Classes Of Ribonucleic Acid Research Articles

Synthesis and cleavage of influenza virus proteins.

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.

Electrophoretic characterization of foot-and-mouth disease virus-specific ribonucleic acid.

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.

Characterization of ribosomes from the myxomycete Physarum rigidum grown in pure culture.

The plasmodial phase of the myxomycete Physarum rigidum, analyzed during the period of rapid growth, attained a ribonucleic acid (RNA) and protein content of 9.8 and 60.0%, respectively, on a dry weight basis. It possessed ribosomes of the 80S class which, especially in the absence of magnesium ions, partially dissociated to 60S and 40S subunit classes. Electron micrographs of ribosomes treated with uranyl acetate-lead citrate revealed a number of surface features. Nucleotide analyses of both ribosomal and total RNA disclosed that they were composed of 51.0 and 52.5% guanylic and cytidylic acids, respectively. Consistent with most reports on other organisms, guanylic acid was the most abundant nucleotide found in the various types of RNA and cytidylic acid was the least abundant. The S(0) (20,w) values of the total RNA classes, in 0.01 sodium acetate (pH 4.6) containing 0.10 m NaCl, were 5.2, 18.1, and 27.3 in S units. Changing the ionic environment of the RNA (0.017 molal potassium phosphate, pH 7.0, containing 0.01 m disodium ethylenediaminetetraacetate) resulted in a reduction of the S(0) (20,w) values to 4.2, 16.6, and 22.6 in S units, which is indicative of molecular conformational transitions. In general, the amino acid composition of the ribosomal proteins was similar to the data available on ribosomal proteins from other biological sources.

Relationship between protein and ribonucleic acid synthesis during outgrowth of spores of Bacillus cereus.

The rate of protein and ribonucleic acid (RNA) synthesis was examined during the outgrowth of spores of Bacillus cereus T in a chemically defined medium. RNA synthesis started 2.5 min after the initiation of germination, and protein synthesis after 4 min. Addition of a complete amino acid supplement and uracil supported high rates of RNA and protein synthesis throughout outgrowth. To determine the relationship between the rate of protein (k) and RNA synthesis, the kinetics of formation of various classes of RNA were followed during outgrowth. Ribosomal RNA (rRNA) comprised a relatively constant fraction of the total RNA throughout outgrowth (71 to 78%). The classes of RNA synthesized during this period were determined by germinating spores in radioactive uracil and then at intervals following their stability to actinomycin D. Initially, labile RNA comprised the largest fraction of newly formed RNA (DeltaRNA), and this proportion decreased during outgrowth. The ratio of k/rRNA or k/Delta stable RNA varied considerably during outgrowth, whereas the ratio of k/labile RNA remained constant. The data suggest that the rate of protein synthesis is not rigidly coupled to either total or newly synthesized rRNA (ribosomes) during the early stages of outgrowth.

Transcription of the genomes of type 1 and type 3 reoviruses.

The double-stranded ribonucleic acid (RNA) genome of type 1 reovirus was fragmented into three size classes on extraction, as has already been shown for reovirus type 3. The relative amounts and molecular weights of the three classes were the same for the two viruses. Cells infected with type 1 virus synthesized three classes of messenger RNA. Each class of messenger RNA hybridized exclusively with a denatured double-stranded RNA fragment of equivalent length, as had also been found for type 3 reovirus. The double-stranded RNA segments thus act as specific units for transcription of messenger RNA in the infected cells. In cells infected with type 3 reovirus, the three classes of messenger RNA are made in equal amounts throughout the course of multiplication. In contrast, cells infected with type 1 virus produced only half as much of the largest messenger RNA as they did of the other two classes at all times during the replicative cycle. This finding suggests that transcription of the largest segments of type 1 viral genome is restricted.

Structural Units of Reovirus Ribonucleic Acid and Their Possible Functional Significance

