Biomedical subjects

K M Chumakov

Publications and source records attributed to K M Chumakov.

At least 37 records · Page 2Linked to original sources

PubMed1993

Quantitative aspects of the mutant analysis by PCR and restriction enzyme cleavage (MAPREC).

Quantitation of virus revertants by PCR and restriction enzyme cleavage may give nonlinear results and, in some cases, produce artifacts caused by nucleotide misincorporation and heteroduplex formation, occurring during PCR. Modifications of the procedure allowed us to overcome these problems and develop a highly sensitive and reliable method of mutant quantitation. This procedure can be used to assess the quality of live vaccines and to study heterogeneity of viral and bacterial populations.

Base Sequence↗

PubMed1992

Bacillus methanolicus sp. nov., a new species of thermotolerant, methanol-utilizing, endospore-forming bacteria.

The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60 degrees C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5S rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16S and 5S rRNA sequences indicated that this new species is distinct from Bacillus brevis but closely related to B. firmus and B. azotoformans. The name proposed for this new species is B. methanolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.

Bacillus↗

PubMed1992

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Bacterial Proteins↗

PubMed1992

RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence.

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) was used to study sequence heterogeneity and stability in attenuated poliovirus type 3 at positions in which the vaccine virus differs from its wild-type progenitor. Of seven genomic positions tested, only two (positions 472 and 2493) show nucleotide heterogeneity. Propagation of the vaccine virus in cell cultures leads to rapid selection of virus with reversions at these two positions of the genome. The relative abundance of reversions at position 472 correlates with the results of monkey neurovirulence tests, while the mutation at position 2493 is not directly associated with neurovirulence of the virus in monkeys. Instead, the abundance of mutations at the latter position correlates with the source of the seed virus and its passage level. These results further indicate that MAPREC at position 472 can be used to assess the quality of poliovirus type 3 vaccine.

Animals↗

PubMed1991

Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine.

Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.

Animal Testing Alternatives↗

PubMed1990

Sulfobacillus thermosulfidooxidans: a new lineage of bacterial evolution?

The nucleotide sequence of 5 S ribosomal RNA (rRNA) of type strain Sulfobacillus thermosulfidooxidans VKM B-1269 was determined. This organism represents a group of moderately thermophilic acidophilic chemolithotrophic bacteria, able to use ferrous and sulfur compounds as the sole energy source. 5 S rRNA of this bacterium is drastically different from all other known bacterial 5 S rRNA sequences. It is suggested that S. thermosulfidooxidans represents a new lineage of bacterial evolution, that diverged from other bacteria at an early step of their evolution.

Base Sequence↗

PubMed1990

Evolutionary relationship of some stalked and budding bacteria (genera Caulobacter, "Hyphobacter", Hyphomonas and Hyphomicrobium) as studied by the new integral taxonomical method.

A new approach was developed for the determination of taxonomic and evolutional relationships among four genera of oligotrophic bacteria. The main idea of this approach is the algorithmized integrative analysis of the morphological and physiological specificity of these bacteria, their 5S rRNA sequences, fatty acid and lipid composition of their membranes, as well as their sensitivity to a large variety of antibiotics. It was shown that the genera Caulobacter and Hyphomonas are closely related to each other, but they are both distant from Hyphomicrobium species. The new genus, "Hyphobacter", is placed between Caulobacter and Hyphomonas. Taxonomic heterogeneity was found to exist within the genera Caulobacter and Hyphomicrobium. Evolutional pathways from Caulobacter to Hyphomicrobium are proposed on the basis of the present data. No correlations were found between the cell morphology of the organisms and their geno- and chemotaxonomy.

Anti-Bacterial Agents↗

PubMed1990

Taxonomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing.

Nucleotide sequences of 5S ribosomal RNA (rRNA) isolated from 19 strains of Gram-negative methylotrophic bacteria were determined. Comparison of these sequences allowed construction of a tentative phylogenetic tree and showed that the bacteria analysed belong to the Proteobacteria and fell into several clusters, including obligate methanotrophs, obligate methylotrophs and several groups of facultative methylotrophs. Taxonomic relations between methylotrophic and non-methylotrophic bacteria are discussed, and the polyphyletic nature of methylotrophy as a taxonomic feature is highlighted.

Base Sequence↗

PubMed1989

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus.

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.

Ascorbic Acid↗

PubMed1989

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases.

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.

Amino Acid Sequence↗

PubMed1989

[Detection of a sodium pump in the terminal segment of the bacterial respiratory chain].

