RESUMEN
Considering the potentials of algae Euglena to constitute a part of biological systems of human life support, effects of low radiation doses on algal cells and radiosensitivity dependence on their genotype were studied. In experiments with gamma-irradiation (60Co) of Euglena gracilis, the highest radioresistance was demonstrated by strain Z. OFL; the chloroplasts lacking Z-derived strain showed hypersensitivity to radiation. E. bacillaris and derived chlorophyll-lacking strains W3 and W10 had intermediate radiosensitivity. Irradiation with the doses of up to 10 Gy produced a hormetic effect in the stock strains. Cells death was observed only after irradiation by doses above 100 Gy. The stimulating effect was exerted both on radioresistance and growth rate. Dyes made possible rapid evaluation of the proportion of living and dead cells. Comparison of two survival tests showed that the classic medium inoculation overestimates cell deaths as it disregards the living non-proliferating cells.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Euglena gracilis/efectos de la radiación , Rayos gamma/efectos adversos , Técnicas de Cultivo de Célula , Relación Dosis-Respuesta en la Radiación , Euglena gracilis/fisiología , Colorantes Fluorescentes , Genotipo , Humanos , Azul de Metileno , Microscopía Fluorescente , Tolerancia a Radiación , Vuelo EspacialRESUMEN
Molecular mechanisms of activation of DNA repair and checkpoint control by double-strand breaks are considered. They include phosphorylation by protein kinases of repair and checkpoint proteins resulting in their activation, alteration of affinity to other proteins, and alteration of their localization.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Proteínas Quinasas/fisiología , Fosforilación , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of injurious agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm shorten the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G2/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in the formation of a cell response to DNA damage, and in checkpoint regulation in particular.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Daño del ADN/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Mutación , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The nanosecond-long molecular dynamics of the movement of the active protein kinase CDK2/ATP complex has been analyzed. The simulations of substitutions CDK2-G16S in the conserved G-loop of a small lobe and CDK2-R274Q in a large lobe showed the importance of the amino acid residues in the conformation of kinase and their effect on the conformation of CDK2, which shows up in an increase in the distance between G- and T-loops in the corresponding mutant forms. The results obtained indicate that the induction of both Gly16-->Ser16 and Arg274-->Gln274 mutations destabilize the local structure of kinase around the T-loop area. The mutation Arg274-->Gln274 has a more pronounced effect and results in a loosening of the structure of kinase and an increase of the distance between G- and T-loops.
Asunto(s)
Arginina/genética , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/química , Glutamina/química , Glicina/genética , Serina/genética , Sustitución de Aminoácidos , Quinasa 2 Dependiente de la Ciclina/genética , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genéticaRESUMEN
Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9delta were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to gamma-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to gamma-radiation and UV light; mutations rad9delta and rad24delta were shown to manifest the additive effect in the first case and epistatic effect in the second.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Rayos gamma , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Tolerancia a Radiación/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Quinasa de Punto de Control 2 , Epistasis Genética , Genes cdc/fisiología , Mutación , Tolerancia a Radiación/efectos de la radiaciónRESUMEN
Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9Delta were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to gamma-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to gamma-radiation and UV light; mutations rad9Delta and rad24Delta were shown to manifest the additive effect in the first case and epistatic effect in the second.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/genética , Epistasis Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Tolerancia a Radiación/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Rayos UltravioletaRESUMEN
Two-nanosecond molecular dynamics modeling of the crystalline lattice of an active complex of kinase pT160-CDK2/cyclin A/ATP-Mg2+ substrate has been performed. The results of modeling indicated that the structures of the nonmutant CDK2 complex and mutant CDK2 complex, which involves the G 16S-CD K2 substitution corresponding to that of yeast, markedly differ, the differences in structural conformations being particularly well pronounced in those regions that play a key role in the functioning of kinase. Based on the results of computations, structural elements are considered that may affect the kinase activity and regulatory phosphorylation, and the binding of protein kinase to cyclins and substrates.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/química , Quinasa 2 Dependiente de la Ciclina/química , Modelos Moleculares , Mutación Puntual , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Magnesio/química , Unión Proteica/genética , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The repair of a double strand gap in plasmid DNA in radiosensitive mutants of Saccharomyces cerevisiae has been studied. The proportion of repair events resulting in the complete doublestrand gap recovery of the plasmid DNA has been found to be close to 100% in Rad+ cells. The mutation rad55 did not interfere in the doublestrand gap repair efficiency and accuracy. The mutant rad57 is capable of the effective doublestrand gap repair without restoration of the DNA sequence deleted by the gap. The mutation rad53 substantially inhibited the efficiency of the doublestrand gap repair but did not influence the accuracy of the repair. Plasmid DNA doublestranded gap repair is completely blocked by mutations rad50 and rad54.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN Bacteriano/efectos de la radiación , Tolerancia a Radiación , Saccharomyces cerevisiae/genética , Electroforesis en Gel de Agar , Mutación , Plásmidos , Saccharomyces cerevisiae/efectos de la radiaciónRESUMEN
Different combinations of modifying genes which enhance the rho- mutability of haploid yeast cells are shown to be suppressible by the srm1, srm2, srm3 mutations and by the disomy for chromosome IV. The srm1 mutation leads to dramatic decrease in both the spontaneous and ethidium-bromide induced rho- mutability. Other srm mutations studied and the disomy appear to cause relatively moderate quantitative changes in the spontaneous rho- mutation rate and to have no significant effect on mutation induction by ethidium bromide. Neither additivity nor synergism was revealed by the analysis of the interaction between the srm mutations. We suggest that in Saccharomyces an efficient mechanism of the rho- mutagenesis operates which can be directly affected by the srm1 mutation and more or less modified by other srm mutations under study and by the disomy for chromosome IV.
