2 A prebiotic RNA apparatus functions within the contemporary ribosome

Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region, which was identified within all contemporary ribosomes. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA molecular apparatus. Hence, we named it the “proto-ribosome”. This prebiotic pocket-like RNA entity is suggested to be capable to accommodate substrates whose stereochemistry enables the creation of chemical bonds. It could have evolved from an earlier catalytic RNA entity that we named the “pre-proto-ribosome”, presumed to be a molecular machine capable of performing various essential tasks in the RNA world, which was snatched by the amino acid invaders for producing proteins.     

The synthesis and turnover of virus-specific polyadenylated RNA in polyoma-infected cells.

Mouse embryo cells infected with the 3049 strain of polyoma virus contain several fold more virus-specific, polyadenylated RNA beginning between 4 and 8 hours after the onset of viral DNA synthesis than do cells infected with wild-type virus (lpS). Following infection with either virus strain, there is an identical small but significant enhancement of the level of total polyadenylated RNA measured by binding of ¹²⁵I-labeled RNA to poly(dT)cellulose. The polyadenylation of “early” virus-specific RNA is inhibited 85–90% by cordycepin resulting in an “early” RNA preparation which competes fully with polyadenylated “early” virus-specific RNA in the ternary complex assay. Utilizing the nonpolyadenylated “early” RNA, competition hybridization demonstrated that approximately 78% of the enlarged pool of “late” 3049 polyadenylated RNA and 72% of the “late” lpS pool consisted of sequences unique to the “late” period. No significant difference in the rate of decay of 3049 and lpS-specific, “late” polyadenylated RNA following actinomycin D block was found. Infection by either strain of polyoma virus did not alter the rate of decay of total polyadenylated RNA.     

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Abstract

Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable “core” of housekeeping genes accompanied by a much more flexible “shell” consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the “shell” genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution.

Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the “shell” genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages.

The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses.

Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.     

Monte Carlo simulation of early molecular evolution in the RNA World

The origin of life remains a highly speculative field, mainly due to the shortage of our knowledge on prebiotic chemistry and basic understanding on the essence of life. In this context, computer simulation is expected to play an important role. For instance, the scenario concerning the genesis of the widely accepted RNA World remains blurry, though we have gathered some circumstantial evidence and fragmented knowledge on several supposed stages, including formation of polynucleotides from a prebiotic nucleotide pool, emergence of RNA replicases (RNA molecules catalyzing their own replication), and evolution of RNA replicases. It is highly valuable to simulate the stages as a continuous process to evaluate the plausibility of the supposition and study the rules involved. Here we construct a computer simulation on the process using Monte Carlo method. It demonstrates that primordial RNA replicases may appear and spread in a nucleotide pool provided they could recognize their own sequence and their complements as catalytic targets, and then may evolve to more efficient RNA replicases. Apart from its indication on the genesis of the RNA World, the vivid simulation of emergence of the “first replicative molecules” and their subsequent evolution is impressive and may help to get insight into “how could self-replication and Darwinian evolution, two key features of life, emerge in a non-life background?” thus improve our understanding of “what is life” when studying origins of life.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

Evolution of Helix Formation in the Ribosomal Internal Transcribed Spacer 2 (ITS2) and Its Significance for RNA Secondary Structures

Helices are the most common elements of RNA secondary structure. Despite intensive investigations of various types of RNAs, the evolutionary history of the formation of new helices (novel helical structures) remains largely elusive. Here, by studying the nuclear ribosomal Internal Transcribed Spacer 2 (ITS2), a fast-evolving part of the eukaryotic nuclear ribosomal operon, we identify two possible types of helix formation: one type is “dichotomous helix formation”—transition from one large helix to two smaller helices by invagination of the apical part of a helix, which significantly changes the shape of the original secondary structure but does not increase its complexity (i.e., the total length of the RNA). An alternative type is “lateral helix formation”—origin of an extra helical region by the extension of a bulge loop or a spacer in a multi-helix loop of the original helix, which does not disrupt the pre-existing structure but increases RNA size. Moreover, we present examples from the RNA sequence literature indicating that both types of helix formation may have implications for RNA evolution beyond ITS2.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

