What is a quasispecies?

The concept of the quasispecies as a society formed from a clone of an asexually reproducing organism is reviewed. A broad spectrum of mutants is generated that compete one with another. Eventually a steady state is formed where each mutant type is represented according to its fitness and its formation by mutation. This quasispecies has a defined wild type sequence, which is the weighted average of all genotypes present. The quasispecies concept has been shown to affect the pathway of evolution and has been studied on RNA viruses which have a particularly high mutation rate. They (and possibly the majority of other species) operate close to the error threshold that allows maximum exploration of sequence space while conserving the information content of the genotype. The consequences of the quasispecies concept for the new 'evolutionary technology' are discussed.

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli.

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.

The natural 6 S RNA found in Q beta-infected cells is derived from host and phage RNA.

The RNA of Escherichia coli infected with RNA bacteriophage Q beta was isolated and screened for replicable short-chained RNA. In contrast to earlier assumptions we show that (i) short-chained replicable RNA is a very minor part of the RNA synthesized in the infection cycle, and (ii) that the replicable RNA isolated from infected cells is derived from cellular RNA, in particular 23 S rRNA and 10 Sa RNA, and from Q beta RNA itself. None of the many RNA species known from in vitro experiments was found. The RNA species isolated were all inefficient templates. No replicable RNA could be isolated from non-infected cells. Even in cells expressing high amounts of Q beta replicase very few RNA species could be isolated. RNA generated in vitro in template-free synthesis is therefore not derived from RNA species found in vivo, and replicable RNA found in vitro is generated by a mechanism fundamentally different from the one operating in vivo.

Molecular evolution of RNA in vitro.

Experimental studies of RNA evolution in vitro are reviewed in the context of Eigen's 1971 theory and its subsequent extensions. Current research activity and future prospects for using automated molecular biology techniques for in vitro evolution experiments are surveyed.

Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase.

Kinetic analysis of pausing and fidelity of human immunodeficiency virus type 1 reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase catalyzes DNA synthesis from RNA and DNA templates by a sequential mechanism. This enzyme is neither processive nor distributive but has a rather intermediate behavior; at any template position, there is a certain probability that the replica strand will be extended, which we define as extensibility. The extensibility depends on the substrate concentration, i.e. on the concentration of the cognate (and to a smaller extent of the noncognate) deoxynucleoside triphosphates, in a typical Michaelis-Menten mode. The extensibility varies from position to position in a sequence-dependent manner, being particularly low at certain sites, accordingly called pause sites. The rate and fidelity of successive incorporation of nucleotides were measured and then compared with numerical integrations of the pertinent rate equations, which were composed to describe a suitable reaction mechanism and parameterized starting starting with rate constants reported in the literature. We found that agreement between stimulation and experiment requires two-step binding of enzyme to the template-primer. In an initial second-order step, an "outer" binary complex is rapidly formed; this is followed by a slower conformational change into an "inner" complex. During multiple rounds of nucleotide incorporation, the complex remains in the inner form; the rate-determining step for enzyme release is the reversion from the inner to the outer complex, with a standard rate constant of 0.2s-1. This rate constant may be significantly increased at pause sites. In order to match the experimental results, the standard rate constants had to be modified for pause sites. At low concentrations or in the absence of the cognate nucleotide, the site-specific misinsertion frequency, a function of the nucleotide pool is bias and of the efficiency to discriminate against a noncognate nucleotide, can be determined from the dependence of extensibility on concentration of cognate and noncognate substrates. The error frequency was found to be somewhat smaller than the misinsertion frequency, because mismatches are extended less efficiently than matched pairs.

The mutant distribution of an RNA species replicated by Q beta replicase.

The mutant spectrum of an RNA species that is replicated by Q beta replicase, MNV-11, was investigated by retrotranscribing the RNA to DNA and cloning it into plasmids. The sequences of several cDNA clones of MNV-11 populations amplified by Q beta replicase under various conditions were determined and compared. A surprisingly broad mutant distribution was found: the consensus sequence never made up more than 40% of the total population and was accompanied by many mutants. Most mutants had several base exchanges, insertions and/or deletions; up to nine of the total 86 nucleotides were changed. The mutants found had replication rates comparable to that of the wild-type and were thus enriched in the population by selection forces. When the growth conditions were changed, the mutant distribution centre was shifted. The published consensus sequence of MNV-11 did not have the highest growth rate of the mutants, but was rather the best adapted to the various selection forces governing the growth phases the replicating RNA went through, i.e. it had found an optimal compromise between the rates of overall replication, enzyme binding and double strand formation.

Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell.

