Hiroshi Kita

Replication of Genetic Information with Self-Encoded Replicase in Liposomes

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 Qβ 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.

Constructing Partial Models of Cells

Understanding the origin of life requires knowledge not only of the origin of biological molecules such as amino acids, nucleotides and their polymers, but also the manner in which those molecules are integrated into the organized systems that characterize cellular life. In this article, we introduce a constructive approach to understand how biological molecules can be arranged to achieve a higher-order biological function: replication of genetic information.

Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase

During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.

Kinetic Analysis of the Entire RNA Amplification Process by Qβ Replicase

The kinetics of the RNA replication reaction by Qβ replicase were investigated. Qβ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qβ and plays a key role in the life cycle of the Qβ phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qβ phage amplification and the applications using Qβ replicase.

Importance of Translation-Replication Balance for Efficient Replication by the Self-Encoded Replicase

In all living systems, the genetic information is replicated by the self‐encoded replicase (Rep); this can be said to be a self‐encoding system. Recently, we constructed a self‐encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qβ Rep and the RNA was replicated by the self‐encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self‐encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self‐encoded system, and we determined the optimum balance.

A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA

We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification.

Quantitative analysis of the bacteriophage Qβ infection cycle

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.

Detection and Analysis of Protein Synthesis and RNA Replication in Giant Liposomes

In living cells, biochemical reaction systems are enclosed in small lipidic compartments. To experimentally simulate various biochemical reactions occurring in extant cells, giant liposomes are used to reconstruct an artificial model cell. We present methods for conducting a protein synthesis reaction, followed by the reaction catalyzed by the synthesized proteins inside liposomes, and for measurement of the in liposome reaction using a fluorescence-activated cell sorter (FACS). These techniques enable us to perform detailed analysis of the biochemical reactions occurring in the microcompartments, and have the potential to reveal the role of compartmentalization in cellular systems.

Constructive Approaches for the Origin of Life

Life is composed of a vast number of molecules. A central question is how these nonliving molecules organized into an integrated system regarded as living. A straightforward method that addresses this question is the “constructive approach,” which is an approach to construct living systems or subsystems in vitro using identified materials. Here, we review studies that constructed several subsystems or systems in the cell including replication of genetic information, translation, and cell membranes. The achievements and limitations of these studies have also been discussed.

[83] 3,4-Dihydroxyphenylacetate-2,3-oxygenase (Pseudomonas ovalis)

Publisher Summary This chapter describes the assay, purification, and properties of 3,4-dihydroxyphenylacetate-2,3-oxygenase. The assay measures the conversion of 3,4-dihydroxyphenylacetic acid to α -hydroxy-δ-carboxymethylmuconic semialdehyde, which absorbs at 380 m μ . One unit of enzyme is the amount of enzyme that forms 1 micromole of the product in 1 minute. All the purification operations are carried out at 0–4° and centrifugations at 10,000 g for 10 minutes. The purified enzyme is homogeneous by the criteria of sedimentation velocity and electrophoresis. The enzyme is very stable and loses no activity over a period of a year when stored under nitrogen in the presence of ferrous ion. The pH optimum for the enzyme in 0.05 M Tris buffer is 8.2. Fe 2+ serves as an activator to enzyme preparations inactivated either by prolonged storage under air or treatment with hydrogen peroxide and as a stabilizer to the native enzyme.