A T-extended vector using a green fluorescent protein as an indicator.

T-extended vector (T-vector) is a useful tool for cloning PCR products directly. We exploited a novel T-vector using a green fluorescent protein (GFP) as an indicator based on insertional inactivation. The brightest GFP mutant was used for easy detection even under daylight. The 100bp and 0.9kb of PCR products were cloned, and the transformant colonies with inserts were adjudged by the fluorescent green-white screening. The GFP system was more sensitive to insertional inactivation than the beta-galactosidase system at the conventional insertion sites.

An in vitro DNA virus for in vitro protein evolution.

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.

In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro.

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

Detection of differences in higher order structure between highly homologous single-stranded DNAs by low-temperature denaturant gradient gel electrophoresis.

Denaturant gradient gel electrophoresis performed at low temperature was shown to be able to detect the mobility change corresponding to the denaturation of the secondary structure of single-stranded(ss)DNA. Mobility transitions observed were determined to correspond to the melting of local structures, since both denaturants and temperature were verified to have similar effects on the mobility transition of single-stranded DNAs as on that of double-stranded DNAs. In this study it was found that point mutations can effectively change the secondary structures of ssDNA and RNA, and that the method adopted here is very sensitive to such alterations. The validity of the method was supported by a computer analysis of the secondary structure of DNA.

"Thermal stability" maps for several double-stranded DNA fragments of known sequence.

The origin of cooperatively melting regions in DNA, which appear as fine structures in the optical melting profile, has been examined for DNA fragments of known base sequences from bacteriophages phiX174 and fd. Thermal stability maps, which indicate the states of base pairs along these DNA strands, were constructed within the established theoretical framework using the parameters which best reproduce the melting profiles obtained by high temperature resolution experiments. By comparing these stability maps with genetic maps, it was found that several cooperatively melting regions which span several hundred bases have some correlation with the gene locations.

Characterization of imidazole as a DNA denaturant by using TGGE of PCR products from a random pool of DNA.

Perpendicular temperature gradient gel electrophoresis (TGGE) profiles were analyzed for PCR products from a random pool of DNA [60 nts random region flanked by two primer (20 nts) sites]. Besides a normal transition profile of a homoduplex, unique mobility transition profiles of two kinds of heteroduplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex band is an estimator of the complexity of a random pool, it will be applicable to monitor the extent of the selection process in the in vitro selection method. When imidazole was added to the electrophoretic buffer, the transition pattern shifted to the low temperature side. At a concentration of 1 M, imidazole lowered the melting temperature (Tm) of DNA by 13+/-2 degrees C for all the three chain separation transitions observed. Thus imidazole is a stronger denaturant than urea, at least at dilute concentration. Dependence of Tm on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.

Stability mapping along the DNA double strand and its relation to the genetic map.

The distribution of the stability of the double helical structure along the whole DNA of fdphage and its restriction fragments is calculated. In this calculation, Poland's method, which has been established as a rigorous algorithm for taking the base sequence explicitly into consideration, is used. The molecular thermodynamic parameters in the calculation have been determined so as to best reproduce the melting profile of the DNA and its fragments. The results, which are presented as melting maps, show fairly good agreement with those experimentally obtained by the present authors earlier. A close correlation with genes in the genetic map is apparent for some cooperatively melting regions observed in the stability map.

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: I. Methodology.

A method to convert the conventional denaturing gradient gel electrophoresis into a highly reproducible experimental system was developed. It was based on the following experimental findings; (i) dyes, which are small molecules, do not exhibit mobility changes attributed to their conformational change while nucleic acids do; and (ii) most of the mobility shifts caused by experimental fluctuations could be cancelled by normalizing the mobility of a sample with respect to the corresponding one of a dye. The method involves co-migration of internal reference dyes with samples (nucleic acids), and computer-aided data processing, allowing us to obtain the relative mobility of nucleic acids with respect to a dye throughout the denaturing gradient. The overall pattern of the relative mobilities thus obtained, named the normalized mobility profile (NMP), corresponded well to conformational changes of a macromolecule induced by denaturing effects. This method provides us with objective data without using internal macromolecular references, which not only guarantees the precision but also extends the range of application of the denaturing gradient method.

