How selection affects phenotypic fluctuation.

The large degree of phenotypic fluctuation among isogenic cells highlighted by recent studies on stochastic gene expression confers fitness on some individuals through a 'bet-hedging' strategy, when faced with different selective environments. Under a single selective environment, the fluctuation may be suppressed through evolution, as it prevents maintenance of individuals around the fittest state and/or function. However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment. To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli. Mutant genotypes possessing broad GFP fluorescence distributions with low average values emerged under strong selection pressure. These 'broad mutants' appeared independently on the phylogenetic tree and increased fluctuations in GFP fluorescence were attributable to the variance in mRNA abundance. In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.

Emergence of polyproline II-like structure at early stages of experimental evolution from random polypeptides.

To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III. The initial and 2 evolved polypeptides (one at the 24th generation on branch III and the other at the 25th generation on branch II) were purified to examine their functional and structural properties. Although binding of the initial polypeptide to the target DNA was not detected by surface plasmon resonance measurements, the 2 evolved polypeptides bound to the DNA with dissociation constants of 1.6 and 1.0 microM, respectively, indicating an increase in affinity during the experimental evolution. Circular dichroism spectra of the evolved polypeptides, but not of the initial polypeptide, showed features characteristic of the polyproline II (PPII)-like structure, a left-handed helical structure commonly found in natural proteins, suggesting that the structure emerged through the experimental evolution. The same structural feature was found in another experimental evolution toward catalytic activity. These results demonstrate that the PPII-like structure is one of the common features that could have appeared in the early evolutionary stages of primordial functional protein.

Effective selection system for experimental evolution of random polypeptides towards DNA-binding protein.

An experimental evolution with selection based on binding affinity to DNA was carried out on a library of phage-displayed random polypeptides of about 140 amino acid residues. First, we constructed a system to artificially evolve phage-displayed random polypeptides toward binding to a target DNA containing a restriction enzyme site, in which random polypeptides capable of binding the DNA were recovered as complexes with the target DNA by digestion with the restriction enzyme. The experimental evolution cycle, including the above selection system and random mutagenesis for generating the next mutant library, was repeated until the fourth generation. The ability to bind to the DNA was enhanced per generation. In the fourth generation, convergence of the selected clones to a dominant sequence was observed. These results indicate that the newly constructed selection system is effective for exploring the evolvability of random polypeptides towards DNA-binding proteins.

Extracting characteristic properties of fitness landscape from in vitro molecular evolution: a case study on infectivity of fd phage to E.coli.

We have developed a methodology for extracting characteristic properties of a fitness landscape of interest by analyzing fitness data on an in vitro molecular evolution. The in vitro evolution is required to be conducted as the following "adaptive walk": a single parent sequence generates N mutant sequences as its offsprings, and the fittest individual among the N offsprings will become a new parent in the next generation. N is the library size of mutants to be screened in a single generation. Our theory of the adaptive walk on the "NK landscape" suggests the following: the adaptive walker starting from a random sequence climbs the landscape easily in an early stage, and then reaches a stationary phase in which the mutation-selection-random drift balance sets in. The stationary fitness value is nearly proportional to square root of ln N. Our analysis is performed from the following points: (1) stationary fitness values, (2) time series of fitness in the transitional state, (3) mutant's fitness distribution, and (4) the strength of selection pressure. Applying our methodology, we analyzed experimental data on the in vitro evolution of a random polypeptide (139 amino acids) toward acquiring infectivity (= ability to infect) of fd phage. As a result, we estimated that k is about 27 in this system, indicating that an arbitrary residue in a sequence is affected from other 23% residues. In this article, we demonstrated that the experimental data is consistent with our theoretical equations quantitatively, and that our methodology for extracting characteristic properties of a fitness landscape may be effective.

Experimental rugged fitness landscape in protein sequence space.

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7x10(4)-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18-24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

Different protofilament-dependence of the microtubule binding between MAP2 and MAP4.

To see a molecular basis of the difference in the microtubule binding between MAP2 and MAP4, we compared the binding of them onto microtubule and Zinc-sheet in the presence of various concentrations of NaCl. The Zinc-sheet is the lateral association of protofilaments arranged in an antiparallel fashion with alternatively exposed opposite surfaces, so that binding requiring adjacent protofilaments is restricted. While the salt-dependence of the MAP2 desorption was not altered between these tubulin polymers, MAP4 dissociated from Zinc-sheet at lower concentrations of NaCl than from microtubule. These results suggest that single protofilament is sufficient for microtubule binding of MAP2 as observed by Al-Bassam et al. [J. Cell Biol. 157 (2002) 1187], but MAP4 appeared to interact with adjacent protofilaments during microtubule-binding. Weakened binding on Zinc-sheets was also observed in the projection domain-deletion mutants of MAP4, so that the difference in the protofilament-dependence would lie in the relatively conserved microtubule-binding domain.