Asunto(s)
ADN Mitocondrial/genética , Genes Fúngicos , Mutación , Factor Rho/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , ADN de Hongos/genética , Genes ReguladoresRESUMEN
The phenotypic trait "starry colony" in Saccharomyces is associated with a high spontaneous rho- petite mutability. Genetic analysis of this trait has shown the high rho- mutability to be caused by several modifying genes present together in the cell genome. Every single modifying gene only produces a relatively small enhancement in the rho- mutability. Mutations in four nuclear srm (spontaneous rho- mutability) loci were isolated after mutagenic treatment of highly rho- mutable haploid cells. In contrast to the modifying genes, each of these mutations has a pronounced effect on the spontaneous rho- mutability, causing significant decrease in it.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Genes Reguladores , Mutación , Saccharomyces cerevisiae/genética , Genes Fúngicos , Ligamiento Genético , Marcadores GenéticosRESUMEN
The sensitivity of the yeast Saccharomyces cerevisiae to nitrous acid (NA) is significantly influenced by various spontaneous mutations of the mitochondrial (mt) genome as well as by the nuclear mutation mmg 1 leading to a decrease in the spontaneous mutability of the mt genome. The mmg 1 locus and the mt genome most probably interact and this nucleo-cytoplasmic interaction plays a role in determining the NA sensitivity of yeast cells. A significant subclonal variation of the NA sensitivity has already been reported for the strains under study. Here we show this variability to decrease significantly when the cells are devoid of the mt DNA or carry the mmg 1 mutation. These data suggest a direct relation between the unstable NA sensitivity and the variability of the mt genome.
Asunto(s)
Genes/efectos de los fármacos , Variación Genética/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mutágenos/farmacología , Nitritos/farmacología , Ácido Nitroso/farmacología , Saccharomyces cerevisiae/genética , Genotipo , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
The disomy for chromosome IV in the strains studied led to: reduction in the red pigmentation of ade1 mutant colonies; a decrease in spontaneous rho- mutant frequency, and impairment of sporulation in hybrids descended from disomic parents. The nuclear srm1 mutation decreasing the spontaneous rho- mutability promoted the spontaneous extra chromosome loss in the disomics for chromosome IV. This result suggests a close connection between the spontaneous rho- mutability and mitotic chromosome stability.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Marcadores Genéticos , Fenotipo , Saccharomyces cerevisiae/fisiología , Esporas FúngicasRESUMEN
The srm5 mutation diminishes the spontaneous rho- mutation rate by an order of magnitude. Frequency of rho- mutations is 500 times lower in homozygous cultures, as compared with those of normal SRM+/SRM+ diploids. The rate of spontaneous loss of extra chromosome IV is about 25 times higher in srm5 disomes, as compared with SRM+ ones. Haploid srm1 srm5 transformants loose recombinant circular minichromosomes spontaneously about 4 times more frequently than srm1SRM5 cells. The data presented suggest that general control of mitotic stability of different (mitochondrial and nuclear, nuclear as well as recombinant) genetic structures operates in Sacch. cerevisiae. Autonomously replicating sequences (ARS elements) seem to be involved in this mechanism.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Genes Fúngicos , Mutación , Recombinación Genética , Saccharomyces cerevisiae/genética , Genotipo , PloidiasRESUMEN
From grown cultures of UV-irradiated Saccharomyces cerevisiae cells with disomy at chromosome IV, clones with nuclear gene mutations were isolated, each of which was suggested to change both mitochondrial spontaneous rho- mutability and the mitotic stability of extra natural chromosomes. Four such nonallelic mutations (srm8, srm12, srm15, and srm17) were isolated, and their phenotypic expression characterized. All four mutations are associated with decreased spontaneous rho- mutability and virtually block sporulation in homozygous mutant diploids. Mutation srm8 is temperature-sensitive and, most probably, involves an essential gene. Double mutants of genotypes srm8 cdc28-srm and srm8 srm12 are nonviable. Mutation srm12 increases the rate of spontaneous loss of extra chromosome XIV by disomics by a factor of about 30. Mutation srm15 induces a small (about twofold) but statistically significant decrease of this rate. Mutations srm8 and srm17 drastically decelerate reproduction of cells with disomy, which prevents quantitative estimations of rates of loss of extra chromosomes.
