30 years later--a new approach to Sol Spiegelman's and Leslie Orgel's in vitro evolutionary studies. Dedicated to Leslie Orgel on the occasion of his 70th birthday.

The conditions necessary for evolution are amplification, mutagenesis and selection. Here we describe the evolutionary response of an in vitro replicating system to the selection pressure for fast growth and show what happens to the amplified molecules within this replication system. Our emphasis is on methodology, on the monitoring and the automation of experiments in molecular evolution. In order to perform in vitro studies on the evolution of RNA molecules, a modified self-sustained sequence replication (3SR) method was used. In the first step of the 3SR reaction, the RNA template is reversely transcribed by HIV-1 reverse transcriptase, followed by a second strand synthesis and the transcription of the resulting dsDNA by T7 RNA polymerase. The selection pressure (fast growth) was achieved by applying the principle of serial transfer pioneered in the laboratories of Sol Spiegelman and Leslie Orgel. At the end of the exponential growth phase of the 3SR reaction, an aliquot of the reaction mixture is transferred into a new sample containing only buffer, nucleotides and enzymes while RNA template molecules are provided by the transfer. The conditions in the exponential growth phase allow the RNA molecules to be amplified in a constant environment; all enzymes (HIV-1 reverse transcriptase and T7 RNA polymerase) and nucleotides are present in large excess. Therefore, transferring reproducibly within the exponential growth phase is equivalent to selecting for fast growth; those molecules which can replicate faster will displace others after several transfers. The experiments were performed using a serial transfer apparatus (STA) which allows the nucleic acid concentration to be monitored on-line by measuring the laser-induced fluorescence caused by intercalation of thiazole orange monomers into the RNA/DNA amplification products. The serial transfer experiments were carried out with an RNA template (220b RNA) that represents a 220-base segment of the HIV-1 genome and comprises the in vivo primer binding site (PBS) for the HIV-1 reverse transcriptase. It could be shown that after only two serial transfers two RNA species (EP1 and EP2) emerged that were much shorter. EP1 (48b) and EP2 (54b) were formed by deletion mutations within the original 220b RNA template in the very beginning of the serial transfer experiment; due to their higher replication rate (calculated from the growth curves derived on-line) these two deletion mutants displaced the original 220b RNA template in the course of the following thirty transfers. We assume that these two RNA species evolved independently of each other. Their formation was probably induced by a strand-transfer reaction of HIV-1 reverse transcriptase. Sequence analyses of these two evolution products seem to confirm such a presented pathway. 30 years after Spiegelman's experiment, the study described here is another answer to the question he posed: 'How do molecules evolve if the only demand is the biblical injunction: multiply?'. The answer, derived from a modified 3SR amplification system (mimicking a part of the HIV-1 replication cycle in vitro), is the same as thirty years ago: The RNA molecules adapt to the new conditions by throwing away any ballast not needed for fast replication. Clearly, this is only one aspect of molecular evolution; however, it shows that we should be careful in designating unidentified genetic material as 'junk DNA'.