Autonomous model protocell division driven by molecular replication

The coupling of compartmentalisation with molecular replication is thought to be crucial for the emergence of the first evolvable chemical systems. Minimal artificial replicators have been designed based on molecular recognition, inspired by the template copying of DNA, but none yet have been coupled to compartmentalisation. Here, we present an oil-in-water droplet system comprising an amphiphilic imine dissolved in chloroform that catalyses its own formation by bringing together a hydrophilic and a hydrophobic precursor, which leads to repeated droplet division. We demonstrate that the presence of the amphiphilic replicator, by lowering the interfacial tension between droplets of the reaction mixture and the aqueous phase, causes them to divide. Periodic sampling by a droplet-robot demonstrates that the extent of fission is increased as the reaction progresses, producing more compartments with increased self-replication. This bridges a divide, showing how replication at the molecular level can be used to drive macroscale droplet fission

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Autonomous Plasmid-like Replication of a Conjugative Transposon

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra-chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid-like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1-encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid-like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.National Institutes of Health (U.S.) (grant GM50895

Autonomous Replication of the Conjugative Transposon Tn916

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916 was dependent on the relaxase encoded by orf20 of Tn916. The origin of transfer of Tn916, oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916 likely interact in a complex and that the Tn916 relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916 that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism. IMPORTANCE Integrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.National Institutes of Health (U.S.) (R01GM050895)National Institute of General Medical Sciences (U.S.) (Grant T32GM007287

The Trans-Contextual Model of Autonomous Motivation in Education: Conceptual and Empirical Issues and Meta-Analysis.

The trans-contextual model outlines the processes by which autonomous motivation toward activities in a physical education context predicts autonomous motivation toward physical activity outside of school, and beliefs about, intentions toward, and actual engagement in, out-of-school physical activity. In the present article, we clarify the fundamental propositions of the model and resolve some outstanding conceptual issues, including its generalizability across multiple educational domains, criteria for its rejection or failed replication, the role of belief-based antecedents of intentions, and the causal ordering of its constructs. We also evaluate the consistency of model relationships in previous tests of the model using path-analytic meta-analysis. The analysis supported model hypotheses but identified substantial heterogeneity in the hypothesized relationships across studies unattributed to sampling and measurement error. Based on our meta-analysis, future research needs to provide further replications of the model in diverse educational settings beyond physical education and test model hypotheses using experimental methods

Management and control of self-replicating systems: A systems model

In 1980, a conceptual engineering approach to self-replicating systems was achieved. The design was based on von Newmann's kinematic version of self-replicating automata. The systems management and control and the organization of the control elements are reported. After developing the functional requirements of such a system, a hierarchy of three management and control levels is described. These are an autonomous, an external, and an intelligent management and control system. Systems recycling, systems specialization, and information replication are discussed