Asunto(s)
Núcleo Celular/genética , Genes Fúngicos , Mitocondrias/genética , Mitosis/genética , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos , Genoma Fúngico , Genotipo , Mutación , FenotipoRESUMEN
The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied. The srm8 mutation caused instability of chromosome maintenance in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation. The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.
Asunto(s)
Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Etidio/farmacología , Rayos gamma , Fenotipo , Tolerancia a Radiación/genética , Saccharomyces cerevisiae/efectos de la radiaciónRESUMEN
A tester system consisting of six isogenic strains was used to study the regularities of the induction of point mutations in the yeast Saccharomyces cerevisiae after exposure to gamma-radiation. This system allowed the identification of all six possible substitutions in the Cys-22 codon of the CYC1 gene encoding iso-1-cytochrome c. The dose dependence of the frequency of these six base-pair substitutions was shown to be linear-quadratic. The pattern of base substitutions did not depend on the doses of gamma-irradiation used (from 125 to 1000 Gy) and predominantly included GC-->AT transitions and AT-->TA transversions. The possible mechanisms of gamma-ray mutagenesis leading to a nonlinear dose dependence were considered, and the spectra of mutations obtained for different yeast genes and for Escherichia coli were compared.
Asunto(s)
Citocromos c , Rayos gamma , Mutación Puntual , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Codón , Cisteína/química , Grupo Citocromo c/genética , Escherichia coli/genética , Saccharomyces cerevisiae/efectos de la radiación , Especificidad de la EspecieRESUMEN
The effects of nuclear gene mutations cdc28-srm and srm1 on the maintenance of various recombinant facultative genetic structures (FGSs) in Saccharomyces cerevisiae were studied. These structures are ARS1 TRP1 mini-coils, noncentromeric circular plasmids containing various ARS elements, and extended linear yeast artificial chromosomes (YAC). These mutations led to an increase in the mitotic stability of some of the FGS tested and the disturbed maintenance of the others. Mutation srm1 imposed a stabilizing effect on the maintenance of various recombinant FGSs with ARS chromosomal elements. Mutation cdc28-srm destabilized the maintenance of only those recombinant FGS that shared full or detectable homology with sequences of the nuclear genome of the yeast cell.
Asunto(s)
Genes Fúngicos , Recombinación Genética , Saccharomyces cerevisiae/genética , Cromosomas Artificiales de Levadura , Mitosis/genética , Mutación , Conformación de Ácido Nucleico , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
The presence in the cell genotype of srm1 and srm5 (cdc28-srm) mutations decreasing the spontaneous rho- mutability was shown to have no effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. Mutation cdc28-srm exerts a marked effect on cell sensitivity to the lethal action of ionizing radiation and on the appearance of homoplasmic segregants generated from heteroplasmic diploids. Additive interactions between mutations cdc28-srm and each of the rad6 and rad52 mutations were revealed by an analysis of double mutants with respect to their sensitivity to radiation. Mutation rad9 was epistatic with mutation cdc28-srm. These data agree with the idea that the p34CDC28 gene product is a target for the RAD9-dependent feedback control operating at the cell cycle checkpoints (checkpoint control) and ensuring an additional amount of time for premitotic repair of chromosomal DNA damage.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Mutación , Proteínas Nucleares/genética , Tolerancia a Radiación/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces/efectos de la radiación , Alelos , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Factores de Intercambio de Guanina Nucleótido , Ligasas/genética , Mitosis , Proteína Recombinante y Reparadora de ADN Rad52 , Recombinación Genética , Saccharomyces/genética , Enzimas Ubiquitina-ConjugadorasRESUMEN
The physico-chemical characteristics of EIA, and in particular sensitivity, accuracy, reproducibility, can undergo essential changes with variations occurring in the course of the experiment, which makes it necessary to optimize conditions at each stage of the assay. For this purpose the human anti-IgG-IgG system has been studied in two modifications of EIA: the sandwich and competitive methods.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Inmunoglobulina G/análisis , Unión Competitiva , Humanos , Técnicas para InmunoenzimasRESUMEN
Norepinephrine (NE), dopamine (DA) and serotonin (5-HT) blood plasma levels were measured in 40 alcoholic patients (2nd stage) during overt manifestations of the alcohol abstinence syndrome (AAS). Twenty-five healthy individuals served as controls. The patients were divided into four groups according to the structure of their AAS-related psychopathology and the progress of the disease. In the fast-progressive group with autonomic disorders prevalent in the AAS structure, the excitability, NE and DA levels were elevated. In the group with prevalent asthenic-neurotic and affective disorders, the 5-HT levels were lower than controls. The group with fast-progressive course of the disease and schizoaffective disorders had both the 5-HT and NE levels lowered. The authors discuss the possible impact of the results on perfectioning of the differential AAS therapies.