Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.     

Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Factors Affecting Immunogenic Activity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate. J. Bacteriol. 91:2139-2145. 1966.-Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 x g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 mug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10(-4)m MgCl(2) to the final diluent increased immunogenic activity, whereas larger concentrations (10(-3)m) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Two-week-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.     

Isolation and partial characterization of an immunogenic moiety obtained from Salmonella typhimurium

Ribosomal preparations obtained from Salmonella typhimurium by differential centrifugation and sodium dodecyl sulfate (SDS) treatment of the bacillary lysate were found to be immunogenic in F(1) hybrid (C(3)H/HeJ x DBA/2J) and albino Swiss mice, as determined by progressive host survival. The immunity obtained was independent of the need for adjuvant and dependent on the dosage of immunogen given. Immunizations with the ribosomal preparations induced an immune response comparable to that obtained by vaccination with living organisms and significantly greater than that obtained by immunization with heat-killed salmonellae, purified lipopolysaccharide, or crude and SDS-treated endotoxin preparations. No effect on the immunogenicity of the ribosomal fraction was observed by enzymatic treatment with trypsin, Pronase, deoxyribonuclease, and pancreatic ribonuclease. Linear sucrose density gradient resolution of the preparations showed that the immunogenicity of the ribosomal fraction was not unique to any one of its subcomponents. Ethyl alcohol-precipitated, crude ribonucleic acid preparations obtained from the ribosomal and sucrose density-resolved ribosomal preparations were found to induce an immune response comparable to that obtained by immunization with the entire ribosomal fraction. Dialysis in doubly distilled demineralized water slightly reduced the immunogenicity of the preparation; however, comparable dialysis in 10(-4)m MgCl(2)-phosphate buffer did not. Chemical assays of the preparations found to be immunogenic were performed.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

The isolation and immunologic study of ribosomal preparations from Shigella flexneri

S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

On the Immunogenicity of Ribosomes and Ribosomal Proteins Isolated from Klebsiella pneumoniae and Streptococcus pneumoniae

The two pathogenic species Streptococcus pneumoniae and Klebsiella pneumoniae were used to analyze the immunogenic role of proteins in ribosomal preparations. The protective activity of ribosomes prepared from either strain and further purified by washing with high-salt concentrations, followed or not by sucrose gradient separation of the particles, was identical to that of crude unwashed ribosomes. Similarly, no substantial alteration of the level of protection was observed after treatment with the antibiotic puromycin. Therefore, the immunizing efficacy of ribosomes does not appear to be due either to the nonribosomal proteins adsorbed at the surface of organelles or to the growing polypeptide chain. It seems rather to be attributable to the structural ribosomal proteins themselves, which were indeed shown to induce alone a significant level of protection.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Stoichiometric analysis of barley plastid ribosomal proteins

We analyzed the protein composition of plastid 70S ribosomes isolated from the stromal fractions of barley plastids by the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR 2D-PAGE). Intactness of the ribosomes was confirmed by the poly(U)-directed phenylalanine polymerization activity and by the reassociation capacity of the subunits into 70S ribosomes. The small and large ribosomal subunits were composed of 23 and 36 proteins, respectively. In addition, one acidic protein associated with ribosomes in low salt buffer but released in high salt buffer was found. The plastid ribosomes contained relatively larger numbers of acidic proteins than prokaryotic ribosomes. Stoichiometric analysis revealed the presence of several ribosomal proteins in low copy numbers, indicating that the ribosomes of plastids were heterogeneous. We also investigated the protein composition of plastid ribosomes from greening barley leaves and found that it did not change during greening.     

A temperature-sensitive mutation affecting the mammalian 60 S ribosome.

