Biomedical subjects

V V Sukhodolets

Publications and source records attributed to V V Sukhodolets.

At least 19 recordsLinked to original sources

PubMed2002

[Study of RecA-independent homologous recombination and a chromosomal rearrangement in the Escherichia coli strain carrying an extended tandem duplication].

A heterozygous tandem duplication in the Escherichia coli deo operon region deoA deoB::Tn5/deoC deoD thr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F- strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec+ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD+ (the DeoD+ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD+ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.

Chromosomes, Bacterial↗

PubMed2002

[Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication].

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes. Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.

Adenosine Triphosphatases↗

PubMed2001

[Genetic explanation for vertical evolution].

The genetic theory of natural selection proposed by Fisher takes into account differential reproduction success of organisms, which may be estimated by using the Malthusian parameter as fitness. However, the minimum possible value of this parameter depends on ecological stability of an organism, which determines the probability of the survival and participation in reproduction for each viable offspring. In the course of vertical evolution, leading to an increase in the level of biological organization, ecological stability of organisms increases, and this might be accompanied by a decrease in their fitness. In the macroevolutionary process, alterations in ecological stability of organisms, including those responsible for an increase in the level of biological organization, are basic and primary changes whereas alterations in fitness are additional and secondary.

Adaptation, Physiological↗

PubMed2001

[Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication].

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.

Alleles↗

PubMed2000

[Adaptation, ecological stability and the evolution of diploid organisms].

The possibility of the existence of an organism under different environmental conditions is determined by its ecological stability. This parameter can be expressed as the product of the average life span corresponding species and the probability of an organism's participation in reproduction. If ecological conditions are not substantially altered, regulatory selection provides an increase in fitness of an organism in a certain direction of adaptation. It is supposed that the process of regulatory selection is accompanied by the accumulation of mutations occurring in regulatory genes and mutations in regulatory regions of structural genes which correct the effect of the former mutations. An alteration in ecological stability occurs when the conditions of population existence are changed and is usually accompanied by a decrease in the fitness level earlier achieved. Thus, an increase in organisms' ecological stability is achieved by hybridization between populations of different origin and is accompanied by a decrease in fitness due to outbreeding depression. Under conditions of inbreeding, ecological stability is decreased due to the segregation, in the homozygous state, of recessive alleles of adaptive genes that have not yet reached the stage of evolutionary fixation. Diploidy is a factor allowing organisms to improve their ecological stability in every new generation.

Adaptation, Biological↗

PubMed2000

[Homologous recombination between direct repeats of chromosomal segments comprising heterozygotic duplications in Escherichia coli].

In conjugational matings between double mutants for the deo operon of Escherichia coli, haploid recombinants and extended tandem duplications deoC deoD/deoA deoB::Tn5 with the DeoC+DeoA+DeoB+DeoD- phenotype are formed (the deoD+ allele is not expressed due to the polar effect of the Tn5 insertion). Selection for the expression of the recessive deoC deoD alleles (in the thyA genome) leads to the segregation of haploid clones by duplications and also of clones that retain the diploidy but that are homozygous for deoC deoD. In addition to haploids, diploid clones retaining the duplications have also been found among the DeoD+ segregants. The phenotype of segregants retaining the duplication shows that they were formed by an unequal exchange between sister chromosomes. A comparison of segregation frequency of haploid and diploid DeoD+ clones in rec+ and recBC sbcB sbcC strains shows that duplications in the rec+ genome are more stable. On this basis, it is assumed that the RecBCD pathway possibly makes a greater contribution than the RecF pathway to the preservation of heterozygous duplications playing an important role in the evolution of prokaryotes.

Chromosomes, Bacterial↗

PubMed1999

[Formation of the heterozygous tandem duplication in the process of conjugation recombination in Escherichia coli: study of the effect of mutations for the recQ, uvrD, and recJ genes].

Stable tandem duplications were shown to originate from conjugational recombination between Escherichia coli HfrH strains carrying mutations for the deo operon. The duplications deoC deoD/deoA deoB::Tn5 usually constitute approximately 5% of the Deo+ offspring. The effect of mutations for the recQ, uvrD, and recJ genes on the frequency of duplications was studied. The CM1563 strain carrying the recQ mutation was shown to give, as a recipient, 20% of duplications in the Deo+ offspring. However, this property of CM1563 seems to depend on the presence of a spontaneous mutation of unknown nature, which also increased UV sensitivity of bacteria. The recQ mutation itself increased the frequency of duplications by less than 50%. The recJ mutation did not virtually affect the frequency of duplications. The uvrD mutation possessing the recombinogenic effect was shown to increase the frequency of deo+ recombinants and simultaneously decrease the frequency of duplications. Tandem duplications are assumed to be normal intermediates of multi-stage conjugational recombination initiated by the integration of the proximal region of the Hfr chromosome into different nonhomologous regions of the recipient chromosome.

Conjugation, Genetic↗

PubMed1998

Isolation and analysis of UV and radio-resistant bacteria from Chernobyl.

