*Department of Genetics
and
Department of Ecology and Evolutionary Biology, Yale University
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Abstract |
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Introduction |
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The frustration with retrospective approaches, coupled with the advent of molecular tools in evolutionary biology, has led to an increasing number of experimental studies of microevolution using both viral (e.g., Bull et al. 1993, 1997
; Cunningham et al. 1997
) and bacterial systems (e.g., Lenski and Travisano 1994
; Nakatsu et al. 1998
; de Visser et al. 1999
).
This study takes an explicitly experimental approach to understanding molecular adaptation and diversification. It belongs to the expanding body of work that uses simplified molecular systems to address evolutionary questions (Ellinger, Ehricht, and McCaskill 1998
; Hanczyc and Dorit 1998
). Using a deliberately simplified experimental system consisting of catalytic RNA molecules (ribozymes) evolving in controlled in vitro environments, we probed the trajectories that lead to novel catalytic function. Our previous work dealt with the emergence of complexity and divergent function in a similar experimental system (Hanczyc and Dorit 1998
). Here, we focus on the recurrence of particular outcomes when replicate molecular populations, identical at the outset, are selected in parallel for the same catalytic function.
In our system, evolving catalytic RNA molecules acquire the ability to cleave a novel substrate (DNA) in response to the experimental conditions we impose. Those ribozymes best able to efficiently cleave this DNA substrate are preferentially replicated. By repeatedly supplying variation to the system and reiterating the selection cycle, we improve the mean DNA cleavage activity of the population. As selectable variance is exhausted, the rate of improvement of the population for the selected phenotype slows and eventually asymptotes: the population reaches an activity plateau and shows no further improvement in the selected activity. In principle, only those ribozymes best able to cleave the DNA substrate will be represented in the population. The individual ribozyme variants present at the end of the experiment embody an adaptive solution to the imposed catalytic challenge.
The evolving entity in this study is the L-21 ribozyme, derived from the self-excising group I intron located in the 23S rRNA gene of Tetrahymena thermophila. This ribozyme readily cleaves an RNA oligonucleotide that is partially complementary to the 5' end of the ribozyme (Zaug and Cech 1986
; kcat/Km = 1.6 x 107/M/min; see Materials and Methods). This same ribozyme can also cleave a DNA oligonucleotide counterpart, but it does so one million times less efficiently (Herschlag and Cech 1990; kcat/Km = 3.6 x 101/M/min). This low but latent ability of the L-21 ribozyme to cleave DNA, coupled with the results of previous studies that demonstrated the selectability of the DNA-cleaving phenotype (Beaudry and Joyce 1992
; Tsang and Joyce 1994
), made this ribozyme the ideal choice for this study.
This study explores the replicability and variety of adaptive responses that evolve under well-defined and concerted directional selection. We established replicate, independent populations drawn from the same initial variable ribozyme pool and selected these populations in parallel for improved DNA cleavage. Single solutions were thus likely to be fixed in each of the populations, but these endpointsand the trajectories that give rise to themcould then be compared among populations. In effect, each of the five evolving populations undertakes an excursion along the functional landscape. The extent to which these independent populations converge on a single outcome, or arrive instead at different functional solutions, sheds light on the structure and topography of the underlying functional landscape.
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Materials and Methods |
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Construction of Independently Evolving Ribozyme Lines
The variant pool of ribozymes created by mutagenic PCR was transcribed by T7 RNA polymerase, and the transcripts were gel-purified as described above. From this pool of mutant transcripts, five samples, each containing approximately 1013 RNA molecules, were used to initiate the five independent evolving lines (lines A, B, C, D, and E).
Evolution In Vitro
The evolution in vitro protocol is outlined below and is based on a procedure originally devised by Joyce and coworkers (Beaudry and Joyce 1992
; Lehman and Joyce 1993) and subsequently modified as described in Hanczyc and Dorit (1998
) with the following changes: Ribozymes were transcribed in vitro by T7 RNA polymerase from PCR templates, gel-purified, and quantitated by spectrophotometry, and approximately 1013 ribozyme molecules were added to the cleavage reactions. Two biotin molecules ("B") are covalently linked to the 3' end of the DNA substrate (STC) (5'-GGCCCTCTAAATAAATAAATAAATAAACAAACAAA-BB-3'). Upon cleavage, the 3' moiety of the DNA oligonucleotide substrate (5'-AAATAAATAAATAAATAAACAAACAAA-BB- 3') covalently attaches to the ribozyme. Catalytically active ribozymes thus end up as tagged, biotinylated RNA-DNA chimeras which can be captured on streptavidin-coated magnetic beads. A second stage of selection used a reverse-transcription primer, complementary to the tag-ribozyme junction, further ensuring that only active ribozyme variants remained in the evolving population.
