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The levels of ribosomes, tRNA molecules, and total protein per genome in Neurospora mycelia have been determined in eight different conditions of exponential growth. By increasing the rate of growth the number of ribosomes per genome increases dramatically while the level of total protein remains almost unchanged and the level of tRNA increases only slightly. The rates of synthesis of each of the macromolecules have been estimated. Increasing the rate of growth (mu) up to 0.5, the ratio between the rates of synthesis of tRNA and rRNA decreases reaching a constant value. The equations that best describe the dependence of the rate of synthesis of the macromolecules on the rate of growth (mu) have been determined. The rate of rRNA synthesis (rr), expressed as nucleotides polymerized, min- minus 1 per genome, is given by the equation: rr equals 6.51 times 10-7 mu-2-19. The rate of protein synthesis (rp), expressed as amino acids polymerized, min- minus 1 per genome is given by the following relationship: rp equals -1.43 times 10-7 + 3.43 times 10-8 mu. The equation describing the tRNA synthesis (rt) expressed as nucleotides, min- minus 1 per genome is rt equals 6.45 times 10-5 times exp 2.30 mu; however, more accurate determinations appear to be required for a firmer assignment of this latter equation. The significance of these equations for the studies on the regulation of rRNA and protein synthesis is discussed. For instance the rate of rRNA synthesis may set the limit for the maximal growth rate attainable by a cell, as the maximal rate of rRNA synthesis that may take place in a given cell is limited by the degree of redundancy of the rRNA genes.
Recombinant viruses derived from coronaviruses mouse hepatitis virus strains JHM and A59, were used to map biologic properties of the virus to viral genes. The 3' portion (about 25%) of the viral genome, including the genes coding for all of the structural proteins, controls biologic properties such as organ tropism of the virus, pattern of the virus-induced central nervous system pathology in mice, plaque morphology, and virus yield in tissue culture.
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Ribonucleic acid extracted from the virions of two human rhinoviruses is infective and is similar in size to poliovirus ribonucleic acid.
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Escherichia coli Phage Qbeta RNA replicase, an RNA-dependent RNA polymerase, is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, are required for initiation of transcription by Qbeta replicase with all templates. Using a previously developed reconstitution system we have examined the effects of modification of EF-Tu on reconstituted replicase activity. The poly(G) polymerase activity of the enzyme can be recovered after pretreatment of the EF-Tu-GDP with either L-1-tosylamido-2-phenylethyl chloromethyl ketone or N-ethylmaleimide, both of which inhibit the aminoacyl-tRNA binding activity of EF-Tu. This suggests that the aminoacyl-tRNA binding site of EF-Tu is not required for Qbeta replicase activity. When Qbeta replicase is treated with kirromycin, an antibiotic which modifies EF-Tu activity by an unknown mechamism, the protein synthetic activity of the EF-Tu in the replicase complex is eliminated but the Qbeta RNA replication activity is only slightly affected. Treatment of pure EF-Tu with kirromycin, however, prevents it from functioning in the renaturation of Qbeta replicase. This antibiotic is not effective against the EF-Tu-Ts complex in the reconstitution assay. Kirromycin at the relatively high concentration used here is found to prevent the formation of the EF-Tu-Ts complex. GDP, which binds to EF-Tu and inhibits formation of the complex with EF-Ts, also inhibits renaturation of Qbeta replicase. It is suggested that the EF-Tu-Ts complex, rather than the individual polypeptides, functions in the renaturation of Qbeta replicase and that the kirromycin and GDP act by preventing formation of this complex.