The accident at the Chernobyl nuclear power station in 1986 led to the dispersal of large amounts of a variety of radioactive materials, most importantly uranium, plutonium, 137Cs, 131I and 90Sr, over very large distances estimated to reach as far as Sweden, Norway, Turkey and possibly the USA. As a consequence, the soil on which the radioactive materials fell was contaminated and the degree of contamination varied with distance from the station, the direction and strength of the wind and the amount of atmospheric scavenging by rainfall at that time. Some of the radioactive materials have left a significant impact on mankind in the form of chromosomal aberrations including trisomy, various forms of cancers and death, whilst others are still in the ground where they will remain for a prolonged period to continue to exert their effects. Likewise, microbes living in the soil and exposed to radioactive materials may have been affected in a number of ways; some perished, and others survived due to the acquisition of advantageous mutation. Six years after the accident, soil samples contaminated with different levels of radioactivity were obtained from five regions within a 30 km radius of the nuclear power plant. From these soil samples spore-forming bacilli were isolated, quantified, identified and tested for resistance to X-rays, UVC and 4-nitroquinoline 1-oxide (4NQO). As a control, spore-forming bacilli were obtained from 'Zeleny mys' (an area 50 km south-east of the power station and emitting basal levels of radioactivity). A mutant of Escherichia coli hyper-resistant to a variety of DNA-damaging agents and its parent strain were also included in the study. Analysis of results reveals that a proportion of isolates of the same species from near the power station and the E. coli mutant SA236 were more resistant to X-rays, UVC and 4NQO compared with isolates from the control site and the E. coli parent strain, KL14, respectively.

4-Nitroquinoline-1-oxide↗

PubMed1998

[The nature of adaptive evolutionary changes: fitness and potential].

Ecological potential of an individual can be defined as its viability in the broad sense including the ability to reproduce in various environments. From the biological viewpoint, ecological potential as a fundamental property of an organism is more important than fitness in the genetic sense, which is estimated as the relative rate of reproduction. In essence, fitness reflects the level of implementation of the biological potential. In the process of evolution, regulatory selection results in an increase of fitness: selected forms reproduce more successfully as the population size increases. By contrast, individuals with high ecological potential are more advantageous when the population size decreases, because the probability of their survival in adverse environments is high. Thus, high levels of fitness and ecological potential are achieved via operation of different types of selection.

Adaptation, Physiological↗

PubMed1998

[Possible function and origin of crossing over].

Crossing over to repair genetic material via exchange probably emerged at the initial stages of organic evolution, earlier than the appearance of the DNA molecule in its present form. In that period, nitrous bases existed as free molecules, although, like in double-helical DNA, they formed aggregates. The electrochemical characteristics of pyrimidines differ from one another to a greater extent than those of purines, which are almost electrically neutral. Therefore, it is possible that the DNA electrostatic balance is accounted for by nonspecific interaction between pyrimidine bases. The data obtained by Nussinov et al. indicate that, in homooligomers, the arrangement of pyrimidine bases in opposite DNA strands exhibits some restrictions with respect to their mutual positions. In functioning DNA, the electrostatic balance of pyrimidine arrangement in complementary strands may favor rapid transitions of the molecule between an open helix and the original state. It is supposed that crossing over restores the electrostatically balanced spatial arrangement of pyrimidine homooligomers and, hence, the functional activity of the entire DNA molecule.

Crossing Over, Genetic↗

PubMed1998

[Further genetic study of tandem duplication formation in the region of the deo operon in the process of Escherichia coli K-12 conjugational recombination].

The formation of heterozygous duplications in the region of the deo operon was studied in conjugational matings (male) HfrH deoC deoD thr::Tn9 thyA x HfrH deoA deoB::Tn5 thyA. When recombinants that inherited the donor marker thr::Tn9 (Cml r) were selected on a medium containing thymine and chloramphenicol, but not threonine, more than 80% of the offspring were heterozygous tandem duplications extending to the region of the deo operon. In matings with a thymidine-dependent HfrH deoA deoB::Tn5 thyA strain as a recipient, when recombinants were selected on a medium containing thymine, i.e., under conditions of thymine starvation of merozygotes, the recombinogenic effect was observed. However, this effect did not change the frequency of duplication formation. The integration of genetic markers via homologous recombination into the chromosomal regions adjacent to duplications occurred at a lower frequency. An analysis of the formation of haploid segregants by duplications showed that, in most cases of duplication formation, the proximal segment of the donor chromosome is integrated into the distal position, i.e., after the homologous segment of the recipient chromosome.

Conjugation, Genetic↗

PubMed1997

[Genetic study of the mechanism of tandem duplication formation in conjugational crosses in Escherichia coli K-12].