Reovirus contains ribonucleic acid (RNA) equivalent in amount to a molecular weight of approximately 10(7) daltons. On isolation, this RNA is invariably broken into fragments of three different sizes. The three pieces have been separated from each other by chromatography on methylated albumin-kieselguhr columns. Denaturation of the three fragments of RNA in dimethyl sulfoxide led to separation of the strands, as suggested by sucrose gradient sedimentation patterns and by the second-order kinetics of reannealing. Molecular weights of 0.8 x 10(6), 1.4 x 10(6), and 2.4 x 10(6) were determined for the double-stranded fragments from the sedimentation rates of the single-stranded RNA obtained by denaturation. There was little or no homology among the three classes of denatured RNA when taken in pairs in hybridization tests. The three pieces of double-stranded RNA, therefore, did not result from random breaks in the original viral RNA molecule. Virusspecific single-stranded RNA formed in infected cells, and previously found to be largely if not entirely messenger RNA transcribed from the viral genome, was also separated into three size classes by sedimentation through sucrose gradients. Each class of single-stranded RNA corresponded in size to one of the three fragments of double-stranded RNA. The largest piece of single-stranded RNA hybridized uniquely with the largest fragment of denatured viral RNA. By whatever means this fragment of double-stranded RNA may be joined into the viral RNA molecule, it seems to act as a specific unit for transcription of an uninterrupted messenger RNA of equivalent length.

Methionine and the regulation of ribonucleic acid synthesis in Escherichia coli.

When cultures of Escherichia coli growing in minimal medium (M-9 salts, 0.5% glycerol, 10 mug/ml of uracil) are shifted to minimal medium supplemented with 50 mug/ml of l-methionine, they exhibit an abrupt increase in their relative rate of synthesis of ribonucleic acid (RNA). The rate of growth of the cells is not increased by such a shift. Sucrose density gradient analyses of cell extract after the shift showed that the effect was exerted equally on all classes of RNA. The initial relative rate of methylation of RNA, as judged by incorporation of radioactivity from l-methionine-methyl-C(14) into RNA, is high in cells recovering from chloramphenicol inhibition, intermediate in cells grown in minimal medium, and low in cells grown in medium containing methionine. In cells recovering from chloramphenicol inhibition, methionine decreases the lag and increases the rate of conversion of ribonucleoprotein particles to mature 70S ribosomes. Turnover of RNA under these conditions, as judged by loss of C(14)-labeled methyl groups, is minimal. These results lead to the inference that methionine increases the relative rate of RNA synthesis by increasing the rate of conversion of ribonucleoprotein particles to ribosomes and thereby decreasing the availability of ribonucleoprotein particles for regulating RNA synthesis by a feedback mechanism.

Effect of interferon on polymerization of single-stranded and double-stranded mengovirus ribonucleic acid.

Gordon, Irving (University of Southern California, Los Angeles), Sara S. Chenault, Douglas Stevenson, and Jean D. Acton. Effect of interferon on polymerization of single-stranded and double-stranded mengovirus ribonucleic acid. J. Bacteriol. 91:1230-1238. 1966.-The effect of interferon on actinomycin-resistant mengovirus ribonucleic acid (RNA) replication in L cells was investigated to determine whether defective or partially polymerized RNA products were made and whether synthesis of any specific class of virus RNA was prevented. RNA labeled with uridine-C(14) was extracted in hot and cold phenol and analyzed by zonal sucrose density centrifugation. Both single- and double-stranded infectious RNA peaks were identified. Interferon treatment caused almost complete depression of uridine-C(14) incorporation throughout linear sucrose gradients except in the 4S region, and no infectivity was detectable in any fraction. These inhibitory effects are attributable to the action of interferon, because they were reversed when cultures were treated with actinomycin D simultaneously with interferon. The results, with those of other investigators, indicate that the step at which interferon interrupts virus multiplication is between the events immediately after uncoating and the formation of template "minus" strands; under the conditions of our experiments, no partially polymerized virus RNA products were made.

NUCLEOTIDE COMPOSITION OF RIBONUCLEIC ACID FROM NEUROSPORA CRASSA.

Henney, H. (The University of Texas, Austin) and R. Storck. Nucleotide composition of ribonucleic acid from Neurospora crassa. J. Bacteriol. 85:822-826. 1963.-The nucleotide composition of total, ribosomal, and soluble ribonucleic acid (RNA) from the conidia and the hyphae of Neurospora crassa was determined. The corresponding RNA classes from the two morphological types had the same base ratio. Total, ribosomal, and soluble RNA from hyphae contained, respectively, 51.0, 50.2, and 61.6% guanylic acid (G) plus cytidylic acid (C), and from conidia, 50.6, 49.9, and 62.1%. The proportion of nucleotides in ribosomal RNA was in close agreement with that reported for ribosomal RNA from other organisms. Soluble RNA contained 2.8% pseudouridylic acid, 36.0% C, and 25.5% G, and differed from soluble RNA from other sources in its high C content. Centrifugation of purified RNA from conidia and hyphae, through a linear sucrose-density gradient, yielded identical sedimentation profiles.