Diphenylhydantoin inhibits cortisol-induced lysis of thymocytes

Diphenylhydantoin (DPH) shares two features with cortisol: immunosuppression and cleft palate formation. We tested the hypothesis that DPH would have effects on lymphocytes in vitro similar to those induced by cortisol, and the corollary that DPH would inhibit those cortisol effects. We found that DPH lysed rat thymocytes, although at higher concentrations than cortisol. When combined, DPH inhibited cortisol lysis of thymocytes. Neither drug lysed human phytohemagglutinin (PHA)-stimulated cells, but both drugs depressed DNA and RNA syntheses in PHA cells. DPH augmented cortisol inhibition of DNA and RNA syntheses in PHA cells and DNA synthesis in rat thymocytes. It had no effect on cortisol inhibition of RNA synthesis in rat thymocytes. It appears that DPH has a cortisol-like action (lysis of rat thymocytes). The actions of this drug enable us to show that cortisol lysis and the inhibition of DNA or RNA synthesis can be associated. These phenomena may explain some immunosuppressive effects of DPH in the human.     

Metabolic complexity in the RNA world and implications for the origin of protein synthesis

Summary A model is presented for the evolution of metabolism and protein synthesis in a primitive, acellular RNA world. It has been argued previously that the ability to perform metabolic functions logically must have preceded the evolution of a message-dependent protein synthetic machinery and that considerable metabolic complexity was achieved by ribo-organisms (i.e., organisms in which both genome and enzymes are comprised of RNA). The model proposed here offers a mechanism to account for the gradual development of sophisticated metabolic activities by ribo-organisms and explains how such metabolic complexity would lead subsequently to the synthesis of genetically encoded polypeptides. RNA structures ancestral to modern ribosomes, here termed metabolosomes, are proposed to have functioned as organizing centers that coordinated, using base-pairing interactions, the order and nature of adaptor-mounted substrate/catalyst interactions in primitive metabolic pathways. In this way an ancient genetic code for metabolism is envisaged to have predated the specialized modern genetic code for protein synthesis. Thus, encoded amino acids initially would have been used, in conjunction with other encoded metabolites, as building blocks for biosynthetic pathways, a role that they retain in the metabolism of contemporary organisms. At a later stage the encoded amino acids would have been condensed together on similar RNA metabolosome structures to form the first genetically determined, and therefore biologically meaningful, polypeptides. On the basis of codon distributions in the modern genetic code it is argued that the first proteins to have been synthesized and used by ribo-organisms were predominantly hydrophobic and likely to have performed membrane-related functions (such as forming simple pore structures), activities essential for the evolution of membrane-enclosed cells.     

A System of Two Polymerases – A Model for the Origin of Life

What was the first living molecule – RNA or protein?This question embodies the major disagreement instudies on the origin of life. The fact that incontemporary cells RNA polymerase is a protein andpeptidyl transferase consists of RNA suggests theexistence of a mutual catalytic dependence betweenthese two kinds of biopolymers. I suggest that thisdependence is a `frozen accident', a remnant from thefirst living system. This system is proposed to be acombination of an RNA molecule capable of catalyzingamino acid polymerization and the resulting proteinfunctioning as an RNA-dependent RNA polymerase. Thespecificity of the protein synthesis is thought to beachieved by the composition of the surrounding mediumand the specificity of the RNA synthesis – by Watson– Crick base pairing. Despite its apparent simplicity,the system possesses a great potential to evolve intoa primitive ribosome and further to life, as it isseen today. This model provides a possible explanationfor the origin of the interaction between nucleicacids and protein. Based on the suggested system, Ipropose a new definition of life as a system ofnucleic acid and protein polymerases with a constantsupply of monomers, energy and protection.     

STRAL: progressive alignment of non-coding RNA using base pairing probability vectors in quadratic time

MOTIVATION: Alignment of RNA has a wide range of applications, for example in phylogeny inference, consensus structure prediction and homology searches. Yet aligning structural or non-coding RNAs (ncRNAs) correctly is notoriously difficult as these RNA sequences may evolve by compensatory mutations, which maintain base pairing but destroy sequence homology. Ideally, alignment programs would take RNA structure into account. The Sankoff algorithm for the simultaneous solution of RNA structure prediction and RNA sequence alignment was proposed 20 years ago but suffers from its exponential complexity. A number of programs implement lightweight versions of the Sankoff algorithm by restricting its application to a limited type of structure and/or only pairwise alignment. Thus, despite recent advances, the proper alignment of multiple structural RNA sequences remains a problem. RESULTS: Here we present StrAl, a heuristic method for alignment of ncRNA that reduces sequence-structure alignment to a two-dimensional problem similar to standard multiple sequence alignment. The scoring function takes into account sequence similarity as well as up- and downstream pairing probability. To test the robustness of the algorithm and the performance of the program, we scored alignments produced by StrAl against a large set of published reference alignments. The quality of alignments predicted by StrAl is far better than that obtained by standard sequence alignment programs, especially when sequence homologies drop below approximately 65%; nevertheless StrAl's runtime is comparable to that of ClustalW.     

Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and of protein in the liver and the lung of the Syrian golden hamster

1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent K(m) and V(max.) values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent K(m) values were very similar in both organs. From the estimated V(max.,) it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time-response and a dose-response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose-response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver.     

