synthesis was decreased more than the synthesis of RNA. Three species of particle accumulated during the incubation. These had sedimentation coefficients of about 44s, 33s and 23s in tris buffer containing 10 mm-magnesium acetate and 100 mm-potassium chloride, but their sedimentation properties were susceptible to changes in buffer composition. The particles contained RNA but were more readily degraded by ribonuclease than were the ribosomes. RNA isolated from the particles differed slightly in sedimentation properties from the major species of ribosomal RNA. The particles are likely to be closely related to ribosome precursors that have been detected in other circumstances. Changes in the polyribosome fraction during inhibition by cobalt chloride, nickel chloride and chloramphenicol provided further evidence that inhibition by Co(2+) involves specific effects on the protein-synthesizing machinery.     

Degradation of heparin in mouse mastocytoma tissue

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) of approx. 60000-70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate-protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [(35)S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating (35)S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting (14)C-labelled chondroitin sulphate to the same procedure.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions

The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.     

A new method for the preparation of uniformly 14C-labelled compounds by using Anacystis nidulans

1. Growth conditions were devised which enabled Anacystis nidulans to be grown on high specific radioactivity (14)CO(2) (at over 98% isotopic abundance of (14)C) in a closed system with greater than 85% substrate utilization. 2. The DNA and RNA content of A. nidulans was estimated by various methods and compared with that of Chlorella pyrenoidosa. 3. A procedure was developed for the quantitative extraction and separation of the DNA and RNA from A. nidulans. 4. The fractionation and analysis of the DNA and RNA includes methods for the preparation of the uniformly (14)C-labelled deoxyribonucleoside monophosphates and the uniformly (14)C-labelled ribonucleoside monophosphates. 5. The base 6-methylaminopurine was identified as a component of the DNA. 6. The preparation and total yield of uniformly (14)C-labelled amino acids from A. nidulans are also briefly described. The results are discussed in relation to the use of other micro-organisms for the preparation of compounds labelled with (14)C.     

Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85-95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [(14)C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction.     

Transfer of free and esterified cholesterol from low-density lipoproteins and high-density lipoproteins to human adipocytes

Cholesterol stored in human adipose tissue is derived from circulating lipoproteins. To delineate the cholesterol transport function of LDL and HDL, the movement of radiolabelled esterified cholesterol and free cholesterol from labelled LDL and HDL to human adipocytes was examined in the present study. LDL and HDL were enriched and labelled in esterified cholesterol with [14C]cholesterol by the action of plasma lipid transfer proteins and lecithin-cholesterol acyltransferase. Doubly labelled (3H,14C) LDL and HDL were prepared by exchanging free [3H]cholesterol into the 14C-labelled lipoproteins. 14C-labelled lipoprotein and 3H-labelled lipoprotein were also prepared separately and mixed to yield a mixed doubly labelled lipoprotein. Relative to the total amount added, proportionally more free than esterified cholesterol was transferred to the adipocytes upon incubation with any doubly labelled LDL and HDL. The calculated mass of free and esterified cholesterol transferred, however, varied with different labelled lipoproteins. 3H- and 14C-labelled LDL or HDL transferred 2-3-fold more esterified than free cholesterol while the reverse occurred with the mixed doubly labelled LDL or HDL. Thus, free cholesterol-depleted particles preferentially transferred cholesterol ester to the fat cells. In the presence of the homologous unlabelled native lipoprotein, the transfers of free and esterified cholesterol from labelled LDL or HDL were specifically inhibited. Selective transfer of esterified cholesterol relative to apoprotein was also observed when esterified cholesterol uptake from both LDL and HDL was assayed along with the binding of 125I-labelled lipoprotein. The cellular accumulation of cholesterol ether-labelled HDL (a non-hydrolyzable analogue of cholesterol ester) exceeded that of cholesterol ester consistent with significant hydrolysis of the latter physiological substrate. These results demonstrate preferential transfer of free cholesterol and esterified cholesterol over apoprotein for both LDL and HDL in human adipocytes. Furthermore, the data suggest that the cholesterol ester transport function of LDL and HDL can be enhanced by free cholesterol depletion and cholesterol ester enrichment of the particles, and affirms a role for adipose tissue in the metabolism of lipid-modified lipoproteins.     

The biosynthesis of basement-membrane collagen by isolated rat glomeruli.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).     

Translation of rat kidney mRNA after cadmium administration

1. Twenty-four hours after the administration of Cd2+ (11 mumol/kg body weight) to rats, the kidneys were removed and the RNA was extracted from the polysomes and used to prepare poly(A) RNA. 2. The poly(A)+ RNA was translated in rabbit reticulocyte lysates containing different labelled amino acids as precursors and the resultant proteins were separated by polyacrylamide gel electrophoresis. 3. The labelling of the proteins was similar using poly(A)+ RNA obtained from control and Cd2+ treated rats except for two proteins. 4. Regardless of labelled precursor used, proteins of mobility in sodium dodecylsulphate electrophoresis of mol. wt 50,000 contained approx twice as much radioactivity using the RNA from the kidney of treated rats. 5. Using labelled leucine, lysine, and cysteine, but not labelled phenylalanine or histidine, proteins of mobility in sodium dodecylsulphate electrophoresis of mol. wt 10,000 contained approx twice as much radioactivity using the RNA from the kidney of the Cd2+ treated rats. These results and the results following carboxymethylation of the proteins prior to electrophoresis, together with the results from co-electrophoresis of the products [125-I]-labelled liver metallothionein support the view that the poly(A)+ RNA contains kidney mRNA for metallothionein.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

The breakdown of Tetrahymena ribosomes in vivo. The effects of inhibitors.