Strand dissociation and cooperative melting of double-stranded DNAs detected by denaturant gradient gel electrophoresis.

Precise analysis using the denaturant gradient gel electrophoresis which was devised by Fischer and Lerman (Cell 16, 191-200, 1979), was found to present specific patterns of fine structures for double stranded DNA fragments. These seem to be a mobility change of DNA fragments caused by specific denaturation processes. The effect of denaturants on DNA melting was ascertained to be similar to the effect of temperature. The observed patterns, in comparison with the melting processes of DNAs theoretically obtained, were closely related to DNA meltings and strand dissociations. A number of electrophoretic mobility transitions showed the retardation correspondent to each of the cooperative meltings. Strand dissociations occurred under the conditions theoretically predicted. The degree of retardation in electrophoresis for DNA fragments seemed to correspond to the size of melted regions. Methods presented here were proved to have advantages over the conventional ones for the study of DNA stability maps.

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.

Adaptive Walks by the Fittest among Finite Random Mutants on a Mt. Fuji-type Fitness Landscape.

Based on the theory of fitness distributions on a Mt. Fuji-type fitness landscape in a multivalued sequence space (Aita & Husimi, 1996 J. theor. Biol. 182, 469-485), we investigated the properties of adaptive walks on the ideal landscape in the case of a cloning-screening-type evolution experiment. We modeled that an adaptive walk is performed by repetition of the evolution cycle composed of the mutagenesis process generating random d-fold point mutants of population size N and the selection process looking for the fittest mutant among them. While an adaptive walk is described in a sequence space, we simplified the description as follows. We mapped the landscape in an x-y plane, where x and y represent a normalized Hamming distance from the global peak and a scaled fitness, respectively. An adaptive walk is described as a trajectory in the plane. The most certain step for a walker to move in a single evolution cycle is represented by a vector in the plane. Then, a walker moves along the streams in the vector field determined by d and N. The walker performs fast hill-climbing until a "trap-line", which traverses the plane. Subsequently, the walker is likely to get trapped in an "apparent local optimum". To continue the walk, apparent local optima must be eliminated by resetting d and N larger. Therefore, for the fastest walk, the optimal schedule of the d-values (initially large d, then small d) is effective, although the economical walk with high cost-performance is different. If a real landscape is just of the Mt. Fuji-type, the walk with the highest cost-performance will be performed by scanning site-directed optimization through all sites. However, in the case of the rough Mt. Fuji-type, which seems to be more realistic, the walking method we have examined will be effective for a walker to sidestep true local optima.Copyright 1998 Academic Press

Kinetics of the polymerization reaction of tobacco mosaic virus protein: transient-saturation type polymerization reaction.

The kinetics of the endothermic polymerization reaction of tobacco mosaic virus protein in the mild acid region was studied by means of temperature-jump (rising time of 6 sec)-turbidimetry, electron microscopy, and computer simulation. The time course profile of the turbidity increase changed from a normal one to an anomalous one as the size of the temperature-jump was made greater. The anomalous type polymerization profile, which we named the "transient-saturation" type, could be characterized by a rapid increase of turbidity and its transient saturation, and a slow increase to the final level. At a higher concentration of the protein, this transient-saturation effect was more marked, whereas the slow turbidity in the second phase occurred with a higher rate. This transient-saturation type polymerization profile was observed also in a pH-induced polymerization reaction. It was not observed in the case of the N-bromosuccinimide modified tobacco mosaic virus protein under a similar environmental change. By an electron microscopic study and computer simulation, it was revealed that in the first phase, a large number of short polymers were formed, and the concentration of the polymerizing units was rapidly reduced to the equilibrium value, and the polymerization reaction stopped transiently. In the second phase, polymer-polymer associations took place slowly and longer polymers were formed. The revlevance of the present study to the polymerization reaction of actin, myosin, and to a transient-overshoot type polymerization are discussed.