Die Eh da‐Initiative

The “Eh da”‐Initiative: more space for Biological Diversity in Cultural Landscapes The “Eh da”‐initiative is based on the principle that definite land in cultural landscapes is “available anyway” (what ?eh da“ means in German) and has the potential for ecological upgrading without relevant limitations of land use. “Eh da” could be the acronym “Ecological habitat development areas.” This land is located in open landscapes: along waysides, on uncropped plots in farmland, it could be communal lawn and other land categories. The initiative uses geodata in order to detect and quantify “Eh da”‐sites. According to an analysis based on geodata in selected landscapes “Eh da”‐land constitutes 2–6 per cent of the total area of Germany. “Eh da“‐sites are mostly narrow, longitudinal and spread like a net all over the landscape. Mainly insects and other invertebrate animals can be supported by upgrading of “Eh da”‐land. Since these sites often form corridors, they may be part of communal biodiversity protection initiatives under the perspective of ecological networks, or they may be used for distinct projects. Communication is a key element of any local initiative in which not only the ecological upgrading options, but also potential trade‐offs (like increase of agricultural pests and weeds, neophyta and pyrrolizidine containing or allergenic plants) should be discussed.     

Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

Nature of the ribosomal RNA transcribed from the X and Y chromosomes of Drosophila melanogaster

In Drosophila melanogaster there is one nucleolar organizer (NO) on each X and Y chromosome. Experiments were carried out to compare the ribosomal RNAs derived from the two nucleolar organizers. ³²PO₄-labelled ribosomal RNA was isolated from two strains of D. melanogaster, one containing only the X chromosome NO, the other containing only the Y chromosome NO. 28 S and 18 S RNA from the two strains were subjected to a variety of “fingerprinting” and sequencing procedures. Fingerprints of 28 S RNA were very different from those of 18 S RNA. Fingerprints of “X” and “Y” 28 S RNA were indistinguishable from each other, as also were fingerprints of “X” and “Y” 18 S RNA. In combined “T₁ plus pancreatic” RNAase fingerprints several distinctive products were characterized and quantitated. Identical products were obtained from X and Y RNA, and the molar yields of the products were indistinguishable. Together these findings imply that the rRNA sequences encoded by the X and Y NOs are closely similar and probably identical to each other.Two further findings were of interest in “T₁ plus pancreatic” RNAase fingerprints: (1) in 28 S (as well as in 18 S) fingerprints several distinctive products were recovered in approximately unimolar yields. This indicates that 28 S RNA does not consist of two identical half molecules, though it does consist of two non-identical half molecules together with a “5.8 S” fragment. (2) Several methylated components in Drosophila rRNA also occur in rRNA from HeLa cells and yeast. This suggests that certain features of rRNA structure involving methylated nucleotides may be highly conserved in eukaryotic evolution.     

The interpretation of oligonucleotide maps. A theoretical study of nucleic acid digests with special reference to repeated diverged sequences

Oligonucleotide mixtures produced by the digestion of RNA by specific nucleases can be defined in terms of isostichs (sets of common chain length), compositional isomers, and sequence isomers. Equations are derived to express the distribution of radioactivity on the isostich length, the distribution of compositional isomers on the isostich length, and the distribution of compositional isomers on the proportion of total radioactivity (“intensity”). It is shown how the properties of a “fingerprint” may be calculated from first principles, and conversely how the complexity of a sequence may be estimated from its fingerprint. The equations are tested by means of computer simulations of RNA digestions and their range of applicability is determined. The three distributions are used to analyze the digests of repetitious sequences. It is shown how the parameters of a diverged sequence family can, in a favorable case, be deduced from its fingerprint.     

Poly(A) synthesis by RNA polymerase II from rat liver

Multiple forms of DNA-dependent RNA polymerase were resolved by DEAE-Sephadex chromatography. In addition to RNA polymerases, an active poly(A) polymerase was also fractionated. RNA polymerases were examined for their capacity to synthesize poly(A). None of the freshly prepared enzymes could efficiently make poly(A) in presence or absence of exogenous primers. However, “aging” of polymerase II by simple incubation at 37°C resulted in the loss of RNA polymerizing activity with a corresponding increase in poly(A) synthesizing activity. Transformation of RNA polymerase to poly(A) polymerase resulted in reduced capacity to transcribe native DNA and altered chromatographic behavior. The results suggest that subunits of polymerase II obligatory to DNA-dependent RNA synthesis were degraded by “aging” and that a stable subunit of the RNA polymerase could preferentially make poly(A).     

Can tumorigenesis result from in vivo “transfection” of a normal cell with DNA escaped from disintegrating nontransformed cells?

