Allergenicity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations in Mice and Guinea Pigs

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 μg (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 μg of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.     

Preparation and Effect of Different Adjuvants on the Immunogenic Activity of Mycobacterial Ribosomal Fraction

Several emulsified and two nonemulsified incomplete adjuvants were examined for their adjuvant activity by use of mycobacterial ribosomal fractions as a substrate. A good adjuvant is defined as one which produces a high immunological response with the ribosomal fraction in mice to infection with virulent tubercle bacilli. Freund's incomplete adjuvant, consisting of Aquaphor and heavy mineral oil, and Arlacel A plus hexadecane were the best adjuvants tested. Aquaphor plus light mineral oil and Arlacel A plus 7-n-hexyloctadecane were not quite as effective. Peanut oil was not satisfactory when emulsified with either Aquaphor or Arlacel A. A moderate degree of immunity was produced in mice vaccinated with ribosomal fraction mixed with aluminum hydroxide gel. Sodium alginate mixed with ribosomal fraction produced a low degree of immunity only with the highest vaccinating dose. It was found that the effectiveness of the emulsified type of adjuvant depended upon the method of preparation. Careful standardization of technique to produce uniform and complete emulsification was essential for maximal adjuvant activity using minimal vaccinating doses. A rapid and practical method of preparing emulsified adjuvants is given. The mode of action of incomplete adjuvants as employed in these experiments is discussed, and it is thought that they acted primarily by protecting the ribosomes from being inactivated by host ribonuclease before they were engulfed by the macrophages.     

Ribosomal Ribonucleic Acids of Chloroplastic and Mitochondrial Preparations

RNA prepared from fractions of chloroplasts and mitochondria sedimented at rates characteristic of ribosomal RNA. A predominance of the 18S species was frequently observed in preparations from chloroplasts from romaine lettuce and endive. The usual distribution, a preponderance of the 28S species, was observed in studies on tomato and spinach chloroplasts and mitochondria from mushroom and cauliflower. Comparisons of the base composition of RNA from organelles with their cytoplasmic ribosomal counterparts revealed that the 18S component from romaine lettuce chloroplast was different. A marginally significant difference was observed in the 28S particle from mushroom mitochondria preparations whereas distinct differences, reflected in all the bases, were seen when the 18S component of cauliflower mitochondria preparations was compared with cytoplasmic RNA.     

Relationship Between Sporulation-Specific 20S Ribonucleic Acid and Ribosomal Ribonucleic Acid Processing in Saccharomyces cerevisiae

The nature and properties of the 20S ribonucleic acid which accumulates only during the sporulation of Saccharomyces cerevisiae were examined. The 20S ribonucleic acid (RNA) has a base composition considerably different from ribosomal RNA species and is virtually unmethylated. The 20S RNA did, however, exhibit approximately 70% homology with 18S RNA by RNA-deoxyribonucleic acid filter hybridization competitions. The 20S RNA showed a hybridization saturation plateau level 30 to 40% higher than 18S, consistent with measurements of the size difference in polyacrylamide gels. Pulse-chase experiments in the presence and absence of cycloheximide indicate that the 20S RNA has a presumptive relationship to the 20S ribosomal RNA precursor normally observed only in short pulse-labeling in vegetative cells.     

Thermally Induced Intracellular Alteration of Ribosomal Ribonucleic Acid

Heating at 55 C causes the intracellular degradation of ribosomes of Staphylococcus aureus MF-31. The 30S subunit of heated cells appears to be selectively attacked. Analysis of ribonucleic acid (RNA) showed that 16S RNA was destroyed and that the secondary structure of 23S RNA was altered.     

Ribosome Development and the Methylation of Ribosomal Ribonucleic Acid

Immature ribonucleoprotein particles accumulate in amino acid-starved cells of a relaxed mutant of Escherichia coli. The ribonucleic acid (RNA) of these particles is nonmethylated in cells starved for methionine. However, in bacteria starved for arginine, lysine, or histidine, the RNA of these particles is one-half methylated. The relationship of submethylation to a structural alteration in the same RNA was studied. The results of kinetic studies showed that submethylation and the structural transition are not causally related, since they are described by different rate constants. Moreover, it was possible to accumulate fully methylated immature-particle RNA that possessed the sedimentation and chromatographic properties of nonmethylated RNA. It was concluded that, during the normal course of ribosome development, methylation of ribosomal RNA is completed prior to the final maturation steps.     

