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We describe a new high-throughput method of quantifying the structural properties of individual cell-sized liposomes. An internal aqueous solution of liposomes was labeled with a green fluorescent marker and the membrane with a red marker. The double-labeled liposomes were analyzed using flow cytometry, and the internal aqueous volume and lipid membrane volume of each liposome were measured. The experimental results indicate that both the internal aqueous and lipid membrane volumes positively correlate with the intensity of forward-scatter (FS) and side-scatter (SS) signals in a logarithmic scale. In addition, liposomes in 18 small areas gated by log(FS) and log(SS) were sorted by fluorescence-activated cell sorting (FACS), and observed by optical microscopy. Structural characteristics observed in the microscopy images of heterogeneous liposomes correlated with FACS data. Because this method does not employ any particular assumption about the shape and structure of liposomes, flow cytometry is a powerful tool for estimating the internal and membrane volumes of individual cell-sized liposomes with heterogeneous shapes and structures. [Copyright &y& Elsevier]

Qβ replicase, an RNA-dependent RNA polymerase of RNA coliphage Qβ, is a heterotetramer composed of a phage-encoded β-subunit and three host-encoded proteins: the ribosomal protein S1 (α-subunit), EF-Tu, and EF-Ts. Several purification methods for Qβ replicase were described previously. However, in our efforts to improve the production of Qβ replicase, a substantial amount of the β-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qβ replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the β-subunit, and in the other kind, the β-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain α-less Qβ replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme. [Copyright &y& Elsevier]

In this study, we investigate the history dependence of the penetrance of a newly emerged gene. Penetrance is defined as the percentage of individuals with a given genotype who exhibit the phenotype associated with that particular genotype. Here, we used the glutamine synthetase gene and its mutants with lower fitness as model genes. They were introduced into host cells of Escherichia coli deprived of the gene, and their penetrance was measured using the host having a different history: either with or without glutamine starvation. Results show that for all genes tested, the value of penetrance was higher when they were introduced into the host cell without starvation than that when introduced into the starved cell, demonstrating the history dependence of the penetrance of a newly emerged gene. In addition, genes with lower fitness showed lower penetrance, and the effect of the difference in fitness on gene penetrance also depended on the history of the host cell. [Copyright &y& Elsevier]

Genome-wide gene expression profiling was performed to investigate the early formation of symbiosis using an artificial symbiosis of Escherichia coli and Dictyostelium discoideum. We have previously reported that when these two species were allowed to grow on minimal agar plates, they achieved a stable state of coexistence, in which the emerging E. coli colonies housing Dictyostelium cells were of a mucoidal nature that was not observed originally. We used this microbiological system as a model to study the initial stages of the development of the symbiotic relationship. The E. coli gene expression profiles of symbiotic cells and non-symbiotic cells captured using GeneChip technology were compared. It was clearly shown that the gene expression profile was substantially altered in E. coli cells undergoing symbiotic transition. The genes responsible for central energy metabolism as well as those responsible for translation apparatus were down-regulated in symbiotic E. coli. The transcriptional patterns of genes coding for the E. coli cell surface structure were drastically altered, and this alteration may determine the mucoidal nature and unique structure of coexisting colonies. General stress inducible genes were expressed at low levels in symbiotic E. coli. These observed changes in the transcription profile indicate that the central metabolism of symbiotic E. coli is attenuated as a whole, and the cells are probably under less stress because of the benefits brought by coexistence with the symbiotic counterpart Dictyostelium. [Copyright &y& Elsevier]

Two well-characterized and phylogeneticaly different species, Escherichia coli and Dictyostelium discoideum, were used as the model organisms. When the two species were mixed and allowed to grow on minimal agar plates at 22 °C, remarkably, the two species achieved a state of coexistence at an average of 2–4 weeks. In addition, the emerged colonies housing the coexisting species had a mucoidal nature that was not observed from its origin. Moreover, the state of coexistence was confirmed to be stable, and so was the mucoidal nature of the emerged colonies. Comparing with the pure E. coli origin, the mucoidal colony showed a significant increase in higher molecular weight extracellular components, with polysaccharides as the major constituent. Qualitative analysis of the monosaccharide contents in the extracellular components of the mucoidal colony revealed not only a significant increase in the glucose content, but also significant amount of additional xylose and galactose. The system permits the initial stages of the development of relationship between two species be captured within a short period of time. This feature, together with being simple and reproducible in laboratory conditions, provides a new model system for the study of symbiosis, especially when initial stages are concerned. [Copyright &y& Elsevier]

The kinetic properties of Qβ replicase, an RNA-dependent RNA polymerase, were investigated experimentally. The reaction at the A-incorporation site was inhibited by UTP and CTP with inhibition constants of 3.2 and 2.7 mM, respectively, while the reactions at the U-, G-, and C-incorporation sites were inhibited by ATP with inhibition constants of 1.09, 1.25, and 1.48 mM, respectively. When nucleotide concentrations were low, C was incorporated at the fastest rate and G at the slowest. Accordingly, the G-incorporation step largely limits the overall reaction rate. From the obtained kinetic parameters, calculations showed that the optimum ratio of the concentrations of the four nucleotides could be achieved by increasing the ratio of GTP concentration with a concomitant decrease in the ratios of CTP and ATP concentrations. Consequently, a 60 to 140% increase in the reaction rate is expected as compared to the rate with equimolar ratio of the four nucleotides. [Copyright &y& Elsevier]

We performed a series of evolution experiments, the results of which illustrated the relationship between mutations and increased carrying capacity (K). Performing an evolution experiment with repeated cycles of mutation by PCR and selection makes it possible to obtain results over shorter culture durations than in methods reported previously relying on spontaneous mutation and selection. We constructed random mutant populations of Escherichia coli in which members differed only in part of the genomic copy of the glutamine synthetase gene and performed daily serial transfer culture where the populations were in K-selected environments. The value of K in this system was increased by 10(5)- to 10(8)-fold relative to the parent clone, which was achieved by four randomly introduced mutations. This method can be applied to any gene and will be useful for analyzing a number of important issues in evolutionary biology.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

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.

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E. coli cells with increased GFP concentration appeared, and when the formation of mucoidal colonies housing the coexisting two species began, most E. coli cells were from the new population. Further experiments suggest that the physiological change is induced by interaction with D. discoideum cells and is reversible, although the processes of the changes in both directions seem to proceed gradually. The observed phenotypic plasticity, together with natural selection under a co-cultivation environment, may be important for leading to the evolution of a new symbiotic system.

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.

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.

Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon. Moreover, the generated complexes were stable at temperatures between 4 and 50 degrees C, and are thus highly stable in contrast to previous assumptions.

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His(6) tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.

To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution.

A directed evolution with phage-displayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the next generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.

A self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.