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Tetsuya Yomo

Publications and source records attributed to Tetsuya Yomo.

At least 19 recordsLinked to original sources
PubMed2006

Adaptive response of a gene network to environmental changes by fitness-induced attractor selection.

Cells switch between various stable genetic programs (attractors) to accommodate environmental conditions. Signal transduction machineries efficiently convey environmental changes to the gene regulation apparatus in order to express the appropriate genetic program. However, since the number of environmental conditions is much larger than that of available genetic programs so that the cell may utilize the same genetic program for a large set of conditions, it may not have evolved a signaling pathway for every environmental condition, notably those that are rarely encountered. Here we show that in the absence of signal transduction, switching to the appropriate attractor state expressing the genes that afford adaptation to the external condition can occur. In a synthetic bistable gene switch in Escherichia coli in which mutually inhibitory operons govern the expression of two genes required in two alternative nutritional environments, cells reliably selected the "adaptive attractor" driven by gene expression noise. A mathematical model suggests that the "non-adaptive attractor" is avoided because in unfavorable conditions, cellular activity is lower, which suppresses mRNA metabolism, leading to larger fluctuations in gene expression. This, in turn, renders the non-adaptive state less stable. Although attractor selection is not as efficient as signal transduction via a dedicated cascade, it is simple and robust, and may represent a primordial mechanism for adaptive responses that preceded the evolution of signaling cascades for the frequently encountered environmental changes.

Adaptation, Biological↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

Nascent chain, mRNA, and ribosome complexes generated by a pure translation system.

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.

Cell-Free System↗
PubMed2006

Femtoliter compartment in liposomes for in vitro selection of proteins.

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.

Cell Compartmentation↗
PubMed2006

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.

BACKGROUND: Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. RESULTS: We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells. CONCLUSION: We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

Adrenal Gland Neoplasms↗
PubMed2006

Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta 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.

Enzyme Activation↗
PubMed2006

In vitro evolution of proteins.

Consecutive rounds of diversification and selection of the fittest is believed to be the main driving force for the evolution of life. For the evolution of life to proceed, all living cells are surrounded by a lipid bilayer that separates their own genes from the external environment and from those of other organisms. In this way, the genetic information of an individual is replicated on the basis of their phenotype; thus the enrichment of the fittest will occur. Hence, evolution is based on linkage between genotype and phenotype owing to the surrounding of the genetic material with a barrier. The linkage between genotype and phenotype is also known to be essential for the directed evolution of proteins. Indeed, systems for molecular evolution, including phage display, ribosome display, and in vitro compartmentalization, all satisfy this requirement in different ways. These systems have been shown to be powerful tools for high-throughput screening for the functions of proteins, screening as many as <10(12) molecules in 1 d. These selection systems in combination with various gene libraries yield proteins with improved or altered biophysical properties, and may even allow the generation of proteins with novel functions.

Amino Acid Sequence↗
PubMed2006

Quantification of structural properties of cell-sized individual liposomes by flow cytometry.

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.

Flow Cytometry↗
PubMed2005

Correlation between evolutionary structural development and protein folding.

Evolution should have played important roles in determining folding mechanisms and structures of proteins. In this article we discuss how the folding mechanisms had been affected by the early stage of evolution through which the uniqueness of structure had developed. Although the process of such early-time evolution has remained a mystery, a plausible scenario is that the evolution of proteins toward the ordered structures was guided by functional selection pressure as demonstrated in vitro and in silico. We examine the in silico functional selection of sequences and show that there is a significant correlation between two different processes toward the unique 3D structure, the evolutionary development of structure through sequence selection, and the folding process of the resultant sequence. This finding could be rephrased as protein folding recapitulates the emergence of topology in the molecular evolution. The correlation suggests a guideline for engineering foldable proteins.

Binding Sites↗
PubMed2004

Expression of a cascading genetic network within liposomes.

Liposomes have long been used as possible compartments for artificial cells, and it has been shown that liposomes can sustain various types of biochemical reactions. To elevate the degree of molecular complexity of the system in liposomes, we have constructed a two-stage genetic network encapsulated in liposomes. This two-stage genetic network was constructed with the plasmid pTH, in which the protein product of the first stage (T7 RNA polymerase) is required to drive the protein synthesis of the second stage (GFP). We show that the two-stage genetic network constructed in a cell-free expression system is functional within liposomes.

Base Sequence↗
PubMed2004

Universality and flexibility in gene expression from bacteria to human.

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from E. coli to human, where gene expression changes are proportional to their expression levels, and show that this "proportional" dynamics or "rich-travel-more" mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels.

Animals↗
PubMed2004

Evolution of an arbitrary sequence in solubility.

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.

Amino Acid Sequence↗
PubMed2004

Global change in Escherichia coli gene expression in initial stage of symbiosis with Dictyostelium cells.

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.

Animals↗
PubMed2004

History dependent effects on phenotypic expression of a newly emerged gene.

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.

Cell Proliferation↗
PubMed2004

Protein folding by the effects of macromolecular crowding.

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.

Animals↗
PubMed2003

On the relation between fluctuation and response in biological systems.

A general relationship between fluctuation and response in a biological system is presented. The fluctuation is given by the variance of some quantity, whereas the response is given as the average change of that quantity for a given parameter change. We propose a relationship where the two are proportional, in a similar way to the fluctuation-dissipation theorem in physics. By studying an evolution experiment where fluorescence of protein in bacteria increases, we confirm our relation by observing a positive correlation between the speed of fluorescence evolution and the phenotypic fluctuation of the fluorescence over clone bacteria. The generality of the relationship as well as its relevance to evolution is discussed.

Biological Evolution↗
PubMed2003

Can an arbitrary sequence evolve towards acquiring a biological function?

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.

Amino Acid Sequence↗