Selection and evolution of bacteriophages in cellstat.

Objectives of this work were as follows: 1. to establish a laboratory experimental system utilizable in a biophysical approach to molecular evolution; and 2. to provide real world parameters to theories of molecular evolution, especially to Eigen's theory of quasi-species. Secretion type bacteriophage fd of E. coli, closely related phages and artificial chimera phages of fd, and a virulent phage Q beta of E. coli were cultured continuously in a specially designed fermenter called a "cellstat". A phage is cultured in a flow of host bacterial cells. Due to its high dilution rate, the mutant cell could not be selected in the cellstat. It was therefore recognized that the cellstat is suitable for study of the selection and evolution process of a bacteriophage under well-defined environmental conditions without interference from host cell mutations. Population dynamics of bacteriophages of various types in the cellstat were studied theoretically by computer simulation and experimentally. A genetically invariable pure population of phage behaves like an open non-linear chemical reaction system. An invariable mixed population shows a selection process, while a variable population generates an evolution process. Kinetic constants describing the dynamics were determined by curve fitting between the theoretical and the experimental curve obtained from competition experiments and from biological relaxation experiments. One of the most important kinetic parameters thus obtained was the selection coefficient, and its dependence on the base sequence of phage DNA. We drew a local landscape of the selection coefficient near the fd sequence on the base sequence space. From this landscape we were able to confirm the importance of slightly deleterious mutants in molecular evolution. We also confirmed the possibility of developing an evolutionary molecular engineering using a cellstat as an evolution reactor and fd phage as a working replicon. Novelties of this work were as follows: 1. the first stable continuous culture of a bacteriophage was achieved with a cellstat; 2. a local landscape of selection coefficient near the fd sequence on the sequence space was the first experimental drawing of such a map; 3. a biological relaxation method was realized to measure kinetic constants of a biological kinetic process, or molecular evolution; and 4. a practical engineering process of evolutionary molecular engineering was proposed.

Period dependent selection in continuous culture of viruses in a periodic environment.

Selection in a cellstat culture of two mutant strains of a bacteriophage under periodically fluctuating temperature was analyzed mathematically and numerically. Each of the two viral strains (P1 and P2) was assumed to have a different Arrhenius activation energy for its reproduction reaction. A phase diagram of the final state in the continuous culture was drawn. The most noticeable was that there were both P1-only and P2-only phases of competitive exclusion depending on the period of oscillation and the dilution rate. The period-dependent selection was proved that it was based on the feedback effect via the host-population change. At a short period of oscillation, the strain with larger arithmetic average fitness ultimately dominated in the population; on the other hand, at a long period of oscillation, the strain with larger geometric average fitness ultimately dominated. There was a co-existence phase between the two exclusion phases. The results suggest the feasibility of a method to escape from trapping at local optima on a fitness landscape in evolutionary molecular engineering.

A model of the virus-type strategy in the early stage of encoded molecular evolution.

Recent advances in evolutionary molecular engineering have revealed that the essential nature of a "virus" in the evolutionary aspect is its bonding strategy for assignment of the phenotype to its genotype. Based on the definition of "virus"-type and "cell"-type of the assignment strategy, we propose a virus-early/cell-late model of the history of life. The first encoded protein is assumed to be a cofactor of replication ribozyme in the RNA world and to be bound to its genetic RNA. As such a virus-type strategy could introduce the Darwinian selection process into the hypercycle with translation, a hypercycle with virus-like members could make the replicase protein and the translation system gradually evolve together out of the RNA world without a proto-cell. Moreover, they could evolve much faster by this virus-type strategy than by a primitive cellular organism.

Fitness spectrum among random mutants on Mt. Fuji-type fitness landscape.

