Non-involvement of nucleolytic activities in the early effect of dimethylnitrosamine on the content of free mRNA in mouse liver.

1. After dimethylnitrosamine (DMNA) administration to mice, the content of poly(A)-containing RNA decreases rapidly in the postmicrosomal fraction of the liver. We report here that the loss of free mRNA is not a result of increased nucleolytic activity. On the contrary, a decreased activity of microsomal endonuclease, assayed by its effect on polyribosomal mRNA, was demonstrated already 15 min after the administration of DMNA at 37.5 mg/kg body wt. The loss of activity was more pronounced in the rough than in the smooth membranes. Total detergent-released microsomal nucleases, as assayed by use of labelled poly(U) as substrate, showed a less rapid decline. No corresponding increase in enzyme activities was observed in the postmicrosomal fraction. 2. The dimethylnitrosamine effect on the microsomal endonuclease was not accounted for by altered lysosomal contamination of the microsomal fraction. 3. No early effect of dimethylnitrosamine administration was found on the cytoplasmic ribonuclease inhibitor.     

Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids.

Homogenates of rat liver obtained 3 or 14 days after partial hepatectomy were used to prepare the postmicrosomal pH5-supernatant fraction and to prepare salt-wash fractions of the 40S ribosomal subunits and the 80S ribosomes. The factor-dependent binding of methionyl-tRNAfMet to ribosomes and the elongation-factor-1-dependent binding of phenylalanyl-tRNA to ribosomes were both increased after 3 days of growth, but not after 14 days of growth. An activity inhibitory to phenylalanyl-tRNA binding that was located in ribosomal wash fractions was decreased after 14 days of growth. Since the decreased inhibitory activity was obtained from the ribosomes and was tested against ribosomes and excess of pH5-supernatant fraction from control rat liver, its action was separate from the phenylalanyl-tRNA binding activities of the pH5-supernatant fractions from sham-operated and regenerating liver.     

RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts

During microcyst germination in the cellular slime mould Polysphondylium pallidum, an immediate rapid increase in the rate of protein synthesis ([3H]leucine incorporation) is observed within 15 min after the initiation of germination. The data, corrected for amino acid pool changes, reveal that the rate of protein synthesis reaches its peak at 1 1/2 h, after which it decreases. A low level of RNA synthesis ([3H]uridine incorporation) is observed after 1 h and this rate increases markedly after 2 h. Analysis of the RNA species shows a low level of synthesis of all ribosomal RNA's which begins between 1 and 2 h and increases after 2 h. The synthesis of a heterogeneously distributed, poly(A)-containing fraction of RNA (presumptive mRNA) is initiated some time after 2 h and the synthesis of a small molecular weight species in the 4-5S region is observed after 3 h. Thus, it seems that Polysphondylium microcysts show sequentially initiated synthesis of RNA during germination.     

Protein chain initiation in vitro by liver cell components from DMNA-treated rats.

The mechanism of inhibition of protein synthesis in rat liver after dimethylnitrosamine (DMNA) administration was studied at the level of peptide-chain initiation by use of initiation-dependent amino acid incorporating systems. Ribosomal monomers, poly(A)-concontaining loss of acticity due to the DMNA treatment. The poly(A) RNA from monosomes and polysomes, and crude initiation factors from microsomes were prepared 2 h after a single dose of DMNA (75 mg/kg), and their activities in the production of new protein chains determined under conditions of nearly linear response. Monosomes and crude initiation factors from DMNA-treated rats were at least as active as those from controls. Preparations of poly(A)-containing RNA had a consistently higher template activity when prepared from polysomes instead of monosomes. However, in neither case was there any ltaining RNA was methylated by DMNA to about the same extent as the 18S and 28S rRNA. The methylation was consistently somewhat higher in the RNA preparations from monosomes than in those from polysomes.     

