The RNA World and Its Evolution

This paper develops Belozersky’s early idea of the precedence of RNA in the origin of life on the Earth. Based on the current knowledge of the functional omnipotence of RNA, three new mechanisms are considered that could be critical for the origin and evolution of the ancient RNA world: (1) the reaction of spontaneous transesterification of polyribonucleotides in aqueous media, which has been recently discovered by A.B. Chetverin and colleagues and could result in elongation of short initial oligoribonucleotides and generate sequence variants for further selection; (2) compartmentation of functional RNA ensembles in the form of mixed molecular colonies on moist mineral surfaces, in the absence of membranes and other envelopes; and (3) systematic exponential enrichment of an RNA population with “ functionally the best” molecules due to alternating dissolution of the colonies upon flooding and formation of new colonies upon drying in ancient pools (“primordial natural SELEX”).__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 550–556.Original Russian Text Copyright © 2005 by Spirin.     

Ancient RNA world

Based on the modern concept of multiplicity of RNA functions, certain new mechanisms, which could have played a key role in the origin and evolution of the ancient RNA world, are discussed. In particular, the reaction of spontaneous transesterification of polyribonucleotides, which was discovered by A.B. Chetverin and colleagues, could result in elongation of short initial oligoribonucleotides and produce sequence variants for further natural selection of accidentally arising functionally active molecules. Further, the formation of mixed molecular colonies of RNA on moist solid media, such as clays, could have provided compartmentation of functional RNA ensembles in the absence of envelopes or membranes, which were necessary for further evolution of the RNA world. The systematic exponential enrichment of the RNA population with functionally the best molecules because of alternating dissolution of colonies due to flooding and formation of new colonies due to drying in ancient pools (“primordial natural SELEX”) could be critical for the evolutionary process in the RNA world.     

Queen polymorphism and queen-morph related facultative polygyny in the ant, <Emphasis Type="Italic">Myrmecina graminicola</Emphasis> (Hymenoptera,Formicidae)

Summary: Polymorphism of the functional queens in Myrmecina graminicola is analyzed. Both gynomorphs (G-§§ G) and a wide range of intermorphs (I-§§ I) occur, which all are usually mated and egg-laying. Colonies having a gynomorphic queen are always monogynous, whereas about 57% of all colonies with intermorphic queens are polygynous, having two or more coexisting functional queens. The female sexual offspring of individual gynomorphic queens either consists of gynomorphs only, or exclusively of intermorphs. Intermorphic queens may have exclusively intermorphic female sexual progeny, or simultaneously both gynomorphs and intermorphs. Single colonies in laboratory culture produce the same kind of female progeny over several subsequent breeding cycles (artificially compressed "years" of 9-10 months). No environmental influence on queen morph determination could be detected. A genetically mediated queen polymorphism, as in Harpagoxenus sublaevis and Leptothorax sp. A, is suggested. Colony sizes vary considerably, with polygynous I-queen colonies being largest (57.2 - 34.3 s.d. workers), followed by G-queen colonies (44.6 - 22.7 s.d.) and monogynous I-queen colonies (34.4 - 23.7 s.d.), suggesting occasional budding of polygynous colonies.     

Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene.

Von Willebrand Factor (vWF) mRNA was identified in fractionated polyA+ RNA preparations isolated from cultured human endothelial cells. Micro-injection of specific polyA+ RNA fractions in Xenopus laevis oocytes provoked the synthesis of a vWF-like product which could be detected with an immunoradiometric assay relying on Sepharose-linked monoclonal anti-vWF IgG and different radiolabeled monoclonal anti-vWF IgGs. A vWF-mRNA-containing polyA+ RNA preparation served as substrate for a size-selected cDNA-expression library of 60 000 colonies which was screened for the synthesis of antigens related to vWF, using polyclonal anti-vWF IgG and a second antibody conjugated with peroxidase. Eight positive colonies were detected of which two reacted strongly in the enzyme-linked assay. Immunoblotting of bacterial extracts of "expression clones" with a monoclonal anti-vWF IgG revealed polypeptides which size fits within the length of the cDNA insertions. Northern blotting of human endothelial RNA, employing fragments of vWF cDNA as probes, showed specific hybridization with a mRNA of about 9000 nucleotides. DNA-sequence analysis of a vWF-cDNA insertion revealed an open reading frame followed by a translation stopcodon. It is argued that the cDNA insertions encode the carboxy-terminal part of the vWF protein. vWF-cDNA probes were employed to map the von Willebrand factor gene on chromosome 12 using a panel of 35 human-rodent somatic cell hybrids.     

