Milan Bezděk

5-Azacytidine-induced hypomethylation of tobacco HRS60 tandem DNA repeats in tissue culture

The methylation status and 5-azacytidine-induced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leaf-derived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine.

Changes in chromatin structure due to hypomethylation induced with 5-azacytidine or DL-ethionine

Changes in chromatin structure of the HRS60 family of repetitive sequences in tobacco DNA were studied after hypomethylation induced with 5‐azacytidine or DL‐ethionine. The TaqI site in the HRS60 units lies in nucleosomal core regions and its cleavage is enhanced in the hypomethylated chromatin. In contrast, the cleavage of the Sau3A1 site located in linker DNA does not depend on the level of methylation of DNA.

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome.

SummaryTwo DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.

Hormonal control of 5-bromodeoxyuridine (BUdR) tolerance in crown-gall tumors and habituated tissues

We have found that the unorganized tobacco crown-gall tumor, isolated in our laboratory, and Brauns teratomatic tumor both exhibit the capacity to grow in the presence of high toxic concentrations of BUdR (up to 10-3 mol·l-1). Double cytokinin-auxin habituated tissues also exhibit a constitutive BUdR-tolerance. In cytokinin-habituated tissues the BUdR-tolerance seems to depend on the degree of cytokinin autonomy. The presence of exogenous cytokinin significantly increases BUdR-tolerance, and the presence of BUdR suppresses the inhibitory effect of exogenous cytokinin upon habituated tissues. The results clearly show that the disposition of a cell for transient BUdR tolerance requires at least active expression of genes for cytokinin synthesis and that the tolerance becomes permanent when the auxin system is turned on. We conclude that BUdR-tolerance is connected with the establishment of a certain hormonal regulator pattern, depending on the state of cellular differentiation.

Thermal decomposition of copolymers of styrene and halogenated monomers

Abstract Copolymers of 1,2,2,2-tetrachloroethyl esters of unsaturated acids and halogenated N-phenyl maleimides with styrene were pyrolyzed; volatile products were analyzed with a mass spectrometer combined with a gas chromatograph. Hydrogen halide and carbon dioxide in the volatile products were determined during the thermal decomposition of copolymers in glass ampoules; the acyl chloride groups were determined in the residues. The thermal decomposition of copolymers of tetrachloroethyl esters with styrene sets in at ca. 230° by the release of chloral from the copolymer and splitting of some of the CCl bonds in the copolymer. The decomposition of copolymers of styrene with halogenated N-phenyl maleimides starts above 300° by depolymerization of the polystyrene chain sections and by splitting of some of the carbon-halogen bonds. At 310 and 500° for copolymers of tetrachloroethyl esters and at 500° for halogenated N-phenyl maleimides, there is radical dehydrohalogenation of the copolymers, with depolymerization of polystyrene blocks and splitting of carbon-carbon bonds in the main chain.

DNA synthesis in cytokinin-autotrophic tobacco cells : Effect of bromodeoxyuridine, fluorodeoxyuridine, and kinetin.

DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in “normal” hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.

Binding of polymer molecules with tertiary amine groups by halogen-containing compounds

Abstract Soluble polymers and copolymers with bound tertiary amine groups react at room temperature with halogens and halogen compounds to give crosslinked systems. Crosslinking occurs in solution, emulsion and in polymer mixtures, in ultra-violet and visible light and in the dark.

The kinetics of the requirement for X gene product in bacteriophage P22

SummaryThe kinetic study of the requirement for X gene product showed that the average burst size of the P22 phage depended on the length of the permissive interval in which the X function was expressed. Results of the temperature shift experiments with the clear plaque recombinants tsX c25and ts 25.1 c25gave a complicated pattern of the phage yield response.It is concluded that X gene product, besides the control function in the initiation of the phage development, is involved directly or indirectly in the control of late functions and is required throughout the entire period of the phage development.