Diminishing-returns epistasis decreases adaptability along an evolutionary trajectory.

Populations evolving in constant environments exhibit declining adaptability. Understanding the basis of this pattern could reveal underlying processes determining the repeatability of evolutionary outcomes. In principle, declining adaptability can be due to a decrease in the effect size of beneficial mutations, a decrease in the rate at which they occur, or some combination of both. By evolving Escherichia coli populations started from different steps along a single evolutionary trajectory, we show that declining adaptability is best explained by a decrease in the size of available beneficial mutations. This pattern reflected the dominant influence of negative genetic interactions that caused new beneficial mutations to confer smaller benefits in fitter genotypes. Genome sequencing revealed that starting genotypes that were more similar to one another did not exhibit greater similarity in terms of new beneficial mutations, supporting the view that epistasis acts globally, having a greater influence on the effect than on the identity of available mutations along an adaptive trajectory. Our findings provide support for a general mechanism that leads to predictable phenotypic evolutionary trajectories.

Genetic background affects epistatic interactions between two beneficial mutations.

The phenotypic effect of mutations can depend on their genetic background, a phenomenon known as epistasis. Many experimental studies have found that epistasis is pervasive, and some indicate that it may follow a general pattern dependent on the fitness effect of the interacting mutations. These studies have, however, typically examined the effect of interactions between a small number of focal mutations in a single genetic background. Here, we extend this approach by considering how the interaction between two beneficial mutations that were isolated from a population of laboratory evolved Escherichia coli changes when they are added to divergent natural isolate strains of E. coli. We find that interactions between the focal mutations and the different genetic backgrounds are common. Moreover, the pair-wise interaction between the focal mutations also depended on their genetic background, being more negative in backgrounds with higher absolute fitness. Together, our results indicate the presence of interactions between focal mutations, but also caution that these interactions depend quantitatively on the wider genetic background.

The continuous evolution in vitro technique.

In vitro experimentation techniques were developed in response to the necessity of exploring new molecular structures and functions and to better understand evolutionary phenomena that shape organismal and molecular populations. The advancement of these techniques has allowed further exploration of more complicated evolutionary dynamics. One such technique is the continuous evolution in vitro (CE) method, to which this unit is devoted. The CE method is characterized by continuous cycles of amplification of RNA molecules that occur without much participation of the researcher. This feature allows us to evolve lineages in which the evolutionary phenomena occurring at the molecular level more closely mimic what happens in organismal populations in the present, or what may have happened in RNA populations during the RNA world stage of life.

Gel purification of radiolabeled nucleic acids via phosphorimaging: Dip-N-Dot.

RNA and DNA oligonucleotides radiolabeled with (32)P or (33)P often require gel electrophoresis to remove undesired side and/or degradation products. Common ways to visualize these molecules after electrophoresis are by ultraviolet (UV) shadowing, which necessarily reduces the specific activity of the oligonucleotide, and by autoradiography using film, which is cumbersome and increases the cost of generating the radiolabeled molecule. A more cost-effective method is to physically inject the gel with a "Dip-N-Dot" solution of dye and radionuclide after electrophoresis but prior to phosphorimaging. The gel can be overlaid on its computer-generated image, allowing the labeled molecules to be visualized quickly.