1. When Tetrahymena were deprived of nutrients 50% of the polysomes disaggregated within 20 min and 20% of the total RNA broke down in 2 h. Ribosomal RNA accounted for 75% of the RNA breakdown. 2. RNA labelled by a long incubation with [14C]uridine was stable in growing cells and in the presence of actinomycin D, but broke down at the same rate as bulk RNA in starved cells. 3. The following substances inhibited the loss of RNA during starvation: cycloheximide (which inhibited both polysome disaggregation and protein synthesis), inhibitors of energy metabolism and puromycin (all of which caused polysome disaggregation and inhibited protein synthesis), and chloroquine and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK') (neither of which affected polysomes or protein synthesis). 4. Starvation appears to activate a ribosome degradation mechanism that may involve lysosomal and non-lysosomal enzymes.     

Structural characterization of polysomal poly(A)-protein particles in rat liver

Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5--13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic ribonuclease treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)-protein particles showed a broad distribution of about 70--290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)-protein particles banded in a density range of 1.30--1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated six polypeptides with molecular weights of 50 000, 54 000, 58 000, 63 000, 76 000 and 90 000 in the poly(A)-protein particles, but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90 000-molecular-weight component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde-fixed particles with ribonuclease T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the electron micrographs the shadowed poly(A)-protein particles appeared in a globular, somewhat elongated form and were mostly 14-18 nm in diameter. On the basis of the results a model for the 'average' 9-S particles was constructed.     

Loss of the polyadenylate segment from mammalian messenger RNA: Selective cleavage of this sequence from polyribosomes

Treatment of mouse sarcoma 180 ascites cell polysomes with low levels of micrococcal nuclease, under conditions that cause relatively little fragmentation of the messenger RNA chains, results in considerable loss of poly(A) from these chains. This treatment generates a substantial amount of functional poly(A)-lacking mRNA. Brief incubation of cytoplasmic extracts of the ascites cells, and of mouse liver extracts, has similar effects on the polysomes present in the extracts and on the generation of poly(A)-lacking mRNA chains.The poly(A) segment is released from the polysomes treated with micrococcal nuclease as a nucleoprotein complex, and is protected from the action of the enzyme because of its association with protein. There is considerable poly(A) hydrolysis in incubated ascites cell extracts, and accumulation of a poly(A)-protein complex does not take place in this case. The liver extracts have little poly(A)-hydrolyzing activity, and free poly(A)-protein complexes are observed in these extracts.The poly(A)-cleavage process shows evidence of considerable selectivity. The newly synthesized mRNA population is more susceptible to this process than is the steady-state population. Moreover, only a portion of the steady-state mRNA loses its poly(A) readily upon incubation with micrococcal nuclease. Two-dimensional gel electrophoresis of translation products from total and poly(A)-lacking polysomal RNA preparations shows that not all mRNA species lose their poly(A) upon incubation of polysomes in ascites cell extracts. The sensitive population resembles the normal population of translatable poly (A)-lacking mRNA that is obtained from untreated polysomes. Individual species within this population show wide differences in their degree of susceptibility to the poly(A)-release process in vitro. Analysis by one-dimensional gel electrophoresis indicates that the same general population is generated by the incubation of cytoplasmic extracts and by the treatment of polysomes with micrococcal nuclease.It is suggested that the 3′ non-coding region of mRNA in polysomes is particularly sensitive to endonucleolytic cleavage, and that loss of poly(A) via this cleavage may be a normal cellular process. The diversity in nucleotide sequence and in overall configuration in this region could provide a basis for the observed differences in susceptibility to cleavage by nucleases.     

Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

1. The incorporation of [5-(3)H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (;milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10-14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.     

The effect of 4-dimethylamino-3′-methylazobenzene on 14C-labelled amino acid incorporation by rat-liver polysome preparations

1. The incorporation of (14)C-labelled amino acids into polysomal protein was studied in a system consisting of polysomes and pH5 enzyme obtained 4 and 40hr. after a single intraperitoneal injection of 4-dimethylamino-3'-methylazobenzene. Labelling of the polysome fraction of preparations of both the 4hr.-treated and 40hr.-treated rats was considerably higher than in the normal control. 2. In further experiments on protein synthesis by polysomes from azo-dye-treated rats, the effect of replacing pH5 enzyme with cell sap was studied. Incorporation of (14)C-labelled amino acids into polysomal protein was similar to that of the control. 3. Aggregate size of polysomes obtained from rats injected previously with 4-dimethylamino-3'-methylazobenzene was studied by sucrose-gradient centrifugation. Polysomes prepared at 4hr. after azo-dye administration contained a considerable amount of large aggregates (approx. 700s), whereas at 40hr. after administration of the azo-dye the amount of large aggregates was less than in the control. 4. Determination of the ultraviolet spectra of polysome preparations from both normal and azo-dye-treated rats revealed no difference between the preparations. On the other hand, the ultraviolet spectra of cell-sap fractions from the different preparations showed that there is a definite shift in the absorption maximum from 272mmu (normal) to 267mmu, 40hr. after treatment, with an intermediate value of 270mmu for the preparation from 4hr.-treated rats. The absorption minimum changes from 250mmu (normal) to 245mmu for the preparation from 40hr.-treated rats.