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A B Chetverin

Publications and source records attributed to A B Chetverin.

At least 19 recordsLinked to original sources
PubMed2000

Cell-free synthesis and affinity isolation of proteins on a nanomole scale.

The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action. The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus. They can be used in translation, transcription-translation or replication-translation reactions. Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein. The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day. The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.

Amino Acid Sequence↗
PubMed1999

The puzzle of RNA recombination.

For more than three decades, RNA recombination remained a puzzle and has only begun to be solved in the last few years. The available data provide evidence for a variety of RNA recombination mechanisms. Non-homologous recombination seems to be the most common for RNA. Recent experiments in both the in vitro and the in vivo systems indicate that this type of recombination may result from various transesterification reactions which are either performed by RNA molecules themselves or are promoted by some proteins. The high frequency of homologous recombination manifested by some RNA viruses can be easier explained by a replicative template switch.

DNA-Directed RNA Polymerases↗
PubMed1999

Spontaneous rearrangements in RNA sequences.

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 Qbeta. The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10(-9) h(-1) per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qbeta replicase, and have a number of biologically important implications.

Allolevivirus↗
PubMed1999

Nonreplicative RNA recombination in poliovirus.

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.

3' Untranslated Regions↗
PubMed1997

Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure.

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.

Base Sequence↗
PubMed1994

Expression and stability of recombinant RQ-mRNAs in cell-free translation systems.

Expression of dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CAT) mRNAs in cell-free Escherichia coli translation systems is greatly enhanced as a result of their insertion into RQ135 RNA, a naturally occurring satellite of phage Q beta. The enhancement is due to protection of the recombinant mRNAs against endogenous ribonucleases and to an increased initial rate of translation in the case of the RQ-CAT mRNA.

Allolevivirus↗
PubMed1994

Oligonucleotide arrays: new concepts and possibilities.

Advances in solid-phase oligonucleotide synthesis and hybridization techniques have led to an incipient technology based on the use of oligonucleotide arrays. The inclusion of a large number of oligonucleotide probes within a single array greatly reduces the cost of their synthesis and allows thousands of hybridizations to be carried out simultaneously. The range of potential applications of oligonucleotide arrays was expanded by the realization that nucleic acids can be sequenced by hybridizing them to all possible oligonucleotides of a given length. Additional possibilities are offered by novel types of oligonucleotide arrays that are capable of parallel sorting, isolating, and manipulating thousands, and even millions, of nucleic acid species. Fields, such as site-directed mutagenesis, protein engineering, and recombinant DNA technology, would benefit from using these arrays. Further, these approaches could enable the analysis of entire genomes by preparing ordered fragment libraries, and by sequencing complex pools of nucleic acids, in a novel approach that provides long-range sequence information by generating nested nucleic acids and then surveying the oligonucleotides contained in the nested strands. This would allow large diploid genomes to be sequenced directly in a completely automated procedure that does not require fragment cloning or chromosome mapping.

Alleles↗
PubMed1993

Synergism in replication and translation of messenger RNA in a cell-free system.

Combination of the Q beta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135(-1) RNA, an efficient naturally occurring Q beta replicase template. The enhancement is associated with a replication asymmetry previously described for the replication of Q beta phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands. This, in turn, results in increased synthesis of the functionally active DHFR. These effects are not observed when DHFR mRNAs or RQ135(-1) RNAs are used as templates, if the translation system is not complete, or if it is inhibited by puromycin. The coupled replication-translation of nonviral mRNA recombinants can serve as a useful model for studying the fundamental aspects of virus amplification and can be implemented for large-scale protein synthesis in vitro.

Base Sequence↗
PubMed1993

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.

Cloning, Molecular↗
PubMed1993

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.

Algorithms↗
PubMed1991

On the nature of spontaneous RNA synthesis by Q beta replicase.

Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.

Air↗
PubMed1991

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.

Base Sequence↗
PubMed1988

An in vivo recombinant RNA capable of autocatalytic synthesis by Q beta replicase.

A variety of small RNAs ranging from tens to hundreds of nucleotides in length grow autocatalytically in a Q beta replicase (Q beta phage RNA-dependent RNA polymerase) reaction in the absence of added template, and similar RNAs are found in Q beta phage-infected Escherichia coli cells. Three such RNAs have been sequenced. One of them that is 221 nucleotides (nt) long ('MDV-1' RNA) has been found to be partially homologous to Q beta phage RNA 8, which might be considered as an indication of its origination from by-products of the Q beta RNA replication. To gain further insight into the origin and function of these RNAs, we have sequenced a new RNA, 120 nt long, isolated from the products of spontaneous synthesis by the nominally RNA-free Q beta replicase preparation. The minus strand of this RNA appeared to be a recombinant RNA, composed of the internal fragment of Q beta RNA (approximately 80 nt long) and the 33-nt-long 3'-terminal fragment of E. coli tRNA(1Asp). This seems to be the first strong indication of RNA recombination in bacterial cells. The various implications of this finding are discussed.

Base Sequence↗
PubMed1988

Electron microscopy study of Q beta replicase.

Purified preparations of Q beta replicase have been studied by electron microscopy using a negative staining technique, and a three-dimensional model of the enzyme molecule has been constructed. The molecule of this four-subunit protein appears to be a compact structure having a size of 100 +/- 10 A; it is subdivided into two unequal bipartite subparticles. The conclusion has been made that all the constituent subunits, including the ribosomal protein Sl, acquire a globular conformation when associated in the replicase complex.

Coliphages↗
PubMed1986

Cooperativity of the alpha beta-protomer structure in Na+,K+-ATPase functioning. A scanning microcalorimetry study.

Heat denaturation of the free and ligand-bound forms of purified Na+,K+-ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two-state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of alpha beta-protomers (Mr approximately equal to 140 000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes approximately equal to 230 kJ/mol higher. The data suggest that in a functionally active enzyme form, the alpha beta-protomers possess a rigid structure with tight association of their subunits and domains, this structural rigidity is essential for the Na+,K+-ATPase functioning and there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation.

Animals↗
PubMed1986

Evidence for a diprotomeric structure of Na,K-ATPase. Accurate determination of protein concentration and quantitative end-group analysis.

Three methods were used to assess protein concentration in membrane-bound Na,K-ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH2-terminal residues of the beta-subunit (NH2-Ala) and one such residue of the alpha-subunit (NH2-Gly) per ligand-binding unit. From the data on equimolarity of the alpha- and beta-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two alpha beta-protomers, one possessing a modified (presumably an N-blocked) alpha-subunit.

Amino Acids↗