5 Efficient and high-fidelity copying of an RNA-like model prebiotic system

Nonenzymatic RNA replication would provide an important bridge to the RNA world. However, the demonstration of efficient and high-fidelity copying chemistry remains a great experimental challenge. It requires an efficient mechanism that can lead to both a high rate of polymerization and a high degree of fidelity in the copying chemistry. Previous experiments concerning nonenzymatic template-directed synthesis of RNA with activated monomers have led to the copying of short RNA templates, but these reactions are generally slow (taking days to weeks) and highly error-prone. Therefore, the ability to efficiently and accurately copy arbitrary template sequences remains frustratingly out of reach. N3′-P5′-linked phosphoramidate DNA is a highly reactive model for self-replicating genetic materials and has been used for studies of nonenzymatic RNA self-replication. It is also an excellent RNA mimic, due to its similar overall duplex structure, rigidity, and level of hydration (Tereshko, Gryaznov, & Egli, 1998). Our experiments show that the high reactivity imparted by the presence of an amino nucleophile allows rapid and efficient copying of all four nucleobases on both homopolymeric and mixed templates. On the other hand, G:T wobble pairing leads to a high error rate. We have, therefore, investigated the use of the modified nucleobase, 2-thio T (Ts) (Sintim & Kool, 2006), to suppress formation of the G:T wobble base-pair. Our results illustrate that the 2-thio modification can both increase polymerization rate and enhance fidelity in this self-replicating N3′-P5′-DNA system. These results suggest that this simple nucleobase modification may have played a role in primordial RNA (or proto-RNA) replication. In addition to suppressing the G:T mismatch, an additional benefit gained from its stronger base-pairing with A is that it also reduces A:C mismatch formation. Thus, simple modifications of nucleobases might provide a means of suppressing mismatches to yield better fidelity. Taken together, our results show that a high rate of polymerization and a high degree of fidelity are not mutually exclusive, but can be achieved simultaneously in nonenzymatic copying of N3′-P5′-linked phosphoramidate DNA. The structural similarity of NP-DNA to RNA suggests that these results could be translated to an RNA-only system.     

Error-prone replication for better or worse

Precise genome duplication requires accurate copying by DNA polymerases and the elimination of occasional mistakes by proofreading exonucleases and mismatch repair enzymes. The commonly held belief that 'if something is worth doing, then it's worth doing well' normally applies to DNA replication and repair, however, there are exceptions. This review describes elements that are crucial to cell fitness, evolution and survival in the recently discovered error-prone DNA polymerases. Large numbers of errant DNA polymerases, spanning microorganisms to humans, are used to rescue stalled replication forks by copying damaged DNA and even undamaged DNA to generate 'purposeful' mutations that generate genetic diversity in times of stress. Here we focus on low-fidelity polymerases from bacteria, comparing Escherichia coli, archeabacteria and those most recently discovered in Gram-positive Bacilli, Streptococcus, pathogenic Mycobacterium and intein-containing cyanobacteria.     

Helicases that underpin replication of protein-bound DNA in Escherichia coli

A pre-requisite for successful cell division in any organism is synthesis of an accurate copy of the genetic information needed for survival. This copying process is a mammoth task, given the amount of DNA that must be duplicated, but potential blocks to replication fork movement also pose a challenge for genome duplication. Damage to the template inhibits the replication machinery but proteins bound to the template such as RNA polymerases also present barriers to replication. This review discusses recent results from Escherichia coli that shed light on the roles of helicases in overcoming protein-DNA barriers to replication and that may illustrate fundamental aspects of how duplication of protein-bound DNA is underpinned in all organisms.     

