Cloning of RNA molecules in vitro.

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.     

Isolation and crystallization of a chimeric Qβ replicase containing <Emphasis Type="Italic">Thermus thermophilus</Emphasis> EF-Ts

Qβ replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qβ. In addition to the phage-encoded catalytic β subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qβ replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the β subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qβ replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.     

Express hybridization of molecular colonies with fluorescent probes

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qβ replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and shorten the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.     

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB_1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB₂ domain, and use NMR spectroscopy to demonstrate for the first time that OB₂ is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.     

Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta 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 Qbeta 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 Qbeta 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 Qbeta 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 Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Paradoxes of replication of RNA of a bacterial virus

The extraordinary ability of the bacteriophage Qβ replicase to amplify RNA outside the cell attracted the attention of molecular biologists in the late 1960s-early 1970s. However, at that time, a number of puzzling properties of the enzyme was not explained. Not until recently has Qβ replicase begun to reveal its secrets, promising to give a key not only to understanding the mechanism of replication of the bacterial virus genome but also to the solution of more general fundamental and applied problems.     

Sequencing of pools of nucleic acids on oligonucleotide arrays

In sequencing-by-hybridization methods, the nucleotide sequence of a nucleic acid is reconstructed by overlapping oligonucleotides capable of hybridizing with the nucleic acid. In their present form, the methods are hardly suitable for sequencing of long nucleic acid molecules because of the occurrence of non-unique overlaps between the oligonucleotides, and similarly to the conventional sequencing methods, it is necessary to obtain an individual molecule. In the method described here, most ambiguities in reconstruction of a sequence from the constituent oligonucleotides are eliminated by preparing on oligonucleotide arrays and separate surveying of the nucleic acid nested partials. This enables longer nucleic acids to be sequenced, and results in a high redundancy of the input data allowing most hybridization errors to be eliminated by algorithmic means. Furthermore, large pools of nucleic acid strands can be sequenced directly, without isolating individual strands.     

Do sodium and potassium forms of Na,K-ATPase differ in their secondary structure?

Infrared spectroscopy in the amide I region of purified membrane-bound Na,K-ATPase preparation shows that Na+- and K+-bound forms of the enzyme have almost the same secondary structure. No difference is detected in the beta-structure (pleated sheets) content. This is contrary to the statement of the recent paper (Gresalfi, T. J., and Wallace, B. A. (1984) J. Biol. Chem. 259, 2622-2628) where a similar preparation was examined by circular dichroism spectroscopy and it was claimed that net 7% of protein peptide groups undergo a beta-sheet to alpha-helix conformational change upon Na,K-ATPase conversion from the K+ to the Na+ form. The discrepancy of the results is most likely caused by the particulate nature of the enzyme preparations used that could lead to optical artifacts in CD but not in IR measurements. A thorough comparison of IR spectra of these enzyme forms has revealed a very minor spectral difference which could suggest conformational perturbations, if any, of a much lower scale and another type than that claimed by Gresalfi and Wallace. The K+ form tends to absorb slightly more in the region of the alpha-helix band. This could reflect some distortion or a transition to a random coil structure of a small fraction of alpha-helical segments (less than or equal to 2% protein peptide groups) upon the enzyme conversion from the K+ to the Na+ form.     

Efficient templates for Q beta replicase are formed by recombination from heterologous sequences.

A very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication. The sequence segments are unrelated to the sequence of Q beta bacteriophage genomic RNA. Nonetheless, this natural recombinant is replicated in vitro at a rate equal to the most efficient of the known Q beta RNA variants. Apparently, the structural properties that ensure recognition of an RNA template by Q beta replicase are not confined to viral RNA, but can appear as a result of recombination among other RNAs that usually occur in cells.