Three helical domains form a protein binding site in the 5S RNA-protein complex from eukaryotic ribosomes.

A ribosomal protein binding site in the eukaryotic 5S rRNA has been delineated by examining the effect of sequence variation and nucleotide modification on the RNA's ability to exchange into the EDTA-released, yeast ribosomal 5S RNA-protein complex. 5S RNAs of divergent sequence from a variety of eukaryotic origins could be readily exchanged into the yeast complex but RNA from bacterial origins was rejected. Nucleotide modifications in any of three analogous helical regions in eukaryotic 5S RNAs of differing origin reduced the ability of this RNA molecule to form homologous or heterologous RNA-protein complexes. Because sequence comparisons did not indicate common nucleotide sequences in the interacting helical regions, a model is suggested in which the eukaryotic 5S RNA binding protein does not simply recognize specific nucleotide sequences but interacts with three strategically oriented helical domains or functional groups within these domains. Two of the domains bear a limited sequence homology with each other and contain an unpaired nucleotide or "bulge" similar to that recently reported for one of the 5S RNA binding proteins in Escherichia coli (Peattie, D.A., Douthwaite, S., Garrett, R.A. and Noller, H.F. (1981) Proc. Natl. Acad. Sci. 78, 7331-7335). The results further indicate that the single ribosomal protein of eukaryotic 5S RNA-protein complexes interacts with the same region of the 5S rRNA molecule as do the multiple protein components in complexes of prokaryotic origin.     

Molecular weights,poly(A)-content,and partial separation of chick globin mRNAs

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000±10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% -chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for -chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the and mRNAs and differences in the poly(A) lengths.     

Lagged Population Growth in a Termite Host Colony: Cause or Consequence of Inquilinism?

The presence of foreign organisms in the colonies of social insects could affect energy allocation to growth and reproduction of these hosts. Highly specialized invaders of such long-lived hosts, however, can be selected to be less harmful. After all, it pays for these symbionts to keep their host’s good health thereby prolonging cohabitation in the homeostatic environment of the termite colony. Here, we investigated such a hypothesis, focusing on populational parameters of a termite host sharing its nest with an obligatory termite inquiline. To this end, 19 natural colonies of Constrictotermes cyphergaster (Silvestri, 1901) (Termitidae: Nasutitermitinae) were sampled and the (i) number of individuals, (ii) proportion of soldier/workers in the colonies, and (iii) presence/absence of obligatory inquiline Inquilinitermes microcerus (Silvestri, 1901) (Termitidae: Termitinae) were measured. Results revealed a negative correlation between the number of individuals and the proportion of soldier/workers in the host colonies with the presence of I. microcerus colonies. In search of causal mechanisms for such a correlation, we inspected life history traits of both, inquilines and hosts, hypothesizing that such a result could indicate either (i) a dampening effect of the inquiline upon its host population or (ii) the coincidence of the moment of inquiline infiltration with the natural reduction of C. cyphergaster populational growth at the onset of its reproductive phase.     

Scientific and practical applications of molecular colonies

Reviewed are the history of invention of the molecular colony technique, also known under name "polony technology", applications of this method to studies of reactions between single RNA molecules, ultrasensitive diagnostics, gene cloning and screening in vitro, and also concepts on the origin of life that consider molecular colonies as a prototype of living organisms.     

Omnipotent RNA

The capability of polyribonucleotide chains to form unique, compactly folded structures is considered the basis for diverse non-genetic functions of RNA, including the function of recognition of various ligands and the catalytic function. Together with well-known genetic functions of RNA – coding and complementary replication – this has led to the concept of the functional omnipotence of RNA and the hypothesis that an ancient RNA world supposedly preceded the contemporary DNA–RNA–protein life. It is proposed that the Woese universal precursor in the ancient RNA world could be a cell-free community of mixed RNA colonies growing and multiplying on solid surfaces.     

The dawn of the RNA World: Toward functional complexity through ligation of random RNA oligomers

A main unsolved problem in the RNA World scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers obtained by random polymerization on mineral surfaces. A number of computational studies have shown that the structural repertoire yielded by that process is dominated by topologically simple structures, notably hairpin-like ones. A fraction of these could display RNA ligase activity and catalyze the assembly of larger, eventually functional RNA molecules retaining their previous modular structure: molecular complexity increases but template replication is absent. This allows us to build up a stepwise model of ligation-based, modular evolution that could pave the way to the emergence of a ribozyme with RNA replicase activity, step at which information-driven Darwinian evolution would be triggered.     

Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light

Background  

A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world"). However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth.     

Virulence plasmid stability in environmentally occurring <Emphasis Type="Italic">Bacillus anthracis</Emphasis> from North East Turkey

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule ?ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.     

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

A replication origin is turned off by an origin-"silencer" sequence

The chromosome of R6K contains multiple origins of replication. The origin gamma is infrequently used in the original plasmid and remains "silent" in certain miniplasmid derivatives. The inactivation of the origin is caused by a natural origin silencer located adjacent to the minimal ori gamma sequence. The silencer functions in cis and has no trans activity. It has functional polarity and works only in one orientation when present immediately downstream from ori gamma. The silencer apparently initiates an RNA that invades ori gamma and turns it off either by competing with a primer RNA or by disrupting ori gamma structure. As predicted, removal of the silencer blocks the synthesis of silencer RNA and derepresses the origin.     

A physical origin for functional domain structure in nucleic acids as evidenced by cross-linking entropy: II.

In Part I, cross-linking entropy (CLE) was proposed as a mechanism that limits the size of functional domains of RNA. To test this hypothesis, the theory is developed into an RNA secondary structure prediction filter which is applied to nearest-neighbor secondary structure (NNSS) algorithms that utilize a free energy (FE) minimization strategy. (The NNSS strategies are also referred to as the dynamic programming algorithm in the literature.) The cross-linking entropy for RNA is derived from a generalized Gaussian polymer chain model where the entropic contributions caused by the formation of base pairs (stacking) in RNA are analysed globally. Local entropic contributions are associated with the freezing out of degrees of freedom in the links. Both global and local entropic effects are strongly influenced by the persistence length. The cross-linking entropy provides a physical origin for the size of functional domains in long nucleic acid sequences and may go further to explain as to why the majority of the domain regions in typical sequences tend to be less than 600 nucleotides in length. In addition, improvements were observed in the "best guess" predictive capacity over NNSS prediction strategies. The thermodynamic distribution is more representative of the expected structures and is strongly governed by such physical parameters as the persistence length and the excluded volume. The CLE appears to generalize the tabulated penalties used in NNSS algorithms. The principal parameter influencing this entropy is the persistence length. The model is shown to accomodate a variable persistence length and is capable of describing the folding dynamics of RNA. A two-state kinetic model based on the CLE principle is used to help elucidate the folding kinetics of functional domains in the group I introns.     

A spreading colony formed method for rapid evaluation of dicarboximide fungicides resistance level of field tobacco brown spot disease

A new method (spreading colony formed method) for rapidly identifying and evaluating the dicarboximide fungicides resistance level of field tobacco spot brown disease caused byAlternaria longipes was developed. Two typical colonies with distinct differences in colony morphology on media containing 5 μg/ml dicarboximide fungicides dimethachlon (CAS registration number: 24096-53-5) were discovered by using this method. The two typical colonies were named spreading colony and dense pad colony, respectively. Isolates (250) ofA. longipes were quickly separated by this method, and their growth properties (including the sensitivity to dimethachlon, the cross-resistance to phenylpyrroles fludioxonil and hyphal development) were examined. Our results indicated that (1) monospore isolates from spreading colonies and dense pad colonies were respectively resistant and sensitive to dimethachlon; (2) resistant and sensitive isolates formed respectively spreading colonies and dense pad colonies on dimethachlon media. Furthermore, molecular experiments confirmed the spreading colony formed method reliable. In conclusion, field resistant isolates and resistant situation in population level of field tobacco spot brown disease could be exactly and timely determined and evaluated by spreading colony formed method.     

Evidence of heterokaryosis in streptomyces coelicolor A3(2)

Summary Hyphae from mixed cultures of complementary auxotrophs of Streptomyces coelicolor A3(2) did not grow on minimal media (MM) when fertility plasmids (SCP1 and SCP2) were missing in both strains. The addition of one part per cent of complete medium (CM) to MM allowed growth of vigorous colonies among the tiny colonies of the parental types. The former, amounting to 1%–10% of the total population, turned out to be heterokaryons. They could be propagated on the same medium by plating of hyphal fragments. When five parts per cent of CM were added to MM, beside the heterokaryotic colonies vigorous spindles of aerial mycelium were formed whenever complementary colonies overlapped. When the SCP1 and SCP2 plasmids were present in one or both parents a clear constraint on the outburst of heterokaryotic aerial mycelium was observed.