Small Ribosomal Ribonucleic Acid Species of Saccharomyces cerevisiae

Yeast ribosomes contain two small molecular-weight species of ribonucleic acid (RNA), in addition to transiently associated transfer RNA. The 5S RNA species is part of the large ribosomal subunit and appears to be exactly the same size as 5S RNA from other organisms. There is another RNA molecule, approximately 5.8S or 150 nucleotides in size, which is noncovalently attached to the 25S ribosomal RNA and can be freed by gentle heating or urea treatment. Neither 5 nor 5.8S RNA are methylated. The 5.8S RNA is probably derived from a part of the 35S precursor RNA, whereas the 5S RNA is made de novo. These results substantiate the notion that ribosome biosynthesis in yeast is analogous to that of the higher eukaryotes.     

Ribosomal Precursors and Ribonucleic Acid Synthesis in Escherichia coli

Ribosomes and immature ribonucleoprotein particles were isolated from extracts of log-phase cells grown under various conditions. Quantitative measurements were made to determine the relative amounts of immature particles present in the extracts. The results indicate that the steady-state level of ribosomal precursors accounted for essentially a constant fraction of the total ribonucleic acid (RNA) of the cells. For cells with RNA-protein ratios between 0.43 and 0.65, about 1.6% of the total RNA occurred as immature ribonucleoprotein particles. Further, increased levels of immature particles were shown to be correlated with a reduced rate of RNA synthesis in cells recovering from chloramphenicol inhibition. The reduction was found to vary directly with the duration of pretreatment in chloramphenicol and, consequently, with the level of immature particles present in the cells.     

Ribosomal Ribonucleic Acid Maturation During Bacterial Spore Germination

All the ribosomal ribonucleic acid made during the early stages of germination of spores of Bacillus subtilis is of the "precursor" type, i.e., that type appearing in the incomplete forms of the ribosome. Shortly before the onset of deoxyribonucleic acid synthesis in germination, this precursor ribonucleic acid changed to the mature ribosomal ribonucleic acid characteristic of the 30S and 50S ribosomal subunits.     

Distribution of Ribosomal Ribonucleic Acid Cistrons Among Yeast Chromosomes

High-molecular-weight deoxyribonucleic acid (DNA) of Saccharomyces carls bergensis has been fractionated by sucrose density gradient centrifugation. The main DNA fraction has an average molecular weight of about 500 x 10(6). A major fraction of the DNA molecules containing sequences homologous to ribosomal ribonucleic acid (RNA) sediments as material of this molecular weight. The remainder sediments as material of a molecular weight of about 250 x 10(6). The latter fraction contains relatively more ribosomal RNA cistrons than the former. Studies on the buoyant density of high-molecular-weight DNA homologous to ribosomal RNA have led to the conclusion that the ribosomal RNA cistrons occur in groups attached to a relatively large amount of nonribosomal RNA and suggest that ribosomal RNA cistrons are distributed over a number of yeast chromosomes.     

Synthesis of Ribosomal Ribonucleic Acid During Sporulation of Bacillus subtilis

The incorporation of radioactive uracil into 50s and 30s ribosomal subunits and ribosomal ribonucleic acid (rRNA) was studied during the growth cycle of different sporogenic and asporogenic strains of Bacillus subtilis. It was found that partially synchronized cultures of the strains examined incorporated labeled uracil into the two ribosomal subunit species and rRNA during sporulation and during the stationary phase of the asporogenic strains. Kinetic studies have shown that, compared to vegetative cells, the percentage of uracil incorporated into the ribosomal subunits of cells taken 30 min after the end of exponential growth was decreased by about 25 to 35%. This decrease, however, appeared to be a general characteristic of stationary-phase cells and seems to depend on the nature of the sporulation medium and to some extent on the nature of the strain but not on the sp(+) or sp(-) phenotype of the strain. Moreover, by use of actinomycin D it was shown that the labeled uracil incorporated, in the presence of the drug, during the sporulation period was located in the ribosomal subunits (stable RNA). Based on these results, we concluded that during sporulation ribosomal genes are transcribed and consequently rRNA continues to be synthesized, although to a lesser extent than during vegetative growth. These results are discussed in the light of those obtained by Hussey et al.     

Comparative Study of Ribosomal Ribonucleic Acid Cistrons in Enterobacteria and Myxobacteria

Deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybrids are formed by Escherichia coli 16S or 23S ribosomal RNA or pulse-labeled RNA with the DNA of various species of the Enterobacteriaceae. The relative extent of hybrid formation is always greater for ribosomal RNA. These DNA-RNA hybrids have been further characterized by their stability to increasing temperature, and, in every case, the stability of pulse-labeled RNA hybrids was lower than that of the corresponding ribosomal RNA hybrids, although 16S and 23S ribosomal RNA hybrids had very similar stabilities. Therefore, ribosomal RNA showed a greater degree of apparent conservation in base sequence than pulse-labeled or messenger RNA both in the extent of cross-reaction and in the stability of hybrid structures. Similar results were obtained with Myxococcus xanthus RNA. Since in this case the base composition of the pulse-labeled or messenger RNA is richer in guanine plus cytosine than ribosomal RNA, the higher cross-reaction of ribosomal RNA is more readily attributable to conservation of base sequence in these cistrons than to its base composition. Thus, the base sequence of ribosomal RNA cistrons of bacilli, enteric bacteria, and myxobacteria is conserved relative to those of the rest of the genomes. This conservation is, however, not absolute since the stability of heterologous ribosomal RNA hybrids is always lower than that of homologous hybrids.     

