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.

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Affinity cross-linking of the plasma membrane fraction to an 125 I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization ϭ 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nm. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 m. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).

The filter-feeding crustacean Daphnia is a key organism in freshwater ecosystems. Here, we report the effect of symbiotic bacteria on ecologically important life history traits, such as population dynamics and longevity, in Daphnia magna. By disinfection of the daphniid embryos with glutaraldehyde, aposymbiotic daphniids were prepared and cultured under bacteria-free conditions. Removal of bacteria from the daphniids was monitored by quantitative polymerase chain reaction for bacterial 16S rRNA gene. The population of aposymbiotic daphniids was reduced 10-folds compared with that of the control daphniids. Importantly, re-infection with symbiotic bacteria caused daphniids to regain bacteria and increase their fecundity to the level of the control daphniids, suggesting that symbiotic bacteria regulate Daphnia fecundity. To identify the species of symbiotic bacteria, 16S rRNA genes of bacteria in daphniids were sequenced. This revealed that 50% of sequences belonged to the Limnohabitans sp. of the Betaproteobacteria class and that the diversity of bacterial taxa was relatively low. These results suggested that symbiotic bacteria have a beneficial effect on D. magna, and that aposymbiotic Daphnia are useful tools in understanding the role of symbiotic bacteria in the environmental responses and evolution of their hosts.

The kinetics of the RNA replication reaction by Q␤ replicase were investigated. Q␤ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Q␤ and plays a key role in the life cycle of the Q␤ 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 Q␤ phage amplification and the applications using Q␤ replicase.Q␤ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Q␤ (1) that plays a key role in the life cycle of the Q␤ phage (2, 3). This enzyme is a heterotetramer composed of a ␤ subunit encoded on the phage genome and three host proteins: ribosomal protein S1, elongation factor Tu (EF-Tu), and Ts (EF-Ts) (4). The replication reaction proceeds first by binding of the enzyme to the 3Ј-end of single-stranded RNA (plus strand), and synthesizes its complementary single-stranded RNA (minus strand), which then becomes the template for synthesis of another plus strand (5). Not all RNAs can be amplified by the Q␤ replicase. The requirements for amplifiable RNAs are known to be the presence of a poly(C) sequence at the 3Ј-end of the RNA (6, 7) and some unique secondary structural features (8), whereas these are not sufficient for designing amplifiable RNAs (9, 10). Using the replicase, RNA can be amplified exponentially from a single copy to 10 12 copies in less than 30 min without the need for any oligonucleotide primer (de novo initiation) (3).Q␤ replicase has been recognized as a representative singlestranded RNA replicase and has been used for various purposes, such as to investigate the kinetics of single-stranded RNA replication (11) and nonhomologous RNA recombination (12) and to study various aspects of molecular evolution (13), hypercycle (14), and the origin of life (15). In addition, it has also been used as a tool for molecular biology (e.g. for the amplification of a particular RNA (16, 17), for virus detection (18), for RNA sequencing (19), and for introducing mutations (20)). Therefore, it is important to understand the basis of the replication reaction.However, at present, there is no kinetic model to account for the entire RNA amplification process by Q␤ replicase, whereas the requirements for ampl...