Principles and Concepts of DNA Replication in Bacteria,Archaea, and Eukarya

The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle.The genetic information within the cells of our body is stored in the double helix of DNA, a long cylinderlike structure with a radius that is only 10 Å or one billionth of a meter but can be of considerable length. A single DNA molecule within a bacterium that grows in our gut flora is approximately 5 million base pairs in length and when stretched out, is about 1.6 mm in length, roughly the diameter of a pinhead. In contrast, the single DNA molecule in the largest human chromosome is 245,203,898 base pairs or about 8.33 cm long. The entire human genome, consisting of its 24 different chromosomes in a male is about 3 billion base pairs or 1 m long. Each cell in our body, with rare exceptions, contains two copies of the genome and thus 2 m of total DNA. Thus the scale and complexity of duplicating genomes is remarkable. For example, ∼2200 human cells can sit on the top of a 1.5 mm pinhead and when extracted and laid out in a line, the DNA from these cells would be ∼4.5 km (2.8 miles) long. In our body, about 500–700 million new blood cells are born every minute in the bone marrow (Doulatov et al. 2012), containing a total of about 1 million km of DNA, or enough DNA to wrap around the equator of the earth 25 times. Thus DNA replication is a serious business in our body, occurring from the time that a fertilized egg first begins duplicating DNA to yield the many trillions of cells that make up an adult body and continuing in all tissues of the adult body throughout our life. The amount of DNA duplicated in an entire human body represents an unimaginable amount of information transfer. Moreover, each round of duplication needs to be highly accurate, making one mistake in less than 100 million bases copied per cell division. How copying of the double helix occurs and how it is so highly accurate is the topic of this collection. Inevitably the processes of accurate copying of the genome can go awry, yielding mutations that affect our lives, and thus the collection outlines the disorders that accelerate human disease.However, the problem of copying DNA is much more complicated than indicated above. The 2 m of DNA in each human cell is wrapped up with histone proteins within the cell’s nucleus that is only about 5 μm wide, presenting a compaction in DNA length of about 2 million-fold. How can the copying process deal with the fact that the DNA is wrapped around proteins and scrunched into a volume that creates a spatial organization problem of enormous magnitude? Not only is the DNA copied, but the proteins associated with the DNA need to be duplicated, along with all the chemical modifications attached to DNA and histones that greatly influence developmental patterning of gene expression. The protein machineries that replicate DNA and duplicate proteins within the chromosomes are some of the most complex and intriguing machineries known. Furthermore, the regulations of the processes are some of the most complex because they need to ensure that each DNA molecule in each chromosome is copied once, and only once each time before a cell divides. Errors in the regulation of DNA replication lead to accelerated mutation rates, often associated with increased rates of cancer and other diseases.The process of accurately copying a genome can be broken down into various subprocesses that combine to provide efficient genome duplication. Central to the entire process is the machinery that actually copies the DNA with high fidelity, including proteins that start the entire process and the proteins that actually copy one helix to produce two. Superimposed on this fundamental process are mechanisms that detect and repair errors and damage to the DNA. Also associated with the DNA replication apparatus are the proteins that ensure that the histone proteins and their modifications in chromatin are inherited along with the DNA. Finally, other machineries cooperate with the DNA replication apparatus to ensure that the resulting two DNA molecules, the sister chromatids, are tethered together until the cell completes duplicating all of its DNA and segregates the sister chromatids evenly to the two daughter cells. Only by combining all of these processes can genetic inheritance ensure that each cell has a faithful copy of its parent’s genome.     

Viral RNA mutations are region specific and increased by ribavirin in a full-length hepatitis C virus replication system

High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid "error catastrophe." Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5' untranslated region (5' UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3' cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5' UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.     

Biological asymmetries and the fidelity of eukaryotic DNA replication.

A diploid human genome contains approximately six billion nucleotides. This enormous amount of genetic information can be replicated with great accuracy in only a few hours. However, because DNA strands are oriented antiparallel while DNA polymerization only occurs in the 5'----3' direction, semi-conservative replication of double-stranded DNA is an asymmetric process, i.e., there is a leading and a lagging strand. This provides a considerable opportunity for non-random error rates, because the architecture of the two strands as well as the DNA polymerases that replicate them may be different. In addition, the proteins that start or finish chains may well be different from those that perform the bulk of chain elongation. Furthermore, while replication fidelity depends on the absolute and relative concentrations of the four deoxyribonucleotide precursors, these are not equal in vivo, not constant throughout the cell cycle, and not necessarily equivalent in all cell types. Finally, the fidelity of DNA synthesis is sequence-dependent and the eukaryotic nuclear genome is a heterogeneous substrate. It contains repetitive and non-repetitive sequences and can actually be considered as two subgenomes that differ in nucleotide composition and gene content and that replicate at different times. The effects that each of these asymmetries may have on error rates during replication of the eukaryotic genome are discussed.     