The replication of a RNA template catalyzed by Q beta replicase was obtained in oleic acid/oleate vesicles simultaneously with the self-reproduction of the vesicles themselves. This was accomplished by entrapping the enzyme Q beta replicase, the RNA template, and the ribonucleotides ATP, CTP, GTP, and UTP inside the vesicles. The water-insoluble oleic anhydride was then added externally. It binds to the vesicle bilayer where it is catalytically hydrolyzed yielding the carboxylate surfactant in situ, which then brings about growth and reproduction of the vesicles themselves. This experiment is presented as a first approach to a synthetic minimal cell, in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously.

Design of artificial short-chained RNA species that are replicated by Q beta replicase.

Different RNA species that are replicated by Q beta replicase have related secondary structures: for both plus and minus strands, "leader" stem structures were found at their 5' termini, while their 3' termini were unpaired. Parallel structures in complementary strands rather than antiparallel ones require the occurrence of wobble pairs and other imperfections in the stem regions. To test whether the leader structures are required for replication, artificial RNA sequences were synthesized by transcription from synthetic oligodeoxynucleotides with T7 RNA polymerase and assayed for their ability to be replicated by Q beta replicase. A synthetic short RNA species known to be replicated was amplified, forming a stable quasi-species; i.e., its sequence was conserved during hundreds of replication rounds. A synthetic mutant of this sequence that stabilized the leader in one strand but favored a 3'-terminal stem in the other one led to the complete loss of template activity. When new RNA sequences with the described structural requirements were designed and synthesized, their template activity was too low to be directly measurable; however, incubation with replicase produced replicating RNA whose sequence was closely related to the synthesized RNA species. The most likely interpretation is that the designed sequences were in a low montainous region in the replication fitness landscape and were optimized during amplification by Q beta replicase to a nearby fitness peak. The structural features postulated to be required for replication were not only conserved but even improved in the outgrowing mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Template-directed and template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added templates, Q beta replicase produces different RNA species after long lag times spontaneously in vitro. The sequences of the spontaneous products are short (30 to 45 nucleotides) and bear little sequence relation to one another and no detectable sequence homology to Q beta virus RNA or to the host. Their replication rates are much lower than those of optimized products. Incubation without template in long closed capillaries produces after long lag times many separate RNA growth foci with heterogeneous kinetics. The template-free reaction is strongly dependent on the conditions: lowering the enzyme or the triphosphate concentrations abolishes the template-free RNA synthesis without affecting the template-dependent synthesis. An explanation of the emerging RNA species in template-free reactions by residual RNA contaminants in the incubation mixture is very unlikely in the light of the experimental evidence; however, the experimental evidence is fully compatible with a de novo mechanism (which may include instruction by non-replicatable oligonucleotides).

Sequence analysis of RNA species synthesized by Q beta replicase without template.

Q beta replicase amplifies certain short-chained RNA templates autocatalytically with high efficiency. In the absence of extraneously added template, synthesis of new RNA species by Q beta replicase is observed under conditions of high enzyme and substrate concentrations and after long lag times. Even under identical conditions, different RNA species are produced in different experiments. The sequences of several independent template-free products have been determined by cloning their cDNAs into plasmids by a novel cloning procedure. Their nucleotide chain lengths are small, ranging from 25 to about 50 nucleotides. While their primary sequences are unrelated except for the invariant 5'-terminal G and 3'-terminal C clusters, their tentative secondary structures show a common principle: both their plus and minus strands have a stem at the 5' terminus, while the 3' terminus is unpaired. Direct accumulation of sufficient quantities of early template-free synthesis products by Q beta replicase is prevented by the inherent irreproducibility of the synthesis process and by the rapid change of the products during amplification by evolution processes, but large amounts of such RNA can be synthesized in vitro by transcription from the cDNA clones. RNA species produced in template-free reactions replicate much more slowly than the optimized RNA species characterized previously. These experimental results illustrate how biological information can be gained in small bits by trial and error.

In vitro recombination and terminal elongation of RNA by Q beta replicase.

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.

Quantitative analysis of mutation and selection in self-replicating RNA.