In this paper I present a hypothetical step on the route to turmorigenesis which may be common to many of the tumorogenic events taking place in vivo. According to this hypothesis portions of DNA from normal nontransformed dying cells may escape degradation, “transfect” other cells and under appropriate conditions also induce transformation. When various high risk factors for carcinogenesis are examined this hypothesis may easily fit into each of them. In addition, recent reports of experimental “transfection” indirectly support this hypothesis. It could now be argued that aging, ionizing radiation, immunosuppressive drugs, genetic errors, defects in DNA repair, immunodeficiency states, chronic infections and chronic inflammatory diseases may all increase the availability of free DNA or the readiness of cells to accommodate “transfection” DNA, or both. Thus the risk of cancer associated with these situations may be explained by increased chance for “transfection” with DNA.It is suggested that the minimal requirements for cell transformation consist of a certain sequence of DNA which does not have to be integrated into the nucleus at once, it may instead accumulate piece by piece so that the first “transfection” of a cell may occur long before transformation takes place. If the “transfecting” DNA sequence does not fulfil the minimal requirements for maintaining a state of repetitive uncontrolled mitosis, then this cell may wait silently or remain a benign tumor until “boosted” with an ultimate complementary DNA sequence to develop into a fully fledged cancer.     

Cellular Changes Accompanying the Transition from Minimal to Maximal Rate of Extracellular Enzyme Secretion by Bacillus amyloliquefaciens

S ummary . During exponential growth of Bacillus amyloliquefaciens in a maltose–L-amino acids medium at 30°, cellular protein, RNA and DNA increased in parallel. After the exponential phase, extracellular protein, including α-amylase, was secreted into the medium at a quarter of the maximum rate of total cellular protein synthesis. The free amino acid pool for protein and the 'nucleotide'pool for RNA both increased fourfold during the transition to the post-exponential phase to 4.0 and 1.6%, respectively, of the cellular dry weight. Subsequently, the nucleotide pool did not change significantly whilst the free amino acid pool was reduced to 2/3 of its maximum size. When post-exponential phase, exoprotein-secreting bacteria were transferred to fresh culture medium, growth was re-established and there was a 4–5 fold reduction in nucleotide pool size accompanied by a loss of exoenzyme-forming ability.     

A hypothesis on the role of immune RNA in antibody variability

The present hypothesis on the mechanism of antibody variability considers immune RNA (IMRNA) as a relatively independent “entity”. After having been transcribed in ontogenesis, as a result of stimulation of primordial multipotent “master cells” by primordial antigens, IMRNA behaves like an RNA virus genome. It controls only the variable part of immunoglobulin chains, proliferates and is transferred from committed to uncommitted cells, directly or using the macrophage as an “intermediate host”. During IMRNA proliferation “mutant” IMRNAs appear and control the synthesis of antibody variable regions of new specificity. The IMRNAs are reverse transcribed and inserted into DNA, chromosomal or extrachromosomal, near the gene controlling the constant part, and a complete CV gene is formed. The selective pressures which assure a relative constancy of IMRNA, so that only changes in the so-called hypervariable region are “tolerated” might be: the recognition by replicase, the insertion device, and the antigen-surface immunoglobulin-membrane receptor interaction. Arguments from immunology and from other fields of biology are brought in support at this hypothesis, and experimental approaches are suggested.     

A new conception of the shoot of higher plants

According to the classical model, the “shoot” consists only of the categories “caulome” (“stem” sensu lato) and “phyllome” (“leaf” sensu lato), (and “root” in cases of “adventitious” root formation). If lateral shoots are present, their position is axillary. Consequently, caulome as well as phyllome are inserted on the caulome and only on the caulome. This classical model of the shoot has two disadvantages of great consequence: (1) Intermediate organs cannot be accepted as such, but have to be interpreted (i.e. categorized) as either caulome or phyllome (or root) by distortion of the actual similarity. (2) Certain positional changes of organs cannot be accepted as such, but have to be “explained” by congenital fusion. The new conception of the shoot will have the advantages of the classical model but not its disadvantages. Hence, the shoot may consist of the following parts: (main and lateral) shoot, caulome, phyllome, root, emergence, and structures intermediate between (i.e. partially homologous to) any of the preceding. Thus, the five categories of the classical model, namely “shoot”, “caulome”, “phyllome”, “root” and “emergence” are no longer mutually exclusive; they may merge into each other due to an actual or potential continuum. Intermediate organs are therefore accepted as such; for example, an organ may be characterized as an intermediate form between a caulome and a phyllome. Besides intermediate forms, all changes in position are accepted as such. Hence, the following positional relations are possible: caulome and phyllome may be inserted on the caulome, caulome and phyllome may be inserted on the phyllome; roots may be inserted on caulome or phyllome; intermediate forms may be inserted on the caulome, phyllome, or other intermediate forms. Consequences of the new conception for morphological research are pointed out, especially for homologization, evolutionary considerations, and the direction in which research progresses.