A temperature-sensitive Chinese hamster cell mutant, ts14, is unable to synthesize protein in tissue culture at 39 degrees. That mutant's protein biosynthetic machinery has been characterized in cell-free, biologically active extracts. Similar to the mutant's phenotype in tissue culture, ts14 extracts cease protein synthesis in vitro within 15 min at 40 degrees. In contrast, at 25 degrees both ts14 and wild type extracts synthesize protein for more than 2 hours. Fractionation of mutant extracts and complementation with comparable wild type preparations indicate that ts14 possesses a thermolabile component associated with its polyribosomes. In preparation of ts14 ribosomes that are free of mRNA and bound protein factors, the defective factor is complemented functionally only by 60 S ribosomal subunits prepared from the wild type parent. Sedimentation analyses in sucrose gradients demonstrate that ts14's mutation specifically affects stability of the mutant's 60 S ribosome. Treatment with high ionic strength buffers preferentially disrupts the mutant's 60 S ribosomal subunit and results in preparations of mutant ribosomes that contain biologically active 40 S subunits only. These studies demonstrate the applicability of a genetic approach to analyzing structure-function relationships in the eukaryotic ribosome.     

Anti-Streptococcus mutans ribosome monoclonal antibodies recognize a cell surface antigen on different serotypes

We have produced a panel of murine monoclonal antibodies to a ribosomal preparation fromStreptococcus mutans NCTC 10449 (serotypec). Ribosomal preparations were isolated from homogenized cells by differential centrifugation and consisted of approximately 17% protein and 83% RNA. Splenocytes from immunized animals were fused to NS-1 myeloma cells using polyethylene glycol and dimethyl sulfoxide. Four stable hybridomas have been currently established, two of which secrete the IgG-k isotype and the other two the IgM-k isotype. All of the monoclonal antibodies react with wholeS. mutans cells representing different serotypes, while not with other bacteria. In Western blot analysis of the ribosomal preparation resolved by one-dimensional electrophoresis, the nmonoclonal antibodies recognize a specific component with an apparent molecular weight of 65,000. These results indicate that theS. mutans ribosomal preparation contains an immunogenic site that is serologically indistinguishable from a species-specific surface determinant common to different serotypes.     

Altered ribosomes after inhibition of Escherichia coli by rifampicin

Addition of rifampicin to growing cells of Escherichia coli affected the ribosomes. The polyribosomes first decayed to 70S ribosomes. These later dissociated to particles distinct from ribosomal subunits. The altered ribosomes sedimented more slowly than the corresponding subunits and had lost some protein; their ribosomal RNA was intact, but they were more susceptible to degradation by ribonuclease than normal ribosomes. The addition of rifampicin to preparations of lysed cells caused no detectable changes in the ribosome fraction.     

The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C

Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.     

Messenger RNA affinity column fractionation of eukaryotic initiation factor and the translation of myosin messenger RNA.

Both myosin mRNA (26 S) and globin mRNA (9 S) have been bound to activated Sepharose 4B. The affinity of initiation factors derived from native 40 S ribosomal subunits from embryonic chick muscle for these messengers has been determined. Although both messengers bind the major components of the muscle factor preparation with the same affinity, some differences are noted in the minor components. There is an enrichment of components which bind myosin mRNA with a high affinity when the 15–18 S initiation factor complex is prepared from initiating 40 S ribosomal subunits found on myosin synthesizing polysomes rather than from total cellular factor preparations. The proteins which have a high binding affinity to myosin mRNA also have a discriminating effect when added to a wheat germ system containing myosin and globin mRNA. This is demonstrated by the fact that the synthesis of myosin heavy chain is specifically stimulated and the number of ribosomes found on myosin mRNA increase five to seven-fold; whereas neither the synthesis of globin nor the number of ribosomes associated with globin mRNA is increased. The components of an impure reticulocyte eukaryotic initiation factor 3 prepared in a similar manner as the muscle factor, do not bind myosin mRNA with the same high affinity, and these fractions separated on the myosin mRNA affinity column did not show a discriminatory effect. These results suggest that specific components of muscle 15–18 S initiation factor preparations have a higher binding affinity for myosin mRNA than globin mRNA and that these proteins may be those factors previously reported to be present which discriminate between mRNAs.     

Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites

The topography of 5.8 rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 S-28 rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gel-sequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the "AU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two -G-A-A-C- sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.