The formation of heterozygous tandem duplications of the deo operon was studied in conjugational matings HfrH deoA deoB::Tn5 thyA x HfrH deoC deoD thyA. When the HfrH deoC deoD thr::Tn9 car::Tn10 thyA donor strain was used, the thr::Tn9 and car::Tn10 transposon insertions linked to the deo operon were integrated into some duplications. Duplications carrying the recipient thr+ and car+ alleles in the duplicated state were not found among duplications that did not include the thr::Tn9 and car::Tn10 donor markers. These data indicate that heterozygous deo operon duplications are primarily formed directly during the recombinational interaction between chromosomes in the merozygote, but not on the basis of preexisting sister duplications in the recipient strain. Thus, genetic recombination occurring during the process of conjugation in E. coli induces both symmetrical and unequal crossing over. Nevertheless, when the HfrH deoA deoB::Tn5 thr::Tn9 car::Tn10 thyA strain was used as a recipient, a deo operon duplication containing thr::Tn9 and car::Tn10 markers was found in the homozygous state. Consequently, some heterozygous duplications can also be formed on the basis of preexisting sister duplications.

Conjugation, Genetic↗

PubMed1996

[Unequal crossing over in Escherichia coli: genetic and physical mapping of duplications isolated in conjugational matings].

We previously demonstrated that stable heterozygous duplications deoA deoB :: Tn5/deoC deoD can be isolated among Deo+ recombinants obtained in conjugational matings between Escherichia coli strains HfrH deoA deoB :: Tn5 and HfrH deoC deoD. In this work, 30 duplications were tested by transduction for the inclusion of a set of genetic markers adjoining the deo operon (99.5 min) at the genetic map of E. coli: cycA :: Tn10 (96 min), zji :: Tn10 (98.2 min), thr :: Tn9 (100/0 min), car :: Tn10 (1 min), leu :: Tn9 (2 min), and proAB :: Tn10 (6 min). The results obtained indicate that only three out of 30 duplications could have originated from unequal crossing over between the rrn operons. In eight strains, duplications were chosen for physical mapping by the use of Not I restriction, pulsed-field electrophoresis, and Southern blot hybridization with DNA of the deo operon. In all these strains, the presence of duplications (once a triplication) was confirmed by corresponding changes in the set of Not I digests, although some strains had additional genetic rearrangements. The order of operon copies in a tandem was determined, and the length of a duplicated chromosomal segment was calculated as equal to 25, 46, 80, 150.5, and 175 kb in duplication D49, D4, D5, D9, and D18, respectively. Thus, the use of the conjugational Hfr x Hfr system allows the generation of unique rearrangements of the E. coli genetic material.

Chromosome Mapping↗

PubMed1995

[Regulatory selection as an alternative to the neutrality theory].

Regulatory selection is the most typical mode of natural selection responsible for the optimization of the regulatory systems of an organism. In addition to regulatory selection, stringent selection of pleiotropic mutants or recombinant forms with altered ecological potential operates in successful species during vertical evolution. Regulatory selection stabilizes alterations in the ecological potential, acting at intervals between larger regulatory genome alterations occurring at the boundaries between evolutionary phases. Fisher's adaptive model can be applied to regulatory selection. At each separate evolutionary phase, the selection coefficients in selected mutants decline gradually. At the final stage of the evolutionary phase, mutations with a minor selective advantage are selected. Hence, regulatory selection may be regarded as an alternative to the neutral evolution theory.

Animals↗

PubMed1993

[Increased frequency of unequal crossing over in recBC mutant cells in conjugational crosses in Escherichia coli K-12].

The formation of heterozygous tandem duplications was studied in conjugational crosses of Escherichia coli using mutations for deo-operon genes. The frequency of tandem duplications in recF::Tn3 and recBC sbcB mutants was not substantially changed from that of the wild type strain. In contrast, the frequency of duplications was increased about ten-fold when using recBC (sbcB+) mutants as recipients. This result can be explained by a participation of the RecBCD protein in the pairing of chromosomes during recombination. We also found that HfrH thyA recBC (sbcB+) bacteria grow very poorly on rich medium and that good growth is restored by conversion from the Hfr to the F+ state, as well as by mutations suppressing the RecBC phenotype; such F+ strains retain sensitivity to UV light and to mitomycin C, which are characteristic of the recBC phenotype.

Conjugation, Genetic↗

PubMed1992

[Principles of prokaryotic genome organization].

The peculiarities of bacterial chromosome organization are discussed, based mainly on the data on Escherichia coli. Highly important for bacterial genome organization is its division into two approx. equal half-genomes undergoing periodically "exchanges" of some kind displayed as continuous inversions including the oriC region of replication initiation. It is believed that short oligonucleotides are comprised in either of half-genomes. The former are predominantly oriented as direct repeats, which ensures the possibility of formation of tandem duplications consisting of identical genes--under conditions when selection for enhancing functions of corresponding genes takes place. Multiple tandem duplications capable of excision of plasmatic gene copies seem to initiate horizontal gene transfer in bacteria. Tandem gene duplications are probably being formed in the process of bacterial genetic recombination as well, when, as a result of non-equal crossing over, gene alleles derived from different strains are united into a tandem.

Alleles↗

PubMed1991

[Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates].

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.

DNA Transposable Elements↗

PubMed1991

[Cloning the gene of beta-galactosidase from the industrial strain of Streptococcus lactis 111 in E. coli cells and conjugated transfer of this gene to Streptococcus thermophilus cells].

The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.

Cloning, Molecular↗