Mutation and selection as principles of Darwinian evolution have contributed a wealth to qualitative insight and understanding of complex biological organizations. However, for quantitative measurements of Darwinian evolution, only model systems are sufficiently simple to allow calculation of values for the relevant evolution parameters. The model system used for our study comprises short-chained RNA species whose self-replication is catalyzed by Q beta replicase. In this system, phenotypic expression of a genotype is reduced to its efficiency in directing its own synthesis. The mechanism of single-stranded RNA reproduction is well understood: RNA synthesis profiles can be described by compact equations. The selection behaviour of competing RNA species can be precisely predicted, using these equations, from kinetic parameters of the species: at low concentrations, RNA species are selected for overall growth rate (fecundity), at higher concentrations, for rapid binding of replicase (selection for competition), and at still higher concentrations, for minimizing losses caused by formation of inactive double strands. Finally, an ecosystem may be established where the different species coexist, their relative concentrations being functions of their kinetic parameters. The analysis of competition and selection can be extended to mutants of a species. Experimental conditions can be found where quantitative measurement of mutation rates and selective values of mutants is possible. The interplay of mutation and selection results in establishing a quasispecies distribution where mutants are represented according to their rates of mutational formation and their selective values. Replicating RNA clones, when amplified, rapidly build up quasispecies distributions containing pronounced "hot spots", produced predominantly by error propagation of nearly neutral mutants. The primitive model system shows the same complex Darwinian behaviour as observed in evolution of biological systems. In the absence of extraneously added template, Q beta replicase synthesizes after long lag times self-replicating RNA de novo. In a first step, nucleoside triphosphates are condensed randomly; self-replicating templates produced by chance are amplified and optimized.

Self-catalysed affinity labeling of Q beta replicase.

The spatial neighbourhood of the active center of Q beta replicase can be selectively modified by the method of self-catalysed affinity labeling. In the template-directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the beta subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N-chlorosuccinimide, we have mapped the attachment site to the region of subunit beta between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.

Template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added template, standard preparations of Q beta replicase spontaneously synthesize RNA in vitro, possibly as a result of RNA contamination. Using special enzyme purifications, Sumper and Luce presented evidence that self-replicating RNA not present ab initio can grow out of 'template-free' incorporation mixtures. In contrast to DNA polymerase I and RNA polymerase, which also show de novo synthesis, the products synthesized 'de novo' by Q beta replicase are RNA species containing nonrepetitive sequences of defined lengths which differ between experiments, even when synthesized under identical conditions, in fingerprints, chain lengths and kinetic parameters. Kinetic analysis of the de novo processes distinguished it from template-instructed synthesis and excluded an assumption of self-replicating RNA contamination. These conclusions were questioned recently by Hill and Blumenthal, who claimed to show that highly purified Q beta replicase preparations cannot produce RNA de novo. We now present evidence that, under the conditions required for de novo synthesis, Q beta replicase prepared according to their method is also capable of de novo synthesis. Furthermore, we show that Q beta replicase condenses nucleoside triphosphates to more or less random oligonucleotides.

Kinetics of RNA replication: competition and selection among self-replicating RNA species.

The process of Darwinian selection in the self-replication of single-stranded RNA by Q beta replicase was investigated by analytical and computer-simulation methods. For this system, the relative population change of the competing species was found to be a useful definition of selection value, calculable from measurable kinetic parameters and concentrations of each species. Critical differences in the criteria for selection were shown to pertain for replicase/RNA ratios greater than or less than 1, for the case that formation of double-stranded RNA occurs and when comparisons are made of closed with open systems. At a large excess of enzyme, RNA species grow exponentially without interfering with each other, and selection depends only on the fecundity of the species, i.e., their overall replication rates. For RNA concentrations greater than the replicase concentration, the selection of species is governed by their abilities to compete for enzyme. Under conditions where formation of double strands occurs, competition leads to a coexistence of the species; the selection values vanish, and the concentration ratios depend only on the template binding and double-strand formation rates. The approach to coexistence is rapid, because when its competitors are in a steady state, a species present in trace amount is amplified exponentially. When formation of hybrid double strands occurs at a substantial rate, coexistence of hybridizing species is essentially limited to cases where the formation rate of heterologous double strands is smaller than the geometric mean of the formation rates of the homologous double strands. At limiting cases, e.g. in the steady states, simple analytical expressions for the main aspects of the selection process were found. Experimental data support the analytical expressions and the simulations.

Kinetics of RNA replication: plus-minus asymmetry and double-strand formation.

The effects of kinetic plus-minus asymmetry and formation of inactive double strands on the self-replication of single-stranded RNA were investigated by analytical and computer simulation methods. It was found that extensions of the analysis developed previously for more restricted models lead to simple formulations that can be used for interpretation of experiments. Relaxation to linear growth or to steady-state conditions for double-strand formation was found to depend upon initial conditions but to be essentially complete for typical laboratory situations. Experimental data confirmed that in the linear growth phase the total nucleotide incorporation rate is about equal in the complementary strands; mostly double strand is formed. However, the enzyme is usually not shared equally, and some steps proceed at different rates in the two strands. The asymmetry is, however, not found to be dramatic for any of the RNA variants studied so far. It appears that the observed prevalence of kinetically rather symmetric self-replication is due to selection of RNA species with similar rate constants during the exponential growth phase.