The cleavage reaction (1 µM ribozyme, 10 µM DNA oligonucleotide substrate, 10 mM MgCl2, and 30 mM EPPS [pH 7.5]) proceeded at 37°C for 180 min in generations 12. This cleavage time was reduced to 10 min in generations 36, and to 5 min in generations 715, effectively increasing the intensity of functional selection for improved cleavage. Following cleavage, the reaction products were incubated (80°C for 30 s) with streptavidin-coated Dynabeads (M-280, Dynal) in order to bind and retain the successful ribozymes. The beads and bound ribozymes were then washed twice with heated (80°C) SDS buffer (10 mM Tris [pH 7.5], 0.15 M LiCl, 1 mM EDTA, 0.3% SDS) and twice with heated (80°C) reverse-transcriptase buffer (25 mM Tris [pH 7.5], 5 mM MgCl2, 25 mM DTT) in order to remove the unbound ribozymes and prepare the bound ribozymes for reverse transcription. The retained ribozymes were amplified from the beads with the primer (TAG1) 5'-TTTGTTTGTTTATTTATTTA-TTTATTTC-3' and AMV reverse transcriptase. The resultant cDNAs were amplified by PCR. No isothermal amplification was required.
Construction and Propagation of the Control Line
L-21 ribozymes were transcribed from PCR product and gel-purified as described above. The control line, begun with approximately 1013 molecules of L-21 ribozyme transcripts, was propagated through the steps of evolution in vitro as outlined above, with two exceptions: no mutagenic PCR was performed on the control line, and the selection step was modified such that successful DNA cleavage was not a precondition for propagation. Specifically, 1013 molecules of the control line were added to a DNA cleavage reaction, and cleavage was allowed to proceed. Following the cleavage reaction, ribozymes were added directly to the reverse-transcription step and subsequently amplified in a reaction containing both T13 and TAG1 primers. The molecules of the control line are therefore replicated independently of their ability to cleave the DNA substrate.
Cloning of Individual Ribozymes
PCR products to be further characterized were cloned into linear T-tailed pBlueScript vector by T4 DNA ligase under standard conditions. The ligated plasmids were transformed into competent cells and grown on ampicillin plates containing X-gal, as described in Hanczyc and Dorit (1998
). Overnight cultures were prepared from a random sampling of single white transformant colonies. Ligated plasmids containing a PCR insert were isolated from overnight cultures using Qiagen miniprep protocols.
Sequencing of Individual Ribozymes
Ribozyme variants were sequenced from plasmid templates by dye-terminator cycle sequencing according to Perkin Elmer protocols, and the genotypes were determined (with a minimum twofold coverage) using a 373A ABI DNA sequencer and software.
DNA Cleavage Assays
The overall DNA cleavage activities of the evolving and control populations were assayed at each generation. In addition, the activity of selected clones was further characterized as follows: 0.5 µM ribozyme was preincubated at 50°C in 50 mM MgCl2 and 150 mM EPPS (pH 7.5) in a 20-µl reaction. 0.1 µM 5' 32P-labeled STC substrate was added to the reaction and incubated at 37°C for 60 min. The products of the reaction were resolved on a 16% acrylamide, 8 M urea gel, visualized using a phosphorimager (Fujix BAS 2000), and quantitated using MacBAS, version 2.0.
RNA Cleavage Assays
The RNA-cleaving activity of the evolved ribozyme populations was determined using a 5' 32P-labeled substrate (RSTC) (5'-GGCCCUCUAAAUAAAUAAAUAAAUAAACAA-ACAAA-BB-3'). Then, 4 nM ribozyme was preincubated at 50°C in 50 mM MgCl2 and 150 mM EPPS (pH 7.5) in a 20-µl reaction. An equivalent of 1 nM labeled RNA substrate was added to the reaction and incubated at 37°C for 1 min. The reaction was stopped with 2 volumes of 7 M urea and 20 mM EDTA with 0.1% xylene cyanol and 0.1% bromophenol blue, and the reaction products were resolved in a 16% acrylamide/8 M urea gel and quantitated as described above.