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Activity of partially purified protein chain initiation factors from the livers of dimethylnitrosamine-treated rats

Partially purified polypeptide chain initiation factors were prepared from the 0.5 M KCl wash of rat liver microsomes. Their activities in connection with dimethylnitrosamine (DMNA)-induced inhibition of protein synthesis were studied by use of the following reactions: (1) poly(U)-directed binding of Phe-tRNA to ribosomes, (2) formation of a GTP-dependent ternary initiation complex with Met-tRNAf, (3) binding of Met-tRNAf to 40-S ribosomal subunits, (4) assembly of a Met-tRNAf containing 80-S ribosomal initiation complex and (5) ribosome-dependent GTPase activity. The inhibition of protein synthesis with DMNA was not associated with a loss of factor activity in any of these reactions. In the binding of Met-tRNAf to 40-S subunits there was a noticeable increase, probably related to the stability of the resulting complex. The Met-tRNA deacylase activity was also increased.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.

The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.     

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.     

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Hypoxanthine-xanthine oxidase-related defect in polypeptide chain initiation by endothelium

After exposure to O2 intermediates generated by the hypoxanthine-xanthine oxidase system (HX-XO), the rate of [3H]phenylalanine incorporation into total proteins in cultured endothelial cells was markedly reduced. This reduction, which was prevented by catalase, could not be explained by 1) changes in amino acid pools, 2) increased rate of degradation of newly synthesized proteins, 3) impaired poly(A)+ RNA synthesis and efficiency, 4) decreased rate of amino acylation. On the other hand, the increase in the monoribosome-to-polyribosome ratio suggested that translation was affected at the level of chain initiation. Further analysis indicated that 40S initiation complex formation was normal, whereas the assembly of 80S initiation complex was inhibited. Results from reconstitution experiments showed that both normal and treated ribosomes could support normal protein synthesis in the presence of normal initiation factors (IFs). In contrast, IFs from HX-XO lysates did not support normal protein synthesis with ribosomes from either source. Thus, the effect of XO treatment on protein synthesis appears to be an initiation defect related to decreased IF activity and/or availability.     

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Lymphocytes, exposed to mitogens in culture, show enhanced protein and RNA synthesis before the onset of DNA synthesis. Inhibition by DL-alpha-difluoromethylornithine of polyamine synthesis in phytohaemagglutinin-activated human lymphocytes resulted in a suppression of protein synthesis, which was evident before the initiation of DNA synthesis. The mitogen-induced increase in the incorporation of [3H]thymidine into DNA was subsequently inhibited in parallel with the activity of thymidine kinase in the polyamine-depleted cells. Ultraviolet absorbance measurement of the ribosomes after sucrose gradient centrifugation revealed a suppression of polyribosome formation that coincided with the decrease in the rate of protein synthesis. The disturbance in the polysomal profiles did not appear to be due to a shortage of mRNA, since the synthesis of poly(A)-rich mRNA was reduced less than that of rRNA after inhibiting polyamine synthesis. Entry of both the pre-existing and newly synthesized ribosomal subunits into polysomal structures was found to be impaired. These results thus suggest an important role for polyamines in the initiation step of protein synthesis.     

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.     

Kinetin-promoted Polyribosome formation in relation to RNA Synthesis in Excised Mung Bean Cotyledons

The relative levels of polyribosomes and total ribosomal materials, the rates of RNA synthesis and the contents of each RNA component were investigated in excised cotyledons of mung bean (Phaseolus radiatus L.) incubated with and without kinetin. 12 h incubation with 50 μmol/ L kinetin markedly increased the levels of polyribosomes and decreased the levels of monoribosome, especially of the ribosomal subunits. In addition, levels of total ribosomal materials (ribosomal subunit+monoribosome+polyribosome) were also increased in cotyledons incubated with kinetin. The kinetin-promoted polyribosome formation could be arrested by the RNA synthesis inhibitor-actinomycin D(ACTD). Kinetin incubation greatly enhanced RNA synthesis and increased that RNA conten. A marked increase was found in the amount of poly(A)+-mRNA, while the levels of other RNA components (25S, 18S rRNA, 4–5S RNA) were also increased to different extent. These results suggest that the promotion of polyribosome formation by kinetin depends upon the de novo synthesis of mRNAs, and the promotion of ribosome con, struction by kinetin may also be related to the synthesis of rRNAs.     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.