Inactivation of Glucose-6-Phosphate Dehydrogenase in Solution by Low- and High-Frequency Ultrasound

We compared the kinetics of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) inactivation in 0.1 M phosphate buffer (pH 7.4) at 36–50° under conditions of exposure to low-frequency (LF, 27 kHz, 60 W/cm²) or high-frequency (HF, 880 kHz, 1.0 W/cm²) ultrasound (USD). The inactivation of G6PDH was characterized by effective first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(usd), ultrasonic inactivation. Dilution of the enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k _in > k _in ^*. The rate constants increased with temperature. The Arrhenius plots of the temperature dependences of k _in and k _in(usd) had an break point at 44°C. The activation energy ( _act) of the total inactivation of G6PDH was higher than _act for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: _act was higher in the case of inactivation with low-frequency ultrasound (LF-USD) than high-frequency ultrasound (HF-USD). The rate of the ultrasonic inactivation of this enzyme substantially decreased in the presence of low concentrations of HO^. radical scavengers (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in solutions exposed to USD.     

Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.     

Increasing the resolution of chromosome analysis using pyrido[1,2-a]benzimidazoles

We studied the influence of three derivatives of pyrido[1,2-a]benzimidazoles (PBIs), which have DNA-intercalating properties, on plant mitotic chromosome condensation, in order to increase the resolution of chromosome analysis. The efficiency of the influence of these agents was assessed using the median chromosome length on chromosome slides, as well as by the number and size of chromosome DAPI bands. We used the third chromosome of Linum grandiflorum Desf. in these experiments. The chromosome was identified on the slides using its DAPI band pattern and a molecular marker, viz., the 5S rDNA site, which is located in the proximal region of the long arm of the chromosome. The influence of the well-known 9-aminoacridine (9-AMA) DNA intercalator, which is widely used in karyotype studies of short-chromosome organisms, was used as a control in all of the experiments. It was found that the influence of each of the three PBIs in the study on the root meristem of L. grandiflorum resulted in an increase in the median length of the third chromosome, the linear centromeric DAPI band size, and the number of intercalary DAPI bands. All three PBIs acted more efficiently than 9-AMA. The median chromosome length was increased by 15?C40% and the number of intercalary bands increased by 1.5?C3 times after PBI treatment, as compared to 9-AMA treatment. At the same time, 7-CF₃-PBI, in a similar manner to 9-AMA, did not change the relative size of the centromeric DAPI band, while 7-NH₂-PBI and 9-NH₂-7-CF₃-PBI gradually increased this parameter. It is concluded that these substances can be used as intercalating agents in cytogenetic studies in order to increase the resolution of chromosome analysis.     

Genetic polymorphism of flax Linum usitatissimum based on the use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flax) and in the k-37 × Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosome rearrangements (chromosome 3 inversions) were detected in the variety Luna and in the k-37 × Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosome analysis in the fiber and oil flax confirm their very close genetic similarity. In spite of this, the combined use of the chromosome and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.     

Karyogenomics of species of the genus <Emphasis Type="Italic">Linum</Emphasis> L.

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were discovered and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.