Temperature as a determinative factor in the evolution of genetic systems

Summary Heat induces a number of premutational lesions (for example, the deamination of cytosine to uracil) in DNA and RNA. These kinds of errors occur in resting as well as replicating polynucleotides. However, an increase in temperature also raises the probability of copying error occurring in nucleic acids because of increased thermal noise in the replicative machinery. In most modern genetic systems, the majority of heat-induced lesions are efficiently repaired. It follows that the importance of heat-induced error increases as the effectiveness of repair declines. We show in this paper that the error rate of enzymatic polynucleotide copying is expected to increase monotonically with temperature. We also explore the effects of temperature variations on the early evolution of biological information transmission mechanisms.     

"Living" under the challenge of information decay: the stochastic corrector model vs. hypercycles

The combined problem of having a large genome size when the accuracy of replication was a limiting factor is probably the most difficult transition to explain at the late stages of RNA world. One solution has been to suggest the existence of a cyclically coupled system of autocatalytic and cross-catalytic molecular mutualists, where each member helps the following member and receives help from the preceding one (i.e., a "hypercycle"). However, such a system is evolutionarily unstable when mutations are taken into account because it lacks individuality. In time, the cooperating networks of genes should have been encapsulated in a cell-like structure. But once the cell was invented, it closely aligned genes' common interests and helped to reduce gene selfishness, so there was no need for hypercycles. A simple package of competing genes, described by the "stochastic corrector model" (SCM), could have provided the solution. Until now, there is no clear demonstration that the proposed mechanisms (compartmentalized hypercycles and the stochastic corrector model) do in fact solve the error threshold problem. Here, we present a Monte Carlo model to test the viability of protocell populations that enclose a hypercyclic (HPC) or a non-hypercyclic (SCM) system when faced with realistic mutation rates before the evolution of efficient enzymic machinery for replication. The numerical results indicate that both systems are efficient information integrators and are able to overcome the danger of information decay in the absence of accurate replication. However, a population of SCM protocells can tolerate higher deleterious mutation rates and reaches an equilibrium mutational load lower than that in a population of HPC protocells.     

The two ages of the RNA world, and the transition to the DNA world: a story of viruses and cells

Most evolutionists agree to consider that our present RNA/DNA/protein world has originated from a simpler world in which RNA played both the role of catalyst and genetic material. Recent findings from structural studies and comparative genomics now allow to get a clearer picture of this transition. These data suggest that evolution occurred in several steps, first from an RNA to an RNA/protein world (defining two ages of the RNA world) and finally to the present world based on DNA. The DNA world itself probably originated in two steps, first the U-DNA world, following the invention of ribonucleotide reductase, and later on the T-DNA world, with the independent invention of at least two thymidylate synthases. Recently, several authors have suggested that evolution from the RNA world up to the Last Universal Cellular Ancestor (LUCA) could have occurred before the invention of cells. On the contrary, I argue here that evolution of the RNA world taken place in a framework of competing cells and viruses (preys, predators and symbionts). I focus on the RNA-to-DNA transition and expand my previous hypothesis that viruses played a critical role in the emergence of DNA. The hypothesis that DNA and associated mechanisms (replication, repair, recombination) first evolved and diversified in a world of DNA viruses infecting RNA cells readily explains the existence of viral-encoded DNA transaction proteins without cellular homologues. It also potentially explains puzzling observations from comparative genomic, such as the existence of two non-homologous DNA replication machineries in the cellular world. I suggest here a specific scenario for the transfer of DNA from viruses to cells and briefly explore the intriguing possibility that several independent transfers of this kind produced the two cell types (prokaryote/eukaryote) and the three cellular domains presently known (Archaea, Bacteria and Eukarya).     