Determination of kcat and Km Values
Assays with the 5' end-labeled RNA substrate were carried out under single-turnover conditions with a substrate concentration at least 10-fold below Km. These assays also involved using four different ribozyme concentrations, which spanned the Km and always exceeded the concentration of substrate. Ribozymes were preincubated at 50°C in 50 mM MgCl2 and 150 mM EPPS (pH 7.5), and the labeled RNA substrate was subsequently added to the reaction. Aliquots (typically 2 µl each) were taken at three time points during the linear portion of the reaction and quenched with 2 volumes of 7 M urea and 20 mM EDTA with 0.1% xylene cyanol and 0.1% bromophenol blue. Reaction products were resolved and quantitated as described above. The kcat/Km values were determined from kobs values according to the equation kobs = (kcat/Km)[E].
Assays with the 5' end-labeled DNA substrate were performed under multiple-turnover conditions using five different excess substrate concentrations. The ribozymes were preincubated at 50°C in 50 mM MgCl2 and 150 mM EPPS (pH 7.5), and the labeled DNA substrate was subsequently added to the reaction. Aliquots were extracted at four time points during the linear portion of the reaction and were resolved and quantitated as described above. Rate constants (Km and kcat) were determined using Lineweaver-Burk plots.
Determination of kobs
The kinetic parameter kobs was estimated for both DNA and RNA substrates from a sampling of individual ribozyme clones in single-turnover reactions. The conditions were modeled after those used above for the determination of kcat and Km. Time points were taken during the linear portion of the reaction, and kobs values were estimated as the absolute value of the slope of the natural log (ln) of substrate remaining over time.
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Results |
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The dynamics of the selective response differ from one population of molecules to the next (fig. 1 ). Line D exhibits the fastest response, followed closely by line B, with both lines reaching a cleavage plateau at approximately 600 amol of DNA substrate cleaved/min/pmol ribozyme. After generation 11, lines C and E eventually evolve to reach the same level of catalytic activity as lines B and D. The response of lines C and E subsequent to generation 15 has not been determined; they may well continue to improve in subsequent generations. Line A shows no response after generation 13, reaching a plateau at 300 amol of DNA substrate cleaved/min/pmol ribozyme. As expected, in the absence of selection the WT control shows no improvement in DNA cleavage activity.
Our replicate runs of experimental evolution thus reveal heterogeneity both in the line-specific responses and in the resultant phenotypic plateaus. We obtained a variety of outcomes: similar trajectories (lines B and D), differing trajectories (line B vs. line C, for example), convergent endpoints (lines B and D), and differing endpoints (line A vs. lines B, C, D, and E).
Correlations with RNA Catalysis
The evolved ribozymes were further characterized by examining the extent to which improvements in the evolved activity (cleavage of DNA) correlated with cleavage of an RNA substrate (the "ancestral" activity). Previous studies had shown a positive correlation between these two activities in evolved variants of the L-21 ribozyme (Tsang and Joyce 1996
), suggesting that increases in DNA activity stemmed largely from overall improvements in nucleic acid cleavage chemistry. A similar pattern emerges in our evolved lines B, C, D, and E: a 100-fold improvement in DNA catalysis is accompanied by a twofold increase in RNA catalysis (fig. 2
). In contrast, line A shows an approximately twofold decrease in RNA catalysis relative to the starting L-21 (wild-type) ribozyme, indicating that line A ribozymes have lost some RNA cleavage ability while evolving enhanced DNA cleavage activity. The association between DNA and RNA catalysis was explored in detail by assaying eight clones (sampled independently of their cleavage phenotype) from each of the five evolved populations. The DNA- and RNA-cleaving abilities of each of these clones are shown in figure 3AE
. As expected from population assays (fig. 2
), clone activity on DNA and RNA substrates is correlated in several of the clones. Clones from lines B, D, and E show a high degree of correlation between DNA and RNA catalysis (Pearson product-moment analysis correlations of 0.90, 0.89, and 0.88 respectively; fig 3F
). Line C clones still show a weak positive correlation (0.43). In contrast, line A clones show a weak negative correlation between DNA and RNA catalysis (-0.31).
