A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.     

Effect of rad 54 mutation on the ability of Saccharomyces cerevisiae cells to recover from radiation and thermal injury

The diploid cells of Saccharomyces cerevisiae carrying rad 54 homozygous mutation do not exhibit an ability for any considerable "rapid" postirradiation and post-hyperthermic recovery. A pretreatment with high temperature (50 degrees C) increases the radiation response of mutant cells. Survival of cells overheated before gamma-irradiation is increased by keeping them in water for 2-6 h at 28 degrees C, while the corresponding value of survival for cells treated by each of the factors delivered separately remains constant in these conditions.     

Recovery of yeast cells following the combined action of hyperthermia and ionizing radiation

Consecutive action of elevated temperature (50 degrees C) and gamma-irradiation on yeast cells Saccharomyces cerevisiae was studied. It was shown that yeast cells can recover from lethal thermal and radiation lesions after the combined action of the two factors. The efficiency of recovery does not depend upon the sequence of treatments. Heating (50 degrees C) before or after gamma-irradiation increases the radiation response of yeast when plating the cells on a nutrient agar containing 1.5 M KCl. The synergistic effect decreases with yeast cells kept in water at 28 degrees C before plating. The influence of one factor on the effectiveness of recovery from damages induced by the other was estimated.     

Macrokinetic basis for the model of microbial growth in a limited volume under constant conditions with a single leading substrate

Within the framework of the macrokinetic approach and continuum and chemical/biochemical gross reaction conceptions, an equation describing the complete dynamics of microbial growth and decline as function of a variable concentration of the leading substrate was deduced. This equation allows us to distinguish quantitatively and qualitatively the stages of microbial growth and the intervals of microbial tolerance to the initial concentration of the leading substrate. Adequacy of the model was confirmed by comparison with experimental dynamics of aerobic microorganisms in the samples of groundwater collected from a region polluted with uranium.     

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.     

Introduction of additional thiol groups into glucoamylase in Aspergillus Awamori and their effect on the thermal stability and catalytic activity of the enzyme

Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using molecular modeling simulations and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth α-helices and that of the loop located close to a catalytic residue—E400—made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.     

Transport of monovalent thallium across the membrane of oocyte of the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using ²⁰⁴Tl. The Tl⁺ transport in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by mechanisms of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl₂, 5 glucose, 10 Tris-HCl-in the presence of ouabain, the coefficient of the ²⁰⁴Tl stationary distribution (cell/medium) was within the range of 2.3–2.5, while the time necessary to reach its 50% value amounted to 40–45 min at 20°C. In potassium-free media, transport of ²⁰⁴Tl via Na/K-pump was described by simple kinetics with saturation and was characterized by the value V _max = 520 pmol/(cell h) and K _M = 0.3 mM. In the presence of 4 mM K⁺ and 0.1 mM/1 Tl⁺, the ouabain-sensitive Tl⁺ flux decreased to 75 pmol/(cell h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na⁺ (in Na-NMDG media of different composition) the total influx of Tl⁺ reached 193 ± 20 pmol/(cell h), while the bumetanide-sensitive component—119 ± 12 pmol/(cell h) with K _M for Na⁺ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl⁻ by NO₃) the total Tl⁺ flux reached 220 ± 21, while via the mechanisms of Na,K,Cl-cotransport—87 ± 8 pmol/(cell h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl⁺ influx. The potassium channels that usually are permeable also to monovalent thallium ions were not revealed.     

Structure and properties of carrageenan-like polysaccharide from the red alga Tichocarpus crinitus (Gmel.) Rupr. (Rhodophyta, Tichocarpaceae)

Sulfated polysaccharides occurring in the red algae Tichocarpus crinitus cell wall were fractionated and purified. NMR and FT-IR spectroscopy analyses revealed that the non-gelling fraction contained a sulfated galactans having a new carrageenan-like structure. It is built with alternatively linked 1,3-linked β-D-galactopyranosyl-2,4-disulphates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl residues. Minor amounts of its biosynthetic precursor were detected in a water-extracted specimen. Brief analysis of rheological and biological properties of the non-gelling fraction was carried out. The carrageenan-like polysaccharide from T. crinitus displayed the properties of “random coil” polymer at high temperature, and possesses high anticoagulant activity at low concentration.     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.