Synthetic polymers and their potential as genetic materials

DNA and RNA are the only known natural genetic materials. Systematic modification of each of their chemical building blocks (nucleobase, sugar, and phosphate) has enabled the study of the key properties that make those nucleic acids genetic materials. All three moieties contribute to replication and, significantly, all three moieties can be replaced by synthetic analogs without loss of function. Synthetic nucleic acid polymers capable of storing and propagating information not only expand the central dogma, but also highlight that DNA and RNA are not unique chemical solutions for genetic information storage. By considering replication as a question of information transfer, we propose that any polymer that can be replicated could serve as a genetic material. Editor's suggested further reading in BioEssays Xenobiology: A new form of life as the ultimate biosafety tool Abstract     

Information catastrophe in RNA viruses through replication thresholds

RNA viruses are known to replicate at very high mutation rates. These rates are actually known to be close to their so-called error threshold. This threshold is in fact a critical point beyond which genetic information is lost through a so-called error catastrophe. However, the transition from a stable quasispecies to genetic drift and loss of information can also occur by crossing replication thresholds, below some replication rates, the viral population is suddenly unable to survive. Available data from hepatitis C virus population analysis [Mas, A., Ulloa, E., Bruguera, M., Furci?, I., Garriga, D., Fábregas, S., Andreu, D., Saiz, J.C., Díez, J., 2004. Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity. J. Gen. Virol. 85, 3619-3626] can be interpreted through this theoretical view, providing evidence for such a replication threshold. Here a simple model is used in order to provide evidence for such a phenomenon, consistent with available data.     

Balancing eukaryotic replication asymmetry with replication fidelity

Coordinated replication of eukaryotic nuclear genomes is asymmetric, with copying of a leading strand template preceding discontinuous copying of the lagging strand template. Replication is catalyzed by DNA polymerases α, δ and ?, enzymes that are related yet differ in physical and biochemical properties, including fidelity. Recent studies suggest that Pol ? is normally the primary leading strand replicase, whereas most synthesis by Pol δ occurs during lagging strand replication. New studies show that replication asymmetry can generate strand-specific genome instability resulting from biased deoxynucleotide pools and unrepaired ribonucleotides incorporated into DNA during replication, and that the eukaryotic replication machinery has evolved to most efficiently correct those replication errors that are made at the highest rates.     

The Paradox of Dual Roles in the RNA World: Resolving the Conflict Between Stable Folding and Templating Ability

The hypothesized dual roles of RNA as both information carrier and biocatalyst during the earliest stages of life require a combination of features: good templating ability (for replication) and stable folding (for ribozymes). However, this poses the following paradox: well-folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes. Here, we describe a strategy to overcome this dilemma through G:U wobble pairing in RNA. Unlike Watson–Crick base pairs, wobble pairs contribute highly to the energetic stability of the folded structure of their sequence, but only slightly, if at all, to the stability of the folded reverse complement. Sequences in the RNA World might thereby combine stable folding of the ribozyme with an unstructured, reverse-complementary genome, resulting in a “division of labor” between the strands. We demonstrate this strategy using computational simulations of RNA folding and an experimental model of early replication, nonenzymatic template-directed RNA primer extension. Additional study is needed to solve other problems associated with a complete replication cycle, including separation of strands after copying. Interestingly, viroid RNA sequences, which have been suggested to be relics of an RNA World (Diener, Proc Natl Acad Sci USA 86:9370–9374, 1989), also show significant asymmetry in folding energy between the infectious (+) and template (?) strands due to G:U pairing, suggesting that this strategy may even be used by replicators in the present day.     

Preextinction viral RNA can interfere with infectivity

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.     

