Populations of short self-replicating RNA variants have been confined to one side of a reaction-diffusion traveling wave front propagating along thin capillary tubes containing the Qfi viral enzyme. The propagation speed is accurately measurable with a magnitude ofabout 1 pm/sec, and the wave persists for hundreds of generations (of duration less than 1 min). Evolution of RNA occurs in the wavefront, as established by front velocity changes and gel electrophoresis of samples drawn from along the capillary. The hih population numbers (.0"11), their well-characterized biochemistry, their short generation time, and the constant conditions make the system ideal for evolution experiments. Growth is monitored continuously by excitation of an added RNA-sensitive fluorescent dye, ethidium bromide. An analytic expression for the front velocity is derived for the multicomponent kinetic scheme that reduces, for a high RNA-enzyme binding constant, to the Fisher form v = 2VcD, whereD isthe diffusion constant ofthe complex and Kc is the low-concentration overall replication rate coefficient. The latter is confirmed as the selective value-determining parameter by numerical solution of a two-species system.It is difficult to provide a constant set of conditions for an explosive reaction over long periods. In a chemical traveling wave, constant reaction conditions are maintained in spite of an intrinsic stability in the homogeneous kinetics (1). The stirred flow reactor (2) and serial transfer method (3) represent laboratory techniques for establishing homogeneous constant chemical conditions. Studies in a constant environment are particularly important to an elementary understanding of molecular evolution. In this work we establish that a constant environment for RNA replication can be attained for a thousand generations or more (corresponding to a day-long experiment) in a traveling concentration wave. The usual difficulties of confinement (1014 molecules, with the reaction catalyzed by a single molecule) are resolved by the physical seal of a fluid-filled capillary.

The nucleotide sequence of a satellite RNA (satRNA) associated with a lilac isolate of arabis mosaic virus (ArMV) was determined from cDNA copies. The sequence was 1104 nucleotides in length excluding the poly(A) tail, contained a long open reading frame which encodes a polypeptide of 360 amino acids, with an Mr of 39K. Nucleotide sequence comparisons revealed that the ArMV-associated satRNA shared 83 % nucleotide identity with a satRNA from grapevine fanleaf nepovirus, but no extensive sequence homology was observed with other nepoviral satRNAs.

Self-replicating molecules set up traveling concentration waves that propagate in an aqueous enzyme solution. The velocity of each wave provides an accurate (±0.1%) noninvasive measure of fitness for the RNA species currently growing in its front. Evolution may be followed from changes in the front velocity, and these differ from wave to wave. Thousands of controlled evolution reactions in traveling waves have been monitored in parallel to obtain quantitative images of the stochastic process of natural selection. An RNA polymerase (RNA-dependent RNA nucleotidyltransferase, EC 2.7.7.6), extracted from bacteria infected by the Qfi RNA virus, catalyzes the replication. The traveling waves that arise spontaneously without added RNA provide a model system for major evolutionary change.An innovative form of spatially inhomogeneous biochemistry permits the simultaneous observation of >1000 separate evolution processes. As a first result, we were able to resolve a phenomenon of spontaneous creation as a process involving rapid evolution: de novo synthesis of self-replicating RNA (1) is shown here to be a statistically reproducible phenomena of natural selection rather than a single enzyme-instructed event. The Qf3 replicase enzyme is an RNA polymerase (Mr 215,000, four subunits) isolated from Qf3 phage-infected Escherichia coli cells (2, 3). The short time (about 30 s) for replication and its kinetically well-characterized mechanism (4-6) make the system an ideal candidate for evolutionary studies. We use massively parallel observations to deal with the stochastic nature of mutational change and to utilize the velocity of traveling concentration waves as an accurate noninvasive measure of fitness.The process of "template-free" or de novo production of RNA by the Q/3 replicase (1) is important for our understanding of evolution because it involves the specific creation of genetic information by a protein enzyme. This would be in defiance of the central dogma of molecular biology (7) data (4-6). However, the mechanism for template-free synthesis of RNA is not known. Self-replicating RNA molecules are produced in vitro in the enzyme solution within hours after the addition of NTP (1). Later, attempts were made to rationalize the phenomenon as contamination by RNA in the enzyme solution or from the air (10-12). Can the templatefree synthesis be purified away or is it an exciting intrinsic property of the enzyme? Biebricher et al. (8,9,11) showed, using kinetic studies, that the mechanism of template-free synthesis must be very different from the well-understood template mechanism. The present work uses measurements of the probability distributions of reaction rates to show that the mechanism involves evolution. Monitoring Evolution in Many Wave Fronts Spatially extended replication reactions are motivated by Darwin's emphasis on the role of geographical isolation for evolution (15) and the requirement of compartmentalization for higher functional organization (16,17). In one dimension, finite diffusion provides str...

Although dideoxy terminated sequencing of RNA, using reverse transcriptase and oligodeoxynucleotide primers, is now a well established method, the accuracy is limited by sequence ambiguities due to unspecific chain termination events. A protocol is described which circumvents these ambiguities by using fluorescence labels tagged to dideoxynucleotides. Only chain terminations caused by dideoxynucleotides were detected while premature terminated cDNA's remain undetectable. In addition, the remaining multiple signals at nucleotide positions can be assigned to sequence heterogeneities within the RNA sequence to be determined.