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After 15 generations of evolution in vitro, we characterized the five evolved lines by sampling 25 individual clones from each line, with no a priori knowledge of their genotypic or phenotypic characteristics. The resulting genotypes were compared with the ancestral L-21 sequence; we define a genotypic difference as a "prevalent mutation" if more than 50% of the sampled sequences from any one line differ from the L-21 sequence at that position. We identify 25 such prevalent mutations in our evolving lines. The identities of these prevalent mutations and their frequencies across all lines at generation 15 are summarized in figure 4 . In addition, we sampled individual clones from lines A, B, and C at generation 10, when the lines appear most divergent in their response to selection (fig. 4AC ).
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Line A shares only two prevalent mutations (positions 87 and 94) with the remaining lines; the other 10 prevalent mutations are unique to line A. The distinct character of line A is evident by generation 10 (fig. 4B ), when the phenotypic divergence between lines is at its maximum (fig. 1 ). The eventual genotypic profile of all lines is foreshadowed well before the activity plateaus are reached.
In all five lines, prevalent mutations occur at positions 87 and 94 (see fig. 4
). The change at position 87, a deletion of a single adenine in stem P2.1, results in the elimination of a bulged nucleotide. The change at position 94 involves an AU change in a putatively single-stranded linker region between stems P2.1 and P3. Of the 125 evolved clones sampled in this study, all exhibit the A
U change at position 94, and all but 2 (123/125) have lost the bulged nucleotide at position 87. Both changes have been observed in previous studies (Beaudry and Joyce 1992
; Tsang and Joyce 1994, 1996
) in which they appear to be associated with bound substrate orientation.
The sequence differences accumulated by the end of the experiment in lines A and B (relative to L-21) are also shown as relative-frequency histograms superimposed on the secondary structure of this ribozyme (fig. 5 ). The differences occur in both the conserved catalytic core of the ribozyme and the periphery; both position and frequency of changes differ significantly between lines A and B.
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Twenty-four clones from the wild-type control line were analyzed after 15 generations to test for the distribution of mutations along the molecule. The molecule was divided into five equivalent (67-nt) intervals, which insured that no interval exhibited fewer than five mutations in our sample. A test of the observed distribution failed to reject the null hypothesis of equal numbers of mutations (including indels) across all intervals (G = 0.769, df = 4, P > 0.9). We noted a significant excess of transitions (54) over transversions (11) and a significant number of insertions (13) and deletions (2), as expected from amplification and reverse- transcription errors. We calculated an inherent mutation rate of 0.066% per position per generation (average = 3.3 mutations per clone; range = 09) and did not detect any pattern of molecular changes evolving solely in response to the experimental design of the system.
Kinetic Analyses
Kinetic analyses quantitate the changes in the kinetic parameters that are responsible for the enhanced phenotype seen in figure 1 . Kinetic dissection can also reveal further disparities among the evolved lines, since similar improvements in catalytic activity (measured as kobs) can obscure different underlying causes (e.g., increased kcat or decreased Km).
Representative molecules from each of the five populations were chosen to insure that the set of characteristic changes for each population was examined. Clone A15.G, for instance, contains all 12 prevalent mutations characteristic of line A. Given the similarity between lines B and D, an additional clone from line D (D15.I), exhibiting a variant 3' end, was also included in this study. The results of the kinetic analysis for all five clones and the wild-type ribozyme are shown in table 1 . Both Km and kcat values changed in response to the selection for DNA catalysis when compared with the L-21 (WT) ribozyme. Km values for most of the clones show about a 10-fold decrease (clone A15.G shows only a fourfold decrease in Km). All clones show 20- to 40-fold increases in kcat over L-21 (WT). Upon comparing kcat/Km values, clone C15.H shows the highest efficiency, a 330-fold increase over L-21 (WT).