Statistical properties of a Mt. Fuji-type fitness landscape on a multi-valued sequence space were analysed. We constructed the model landscape based on additivity of the free energy contributed by each residue on a biopolymer, introducing "tolerance functions" that describe tolerance to residue substitution at each site. The fitness spectrum among a random mutant population around a wild-type sequence was theoretically obtained as the probability density distribution function of fitness. As the Hamming distance from the wild-type to the mutants increases, the mean fitness of the mutant population gradually decreases, and the variance of the fitness increases. These features are originated from the anisotropy of the landscape. On the assumption that the free energy is statistically additive around a wild-type in a sequence space of a real biopolymer, one can estimate the Hamming distance from the wild-type to the optimal biopolymer and the fitness of the optimum. Two sets of experimental data were analysed: (1) a promoter strength spectrum of a mutant population produced by the random mutagenesis of a wild-type lac promoter; (2) four stepwise optimization processes of different peptide mixtures evaluated with ligand binding affinity. Analysis of both experiments showed the compatibility with the hypothesis that local fitness landscapes around contemporary biopolymers are near Mt. Fuji-type. The mean slope of each of the four affinity landscapes for (2) was estimated as delta In K(d)/delta d = 1.3 approximately 2.3, where d denotes the Hamming distance from the optimum and K(d) represents the mean dissociation constant of sequences located at the Hamming distance of d. Mt. Fuji-type landscape can be regarded as a zero-th order approximation to the real local landscape just like an "ideal gas". We showed a method to gauge statistically the shape of a near Mt. Fuji-type landscape by measuring mutant fitness spectra.

Adaptive walks by the fittest among finite random mutants on a Mt. Fuji-type fitness landscape. II. Effect of small non-additivity.

We examined properties of adaptive walks by the fittest on "rough Mt. Fuji-type" fitness landscapes, which are modeled by superposing small uncorrelated random component on an additive fitness landscape. A single adaptive walk is carried out by repetition of the evolution cycle composed of (1) mutagenesis process that produces random d-fold point mutants of population size N and (2) selection process that picks out the fittest mutant among them. To comprehend trajectories of the walkers, the fitness landscape is mapped into a (x, y, z)-space, where x, y and z represent, respectively, normalized Hamming distance from the peak on the additive fitness landscape, scaled additive fitness and scaled nonadditive fitness. Thus a single adaptive walk is expressed as the dynamics of a particle in this space. We drew the "hill-climbing" vector field, where each vector represents the most probable step for a walker in a single step. Almost all of the walkers are expected to move along streams of vectors existing on a particular surface that overlies the (x, y)-plane, toward the neighborhood of a characteristic point at which a mutation-selection-random drift balance is reached. We could theoretically predict this reachable point in the case of random sampling search strategy.

Theory of evolutionary molecular engineering through simultaneous accumulation of advantageous mutations.

We examined the effectiveness of an "adaptive leap" strategy using the "mutation scrambling" method as an efficient optimization technique (Uchiyama, 2000;J. Biochem.128, 441-447) for cases where mutational (rough) additivity holds in fitness. The mutation scrambling method is composed of the following three processes: (1) preliminary selection of several advantageous single-point mutations introduced in a wild-type sequence; (2) preparation of various multiple-point mutants incorporating the advantageous mutant residue or wild-type residue at each of the selected sites, by scrambling the mutant residues and wild-type residues (this process is called mutation scrambling); and (3) selection of the fittest through screening of the mutant pool. The fitness distribution in the mutant pool is controlled by the mixing ratio of the mutant residues to the wild-type residues. We focused on the mutant fitness distribution and obtained the optimal mixing ratio which efficiently generates superior multiple-point mutants with high fitnesses. As a result, we found that the optimal ratio lies between 7/3 and 9/1 in realistic cases. Particularly, this strategy works well in cases where the number of component mutations is large and the size of the population to be screened is small. Analysis of the mutant fitness distributions with various mixing ratios is also useful to explore local fitness landscapes.