An alkalo- and halotolerant aerobic microorganism has been isolated which, according to microbiological data and the ribosomal 5S-RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism termed as Bacillus FTU proved to be resistant to the protonophorous uncoupler CCCP. The fast growth of Bacillus FTU in the presence of CCCP was shown to require high Na+ concentrations in the medium. A procedure has been developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only upon addition of an exogenous respiratory substrate. The exhausted cells were found to oxidize ascorbate in the presence of TMPD in a cyanide-sensitive fashion. Ascorbate oxidation was coupled to the uphill Na+ extrusion stimulated by CCCP and a penetrating weak base, diethylamine (DEA), as well as by valinomycin with or without DEA. The operation of the Bacillus FTU terminal oxidase resulted in the generation of delta psi which, in a Na+ medium, was slightly decreased by CCCP and strongly by CCCP + DEA. In a K+ medium CCCP discharged delta psi even without DEA. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium. CCCP + DEA were inhibitory in both media. The data obtained indicate that there is a Na+-motive terminal oxidase in Bacillus FTU. It is suggested that delta microNa formed by the oxidase can be utilized by an Na+-driven ATP-synthase.

Adenosine Triphosphate↗

PubMed1988

[A principle of maximum topological similarity in molecular systematics].

The paper deals with the problem of phylogenetic reconstruction on the basis of comparative analysis of features. Main attention is paid to comparison and classification of the biopolymer sequences. Different approaches to this task are critically reviewed. The novel principle of construction of tree-like classification schemes permitting subsequent evolutionary analysis is proposed. It concentrates on reconstruction of the tree with a topologic structure that is most close to topologic features, imprinted in the source distance matrix. Realization of this approach was made possible by development of the special formalism, enabling evaluation and comparison of topologic features of distance matrices and trees.

Animals↗

PubMed1988

[Algorithms for constructing phylogenetic trees of maximum topological similarity].

The paper concerns the practical realization of the maximum topologic similarity principle for phylogenetic reconstruction. This novel principle is described in the accompanying paper. Two algorithms that were embodied in the computer program allow one to find out the unique tree in case when source data admit the existence of such tree. In case if numerous parallel mutations make such precise realization impossible, algorithms allow one to obtain approximations to the maximum topologic similarity trees with a high computation efficiency. Examples illustrating use of these algorithms, as well as discussion of biological consistency of the novel concept are presented.

Algorithms↗

PubMed1988

[Evolution of RNA-dependent RNA-polymerases from positive RNA viruses: comparison of phylogenetic trees constructed by different methods].

Presumptive phylogenetic trees of evolutionary conserved fragments of RNA-dependent RNA polymerases of 26 positive strand RNA viruses were generated using a simple clustering procedure or a novel approach based on the so-called maximal topologic similarity principle. The latter methodology involves a quantitative measure of the degree of correspondence between the topology of generated trees and structure of the initial distance matrix. The algorithm for tree construction based on the maximal topologic similarity principle does not include the assumption of evolutionary rate constancy, as opposed to the clustering procedure. Nevertheless, it is demonstrated that the trees generated by the two methods are topologically similar, indicating that no drastic change of evolutionary rate had occurred in evolution of the positive strand RNA virus RNA polymerases. This in turn suggests that RNA-dependent RNA polymerases (or at least their evolutionary conserved core domains used for construction of the phylogenetic trees) are principally functionally equivalent in all positive strand RNA viruses.

Algorithms↗

PubMed1987

[Similarity of Vibrio alginolyticus, V. cholerae and other Vibrio species with respect to the structure of their flagellar apparatus and ribosomal 5S-RNA].

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.

Flagella↗

PubMed1987

[4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus].

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.

Animals↗

PubMed1987

[Evolution of RNA-dependent RNA polymerases of positive riboviruses].

A comparative analysis is presented of 24 known amino acid sequences of RNA-dependent RNA polymerases of positive strand RNA viruses infecting animals, plants and bacteria. Using a newly proposed methodology of group alignment for weakly similar sequences, evolutionary conserved fragments of all these proteins were unambiguously aligned. A unique pattern (consensus) of 7 invariant amino acid residues was revealed which is absent from the sequences of other RNA and DNA polymerases and is thought to unequivocally identify the RNA-dependent RNA polymerases of positive strand RNA viruses. Based on the obtained alignment a tentative phylogenetic tree of viral RNA polymerases was constructed for the first time. The RNA-dependent RNA polymerases of positive strand RNA viruses are concluded to comprise a distinct family of evolutionary related proteins.

Amino Acid Sequence↗

PubMed1986

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures.

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.

Base Sequence↗