Homology of Ribosomal Ribonucleic Acid of Desulfovibrio Species with Desulfovibrio vulgaris

Three species of Desulfovibrio were found to have a high degree of ribosomal ribonucleic acid homology with Desulfovibrio vulgaris. Desulfotomaculum nigrificans, which is also a sulfate-reducing anaerobe, had only 38% ribosomal ribonucleic acid homology with D. vulgaris. The homologies of six other unrelated genera were determined and found to be lower than 50%.     

Ribosomal Ribonucleic Acid Synthesis and Maturation in the Blue-Green Alga Anacystis nidulans

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Chemical Factors Affecting Palmelloid-Forming Activity of Chloroplatinic Acid on Chlamydomonas eugametos

In order to elucidate the physiological mechanism of palmelloid formation of the unicellular green alga Chlamydomonas eugametos by chloroplatinic acid, the factors causing inhibition of the palmelloid formation were studied in various media. Chloroplatinic acid lost its palmelloid-forming effect in the presence of nitrite ion and amines having a second functional group. Nitrate, potassium, and sodium ions and monofunctional amines were without influence on the effect of chloroplatinic acid. These results indicate that the active form of platinum (IV) may undergo ligand substitution, the resulting complex being unable to cause palmelloid formation.     

Methionine-Dependent Synthesis of Ribosomal Ribonucleic Acid During Sporulation and Vegetative Growth of Saccharomyces cerevisiae

Methionine limitation during growth and sporulation of a methionine-requiring diploid of Saccharomyces cerevisiae causes two significant changes in the normal synthesis of ribonucleic acid (RNA). First, whereas 18S ribosomal RNA is produced, there is no significant accumulation of either 26S ribosomal RNA or 5.8S RNA. The effect of methionine on the accumulation of these RNA species occurs after the formation of a common 35S precursor molecule which is still observed in the absence of methionine. During sporulation, diploid strains of S. cerevisiae produce a stable, virtually unmethylated 20S RNA which has previously been shown to be largely homologous to methylated 18S ribosomal RNA. The appearance of this species is not affected by the presence or absence of methionine from sporulation medium. However, when exponentially growing vegetative cells are starved for methionine, unmethylated 20S RNA is found. The 20S RNA, which had previously been observed only in cells undergoing sporulation, accumulates at the same time as a methylated 18S RNA. These effects on ribosomal RNA synthesis are specific for methionine limitation, and are not observed if protein synthesis is inhibited by cycloheximide or if cells are starved for a carbon source or for another amino acid. The phenomena are not marker specific as analogous results have been obtained for both a methionine-requiring diploid homozygous for met13 and a diploid homozygous for met2. The results demonstrate that methylation of ribosomal RNA or other methionine-dependent events plays a critical role in the recognition and processing of ribosomal precursor RNA to the final mature species.     

Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA.     

Precursor of 5S Ribosomal Ribonucleic Acid in the Blue-Green Alga Anacystis nidulans

The maturation of 5S ribosomal ribonucleic acid (rRNA) in the obligately photoautotrophic unicellular blue-green alga Anacystis nidulans has been studied by using polyacrylamide gel electrophoresis and T1 ribonuclease oligonucleotide analysis. A. nidulans mature 5S rRNA (m5) is of approximately the same molecular weight as the 5S rRNA of Escherichia coli, and is derived by cleavage of a precursor (p5) containing a few (three to six) additional nucleotides. Some of these additional nucleotides occur at the 5' end of the precursor molecule; others may occur at the 3' end. Kinetic experiments indicate that precursors of mature 5S rRNA larger than p5 either do not exist or are very transient in A. nidulans. These results are discussed in relation to those obtained with other prokaryotes.     

Gene Dosage for 5S Ribosomal Ribonucleic Acid in Escherichia coli and Bacillus megaterium

The number of gene copies for 5S ribosomal ribonucleic acid (rRNA), relative to that for 16 and 23S rRNA, has been determined by deoxyribonucleic acid (DNA)-RNA hybridization for Escherichia coli and Bacillus megaterium. In both cases, the number of 5S rRNA genes equals the number of 16 or 23S rRNA genes. Rapid procedures for preparing extremely highly purified DNA suitable for DNA-RNA hybridization experiments and chemically pure 5S rRNA are described.