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Discussion |
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We delineate these two solution classes based on the following criteria: (1) the different levels of DNA cleavage activity at which the lines reach a plateau; (2) the differences exhibited by the lines in their RNA cleavage capacities (a related but distinct and unselected phenotype); and (3) the overlapping mutational spectrum of lines B, C, D, and E, in contrast to the distinct mutational profile of line A. Ultimately, the independent character of the two solutions requires an exploration of the genotype space separating them and a demonstration of the incompatible nature of the mutations that define the two solutions (e.g., see Gavrilets and Gravner 1997
).
The Genotypic Basis of Phenotypic Divergence
The solution class that displays the greatest improvement in the selected phenotype evolved more than once (lines B, C, D, and E). These lines have converged phenotypically and genotypically. In contrast, line A also shows enhanced DNA cleavage activity but embodies a different catalytic solution. The phenotypic divergence of line A is accompanied by a comparable divergence at the genotypic level, already evident by generation 10. Despite the heterogeneity in the phenotypic responses at that point, the eventual distribution of prevalent mutations that distinguishes line A from lines B, C, D, and E takes shape early in the experiment.
The presence of distinct solution classes reveals the interplay of stochastic and deterministic forces. Particular mutations responsible for the high activity of lines B, C, D, and E may not have arisen in line A. Eight prevalent mutations present in lines B, C, D, and E are not observed in line A; these mutations could account for the difference in the catalytic activities of the different lines.
Stochastic effects may also be manifested in the differential accumulation of mildly deleterious mutations in the various lines. The activity of line A may be compromised by line-specific deleterious mutations that diminish catalytic activity. In the absence of recombination, hitchhiking of mildly deleterious mutations will continue until mutation-selection equilibrium is reached. Candidates for this mildly deleterious designationprevalent mutations unique to line A (positions 73, 81, and 354; see fig. 4 )show no obvious correlation with reduced DNA cleavage activity in the representative clones. Despite the large effective population sizes (>105) in these experiments, mutations of small deleterious effect (s 1/Ne) are nevertheless unlikely to be eliminated from the population.
Deterministic explanations for the existence of divergent solutions postulate epistatic interactions among the observed point mutations. Because catalytic activity depends on both the sequence and the three-dimensional structure of the ribozyme, such epistatic interactions can be reasonably postulated. The enhanced DNA cleavage activity of lines B, C, D, and E may depend on a combination of mutations that has not arisen in line A. Positions 135, 270, and 312 may represent such an interaction. These individual mutations manifest in line A variants (see fig. 4 ), yet they do not co-occur in a single ribozyme molecule from line A. The absence of recombination again retards the formation of such favorable epistatic combinations, since multiple mutations must arise on the same molecule before a fitness advantage obtains. Finally, the fate of new mutations may depend primarily on the genetic background in which the mutations occur. This context-dependent fate, arising from the type of epistatic interactions described above, sets the stage for the emergence of divergent genotypic and phenotypic classes. As line A and lines B, C, D, and E begin to define distinct genotypic contexts early in the experiment, the fate of any subsequent new point mutation, even if it appears in all lines, is unlikely to be the same. Every new fixation defines a novel context for subsequent mutational events. By generation 10, line A has acquired a suite of line-specific mutations that differ from those seen in lines B, C, D, and E. The two genotypic solutions seen in this experiment are separated by a large Hamming distance, suggesting that line A is not simply an intermediate stage in the evolution of a single convergent solution embodied by lines B, C, D, and E.
Correlated Response: Uncoupling DNA and RNA Catalysis
The line-specific correlation between DNA and RNA cleavage activities reveals further heterogeneity in the phenotypic responses of the replicate lines. In our experiments, there is no selection for retaining activity on an RNA substrate or any counterselection for reduced activity on an RNA target. Despite this, lines B, D, and E show a tight positive correlation between the two cleavage activities, a result expected from previous studies (Tsang and Joyce 1994, 1996
). Line C shows a weaker, but still positive, correlation. In contrast, DNA cleavage appears uncoupled from RNA cleavage in line A, as reflected by the weak negative correlation shown in figure 3F
. This lack of correlation between DNA- and RNA-cleavage activities in line A stands in contrast to previous studies where the correlation between DNA and RNA catalysis could only be weakened by direct counterselection (Tsang and Joyce 1996
). Line A has evolved a distinct solution with greater specificity for the DNA substrate: DNA and RNA cleavage are no longer correlated in this line.