Experimental evolution of RNA versus DNA viruses

Based on their extremely high mutation rates, RNA viruses have been traditionally considered as the fastest evolving entities in nature. However, recent work has revealed that, despite their greater replication fidelity, single-stranded (ss) DNA viruses can evolve fast in a similar way. To further investigate this issue, we have compared the rates of adaptation and molecular evolution of ssRNA and ssDNA viruses under highly controlled laboratory conditions using the bacteriophages ΦX174, G4, f1, Qβ, SP, and MS2 as model systems. Our results indicate that ssRNA phages evolve faster than ssDNA phages under strong selective pressure, and that their extremely high mutation rates appear to be optimal for this kind of scenario. However, their performance becomes similar to that of ssDNA phages over the longer term or when the population is moderately well-adapted. Interestingly, the roughly 100-fold difference between the mutation rates of ssRNA and ssDNA phages yields less than a fivefold difference in adaptation and nucleotide substitution rates. The results are therefore consistent with the observation that, despite their lower mutation rates, ssDNA viruses can sometimes match the evolvability of RNA viruses.     

Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.     

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.     

Ribonucleotides in DNA: Origins,repair and consequences

While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.     

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.     

Quasi-species evolution in subdivided populations favours maximally deleterious mutations

Most models of quasi-species evolution predict that populations will evolve to occupy areas of sequence space with the greatest concentration of neutral sequences, thus minimizing the deleterious mutation rate and creating mutationally 'robust' genomes. In contrast, empirical studies of the principal model of quasi-species evolution, RNA viruses, suggest that the effects of deleterious mutations are more severe than in similar DNA-based microbes. We demonstrate that populations divided into discrete patches connected by dispersal may favour genotypes where the deleterious effect of non-neutral mutations is maximized. This effect is especially strong in the absence of back mutation and when the amount of time spent in hosts prior to dispersal is intermediate. Our results indicate that RNA viruses that produce acute infections initiated by a small number of virions are expected to evolve fragile genetic architectures when compared with other RNA viruses.     

Glyoxylate as a Backbone Linkage for a Prebiotic Ancestor of RNA

The origin of the first RNA polymers is central to most current theories for the origin of life. Difficulties associated with the prebiotic formation of RNA have lead to the general consensus that a simpler polymer preceded RNA. However, polymers proposed as possible ancestors to RNA are not much easier to synthesize than RNA itself. One particular problem with the prebiotic synthesis of RNA is the formation of phosphoester bonds in the absence of chemical activation. Here we demonstrate that glyoxylate (the ionized form of glyoxylic acid), a plausible prebiotic molecule, represents a possible ancestor of the phosphate group in modern RNA. Although in low yields (∼ 1%), acetals are formed from glyoxylate and nucleosides under neutral conditions, provided that metal ions are present (e.g., Mg²⁺), and provided that water is removed by evaporation at moderate temperatures (e.g., 65 ^∘C), i.e. under “drying conditions”. Such acetals are termed ga-dinucleotides and possess a linkage that is analogous to the backbone in RNA in both structure and electrostatic charge. Additionally, an energy-minimized model of a gaRNA duplex predicts a helical structure similar to that of A-form RNA. We propose that glyoxylate-acetal linkages would have had certain advantages over phosphate linkages for early self-replicating polymers, but that the distinct functional properties of phosphoester and phosphodiester bonds would have eventually lead to the replacement of glyoxylate by phosphate.     

THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS : Ultrastructural and Biochemical Studies

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.     

Taming of the shrewd: novel eukaryotic genes from RNA viruses

Genomes of several yeast species contain integrated DNA copies of complete genomes or individual genes of non-retroviral double-strand RNA viruses as reported in a recent BMC Biology article by Taylor and Bruenn. The integrated virus-specific sequences are at least partially expressed and seem to evolve under pressure of purifying selection, indicating that these are functional genes. Together with similar reports on integrated copies of some animal RNA viruses, these results suggest that integration of DNA copies of non-reverse-transcribing RNA viruses might be much more common than previously thought. The integrated copies could contribute to acquired immunity to the respective viruses.     

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3''-deoxyadenosine 5''-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.     

Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration-dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.     

Conversion of Biosynthetic Precursors of RNA to Those of DNA by Photoredox Chemistry

Soon after the origin of RNA-based life, depletion of prebiotically synthesised ribonucleotides would have driven the evolution of a biosynthetic pathway to these key building blocks. Ribozyme-catalysed nucleosidation—the key biosynthetic step—requires that ribose and the nucleobases are produced by abiotic chemistry and are relatively stable to the conditions of their synthesis. The most plausible prebiotic synthesis of sugars involves photoreduction of cyanohydrins by hydrogen sulphide in the presence of copper(I) cyanide, and we therefore subjected ribose to these conditions whereupon it was partially converted to 2-deoxyribose. Furthermore, a derivative of uracil is reduced under similar conditions to thymine. Thus, DNA biosynthetic precursors can be formed abiotically from those of RNA allowing for an early evolutionary transition to life based on RNA and DNA.     

Translation of viral mRNA without active eIF2: the case of picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.