Closing the Circle: Replicating RNA with RNA

How life emerged on this planet is one of the most important and fundamental questions of science. Although nearly all details concerning our origins have been lost in the depths of time, there is compelling evidence to suggest that the earliest life might have exploited the catalytic and self-recognition properties of RNA to survive. If an RNA based replicating system could be constructed in the laboratory, it would be much easier to understand the challenges associated with the very earliest steps in evolution and provide important insight into the establishment of the complex metabolic systems that now dominate this planet. Recent progress into the selection and characterization of ribozymes that promote nucleotide synthesis and RNA polymerization are discussed and outstanding problems in the field of RNA-mediated RNA replication are summarized.Cell division is a fundamental biological process in which genetic information is duplicated and shared between daughter cells. In extant cellular life, DNA serves as the repository of genetic information, but its replication is complicated by the daunting size and complex structural organization of modern genomes. For this reason, multiple enzymes are required to ensure faithful genomic replication in all higher life forms. Notably, simpler replicating systems such as viruses, have smaller genomes and tend to use correspondingly more error-prone replicative machinery (Kunkel and Bebenek 2000; Gago, Elena et al. 2009). Presumably, if the initial organisms on this planet also had small genomes, then the earliest genomic replication could have been a relatively simple and error-prone process compared with the complex replicative strategies of modern life.     

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.     

Modern mRNA proofreading and repair: clues that the last universal common ancestor possessed an RNA genome?

RNA repair has now been demonstrated to be a genuine biological process and appears to be present in all three domains of life. In this article, we consider what this might mean for the transition from an early RNA-dominated world to modern cells possessing genetically encoded proteins and DNA. There are significant gaps in our understanding of how the modern protein-DNA world could have evolved from a simpler system, and it is currently uncertain whether DNA genomes evolved once or twice. Against this backdrop, the discovery of RNA repair in modern cells is timely food for thought and brings us conceptually one step closer to understanding how RNA genomes were replaced by DNA genomes. We have examined the available literature on multisubunit RNA polymerase structure and function and conclude that a strong case can be made that the Last Universal Common Ancestor (LUCA) possessed a repair-competent RNA polymerase, which would have been capable of acting on an RNA genome. However, while this lends credibility to the proposal that the LUCA had an RNA genome, the alternative, that LUCA had a DNA genome, cannot be completely ruled out.     

The Path from the RNA World

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA → RNP → protein, the first proteins in the ``breakthrough organism' (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the ``introns-first' theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a ``eukaryotic-like' genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome → eukaryotic-like DNA genome → prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome → eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA → RNP → protein. A likely molecular mechanism, ``plasmid transfer,' is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history. Received: 14 January 1997 / Accepted: 19 May 1997     

Gene duplication and other evolutionary strategies: from the RNA world to the future

Beginning with a hypothetical RNA world, it is apparent that many evolutionary transitions led to the complexity of extant species. The duplication of genetic material is rooted in the RNA world. One of two major routes of gene amplification, retroposition, originated from mechanisms that facilitated the transition to DNA as hereditary material. Even in modern genomes the process of retroposition leads to genetic novelties including the duplication of protein and RNA coding genes, as well as regulatory elements and their juxtapositon. We examine whether and to what extent known evolutionary principles can be applied to an RNA-based world. We conclude that the major basic Neo-Darwinian principles that include amplification, variation and selection already governed evolution in the RNA and RNP worlds. In this hypothetical RNA world there were few restrictions on the exchange of genetic material and principles that acted as borders at later stages, such as Weismann's Barrier, the Central Dogma of Molecular Biology, or the Darwinian Threshold were absent or rudimentary. RNA was more than a gene: it had a dual role harboring, genotypic and phenotypic capabilities, often in the same molecule. Nuons, any discrete nucleic acid sequences, were selected on an individual basis as well as in groups. The performance and success of an individual nuon was markedly dependent on the type of other nuons in a given cell. In the RNA world the transition may already have begun towards the linkage of nuons to yield a composite linear RNA genome, an arrangement necessitating the origin of RNA processing. A concatenated genome may have curbed unlimited exchange of genetic material; concomitantly, selfish nuons were more difficult to purge. A linked genome may also have constituted the beginning of the phenotype/genotype separation. This division of tasks was expanded when templated protein biosynthesis led to the RNP world, and more so when DNA took over as genetic material. The aforementioned barriers and thresholds increased and the significance and extent of horizontal gene transfer fluctuated over major evolutionary transitions. At the dawn of the most recent transformation, a fast evolutionary transition that we will be witnessing in our life times, a form of Lamarckism is raising its head.