Treponema pallidum (Nichols strain), extracted in medium containing Eagle minimal essential medium 50% fresh, heat-inactivated normal rabbit serum, and 1.0 mM dithiothreitol, was incubated under 3% oxygen in the presence of tritiated nucleic acid precursors. [8-3H]adenine was incorporated with high efficiency into trichloroacetic acid-insoluble material; 2'-deoxyadenosine and uridine were incorporated in lower quantities, and thymine and thymidine were not incorporated. Incorporation of [3H]adenine was inhibited by penicillin G, mitomycin C, actinomycin D, and erythromycin, but was not affected by cycloheximide. Partial purification of nucleic acids from T. pallidum incubated with [8-3H]adenine for 36 to 72 h and subsequent treatment with ribonuclease and deoxyribonuclease revealed that 15 to 20% of the trichloroacetic acid-precipitable counts were resistant to ribonuclease but susceptible to deoxyribonuclease. A simple assay was developed in which NaOH treatment was used to distinguish incorporation into ribonucleic acid and deoxyribonucleic acid. Both ribonucleic acid and deoxyribonucleic acid synthesis continued for 6 days of incubation under 3% O2, whereas incorporation was limited to the first day of incubation in samples incubated under aerobic or anaerobic conditions. T. pallidum thus appears to be capable of significant de novo deoxyribonucleic acid and ribonucleic acid synthesis under microaerobic conditions.
Ribonucleic acid extracted with phenol from Type 1 poliovirus, Coxsackie A-9, Coxsackie B-1, and ECHO 8 viruses infected non-primate cells and animals insusceptible to whole virus as such. Viral RNA was proved infectious for insusceptible cells in test systems of established cell lines, primary monolayer cultures, Maitland type cultures, and living animals inoculated intracerebrally. Cells of rabbit, swine, mouse, guinea pig, chicken, and hamster were infected. Each virus produced was identical with the virus donating RNA, in (a) neutralization by homotypic antiserum, (b) resistance to ribonuclease treatment, and (c) failure to be adsorbed or replicated by nonprimate cells, even of the strain producing the virus from RNA. Produced virus was adsorbed and replicated by susceptible primate cells as usual. Virus in RNA-infected cell cultures was produced in a single cycle unaccompanied by overt cytopathic effect on non-primate cells or disease of intracerebrally inoculated animals. By drastic elution of infective poliovirus associated with rabbit cells exposed to massive inocula of intact virus, intact poliovirus was shown to infect insusceptible non-primate cells to produce progeny indistinguishable from the parent virus population. Under these conditions, infection was accomplished by about 10 virus plaque-forming units per billion inoculated.
The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.
Spontaneous revertants of a temperature-sensitive Escherichia coli strain bearing a thermolabile glutaminyl-transfer ribonucleic acid (tRNA) synthetase have been selected for growth at 45 degrees C. Among 10 revertants still containing the thermolabile enzyme, 2 interesting strains were found. One strain has a fivefold elevated level of the thermolabile glutaminyl-tRNA synthetase; the genetic locus, glnR, responsible for this effect maps at min 24, far from glnS, the structural gene of the enzyme. In the other strain the levels of tRNA Gln and several other tRNAs are twice as high as in the parental strain; the locus responsible, glnU, maps at min 59.5 on the E. coli map.
The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.
Ribonucleic acid (RNA) species from aerobically and anaerobically grown Rhodopseudomonas spheroides were compared via hybridization to deoxyribonucleic acid (DNA). Both long-labeled and stable RNA bound to chromosomal DNA to the same extent, regardless of derivation. About 4% of the chromosomal DNA hybridized with total cell RNA and about 0.08% with stable RNA. About 4% of the mixed satellite DNA could be hybridized to total cell RNA from aerobic or anaerobic cultures, whereas essentially no stable RNA formed a hybrid with this DNA. Hybridization competition experiments with aerobic and anaerobic pulse-labeled RNA and chromosomal or satellite DNA demonstrated that no qualitative differences existed between the RNA species. It is concluded that identical species of RNA in the same relative amounts are synthesized by R. spheroides during aerobic or anaerobic growth on the same medium.
Ribonucleic acid (RNA) synthesis of chick embryo fibroblasts was inhibited by two members of the myxovirus group, Newcastle disease virus (NDV) and fowl plague virus. It was also found that cellular deoxyribonucleic acid-dependent RNA polymerase was inhibited by a cytoplasmic factor induced by NDV infection.
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.