Evolving DNA Cleavage: Necessary and Contingent Changes
Two prevalent mutations, at positions 87 and 94, recurred in every line (fig. 4
). In pursuit of the same objectiveenhanced cleavage of a DNA substratebut utilizing different experimental methods, previous studies also report changes at positions 87 and 94 in response to in vitro selection (Beaudry and Joyce 1992
; Tsang and Joyce 1994
). We note that these changes are not seen in our unselected control line and are thus not a bias or artifact of the evolution in vitro system.
All of the evolved clones from generation 15 sampled in this study (125) exhibit the change at position 94; all but two clones show the deletion at position 87. The prevalence of these two changes does not per se mean that they are the basis for improved DNA cleavage activity. Clone C15.G, for instance, lacks the change at position 87, yet still shows enhanced DNA cleavage (fig. 3
). This deletion is therefore not necessary for improved DNA cleavage. Only one of the 25 prevalent changes described in figure 4
, the AU change at position 94, is truly ubiquitous in our experiment and has previously been observed in similar selections (see Beaudry and Joyce 1992
; Tsang and Joyce 1994
). While the role played by these mutations has yet to be established, we suspect that the changes at positions 87 and 94 are neither necessary nor sufficient to confer enhanced DNA cleavage activity. They may instead represent easily accessible steps in the evolution of enhanced DNA cleavage, and their fixation may set the stage for the emergence of subsequent functional mutations. The diversity of evolved molecules emerging in these experiments suggests that multiple combinations of genotypic changes can confer an enhancement in DNA-cleaving activity.
The multiple evolutionary trajectories seen in this experiment underscore the many-to-one relationship between ribozyme genotypes and catalytic phenotypes. This relationship is reflected in the phylogenetic diversity of many catalytic RNAs (e.g., group I [Michel and Westhof 1990
] or group II introns [Michel and Ferat 1995
]). Such phylogenetic comparisons, however, take place over large Hamming distances and compare very divergent entities from varying contexts.
The results presented here suggest that even on a local scale, where common ancestry is recent and still visible, we are dealing with a rugged adaptive landscape where multiple genotypes give rise to a single convergent function. Such ruggedness is a prediction of a number of theoretical models and arises from assumptions about the role of stochastic events in small populations (Wright 1948, 1949
), the nature of the mutational spectrum (Gillespie 1984
), the prevalence of epistatic interactions (Kaufmann 1995
), the character of the genotype-to-phenotype map (Gavrilets 1997
), and the nature of RNA free-energy landscapes (Schuster et al. 1994
; Hunyen and Fontana 1996; Fontana and Schuster 1998
).
This ruggedness has clear implications for the applied in vitro evolution of novel molecules. Most evolution in vitro is not likely to preserve multiple alternative solutions to a catalytic challenge (but see Ekland, Szostak, and Bartel 1995; Hager and Szostak 1997). In the present case, the solution represented by line A (with its lower overall activity) would not have been recovered had it arisen along with the alternate solution in a single, evolving population. While this would seem to argue for the power of single in vitro runs to produce the "optimal" molecule, it is worth recalling that other aspects of the evolved line A molecules, such as their increased specificity for DNA substrates, may subsequently prove desirable. Replicate in vitro evolution experiments reduce the probability that alternate and potentially desirable solutions will remain undiscovered.
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Conclusions |
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The multiple solutions exhibited by our evolving lines shed light on a fundamental question in evolutionary biology, namely, the relationship between realized solutions and the universe of possible solutions. Our observations suggest that for a single functional phenotype under selection, multiple pathways and multiple endpoints exist and can be reached even on a local scale. Any one of the solutions we have uncovered likely represents an element of a much larger ensemble of functional answers to the catalytic challenge. There is no reason to suspect that a similar relationship between the possible and the actual does not apply for any given novel molecular phenotype we choose to explore.
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Acknowledgements |
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Footnotes |
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1 Keywords: experimental evolution
group I ribozyme
catalytic RNA
directed evolution
adaptive landscape
evolution in vitro
2 Address for correspondence and reprints: Robert L. Dorit, Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, Connecticut 06511. E-mail: rdorit{at}beagle.biology.yale.edu
.
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