Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.

The ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage QL L. The rearrangements are Mg 2+ -dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10 39 h 31 per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of QL L replicase, and have a number of biologically important implications.z 1999 Federation of European Biochemical Societies.

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S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qb replicase, the RNA-directed RNA polymerase of bacteriophage Qb. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qb replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples. All these features provide for an accurate and reliable assay of viruses even at very low amounts, that is, in cases most important to diagnostics.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Q␤ phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Q␤ phage-derived RNA, such that if aligned at complementary 3-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Q␤ replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Q␤ replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Q␤ RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Q␤ replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Q␤ phage.Recombinations (sequence exchange and rearrangements) between and within RNA molecules are rare but biologically important events contributing to the evolution and diversity of RNA viruses (1, 2) and generating defective interfering RNAs that attenuate viral infections (3). In contrast to splicing and other types of regular RNA rearrangements, recombinations occur without apparent sequence or structure specificity (1, 2). There are indications that recombination may occur between cellular RNAs (4 -6), eventually resulting, by means of reverse transcription and integration, in alterations in the chromosomal DNA. Spontaneous Mg 2ϩ -catalyzed rearrangements in RNA sequences (7) might have been a mechanism for evolution in the prebiotic RNA world and might have evolved into contemporary sequencespecific ribozyme-catalyzed reactions (8, 9).RNA recombination was discovered more than 40 years ago as an exchange of genetic markers between polioviruses (10, 11), and since then similar approaches were used to demonstrate that genomes of RNA viruses of animals, plants, and bacteria are all capable of recombination (2, 4, 12, 13). However, such in vivo experiments utilizing living cells, as well as in vitro studies that used crude cell lysates could not uncover the underlying molecular mechanisms or even definitely answer the question if recombining entities were RNA molecules themselves or their cDNA copies, because every living cell contained enzymes capable of reverse transcription and appropriate dNTP substrates. It became evident that further progress in this field depended on the availability of adequate in vitro systems whose composition and other parameters can be strictly controlled by the experimenter (2, 14).The first example of such a sort has been the cell-free system employing purified Q␤ replicase, RNA-directed RNA polymerase (RdRp) 1 of bacteriophage Q␤ (15). The system...