Different DNA methylation pattern in A and B chromosomes of Crepis capillaris detected by in situ nick-translation. Comparison with molecular methods

Experiments were performed on Crepis capillaris callus lines with 0, 1 and 2 B chromosomes and on hairy root lines without or with 1 and 2 B chromosomes. Comparison of HPLC results for DNA from calli differing in number of B chromosomes did not reveal any significant differences in methylation level (30.4 +/- 1.1%, 30.9 +/- 1.2%, 31.7 +/- 1.7% in lines without or with one or two B chromosomes respectively) which could be attributed to the number of B chromosomes. Restriction patterns obtained after DNA digestion with HhaI, HpaII, MspI or HaeIII (i.e. restriction enzymes sensitive to cytosine methylation) were similar in calli and apical root segments and also did not depend on the presence or number of B chromosomes. Methylation of B chromosomes higher than that of A chromosomes was demonstrated by fluorescent in situ nick translation driven by HpaII, MspI or HaeIII in metaphase chromosomes. After short digestion (I and 3 h), B chromosomes, in contrast to A chromosomes, were weakly labelled or not labelled at all, which indicates longer distances between target sequences containing unmethylated cytosine in the former.     

The Correlation between the Chromosome Variation in Callus and Genotype of Explants of Arabidopsis thaliana

Twelve callus lines of Arabidopsis thaliana were derived from four types of explants excised from diploid plants of two ecotypes (Columbia and Wilna) and autotetraploid plants of the Wilna ecotype. Cytogenetic analysis of the chromosome variation in particular callus lines was carried out for primary culture and callus during 5 months of culture. Ploidy levels of interphase nuclei were estimated by counting the number and size of chromocentres and nuclei of interphase cells. The first polyploid cells in all callus lines were observed during callogenesis. In primary culture the ploidy level ranged between 2 and 15x (10-75 chromosomes). The frequency of polyploid cells was higher in the 5-month old callus culture, but the ploidy level was the same. In the callus lines derived from autotetraploid plants, cells with reduced chromosome number appeared quite frequently along with diploid and polyploid cells.     

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.     

Hydrogen bonding in nucleosides and nucleotides

An analysis of the hydrogen bonding in 76 nucleoside and 11 nucleotide crystal structures shows that the hydrogen bond lengths fall into well-defined categories according to the nature of the donor or acceptor groups. The shortest bonds are those involving P---OH or O=P groups. For donor groups, the sequence in bond lengths is

P—OH<C—OH< N−H<O_w(H)—H<N(H)—H<C−H

There are ten examples of two centre

H—H…O

bonds, which are comparable in length with P---OH …O bonds. The acceptor seqeunce is

O=P<OH₂<OH₂<O=CO(H)C<N N(H₂)C<Cl^…<O<S=C

The number of three-centre bonds, about 24%, is comparable to that observed in the carbohydrates and the amino acids. Most hydrogen bonds are involved in short finite chains. Only in the nucleotides are cyclic hydrogen bonding schemes observed.     

The effect of B chromosomes and T-DNA on chromosomal variation in callus cells and regenerated roots of Crepis capillaris

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

A survey of hydrogen bond geometries in the crystal structures of amino acids

An analysis of the geometries of the hydrogen bonds observed by neutron diffraction in thirt-two crystal structures of amino acids shows the following results. Of the 168 hydrogen bonds in the data set, 64 involve the zwitterion groups 

and $\text{C}$O₂. Another 18 are from

to sulphate or carbonyl oxygens. The majority, 46, of these

H … O bonds are three-centered (bifurcated). Nine are four-centered (trifurcated). The geometry in which the three-centered hydrogen bond involves both oxygens of the same carboxylate group is not especially favoured. When it does occur, one hydrogen bond is generally shorter and the other longer, than when the bonding involves oxygens on different carboxylate groups. The shortest hydrogen bonds are the OH … O $\text{C}$, from a carboxylic acid hydroxyl to a carboxylate oxygen, and NH … O$\text{C}$ when the nitrogen is the ring atom in histidine or proline. Carboxylate groups, on average, accept six hydrogen bonds, with no examples of less than four bonds. The reason for the large number of three-centered

H … O$\text{C}$ bonds is therefore a proton deficiency arising from the disparity between the tripled donor property of the

groups and the sextuple, on average, acceptor property of the carboxylate groups. There is good geometrical evidence for the existence of H … O and H … Cl^? hydrogen bonds, especially involving the hydrogen atoms on α-atoms.     

The crystal and molecular structure of 1,6-anhydro-β-d-mannopyranose

The crystal structure of 1,6-anhydro-β-d-mannopyranose, C₆H₁₀O₅, is orthorhombic, P2₁2₁2₁, with a = 10.971(2), b = 13.935(3), c = 9.012(1) Å, V = 1377.76 »³ (MoKα, λ = 0.7107 Å), Z = 8, D_x = 1.563 M.gm⁻³, D_m = 1.565 M.gm⁻³. the structure was solved by MULTAN and refined to R(F) = 0.043 for 2355 reflections. The two symmetry-independent molecules in the unit cell have similar conformations, except for the orientation of one of the three hydroxyl groups. The conformation of the pyranose rings is ¹C₄ distorted towards E_o, and that of the anhydro rings is E. There are significant differences between the two molecules in two of the four C---O bond-lengths. These occur where there are important differences in the hydrogen-bonding environment of the oxygen atoms. The molecules are hydrogen-bonded by three linear and three bifurcated O---H···O interactions which form four-membered loops linked into infinite chains. Empirical force-field calculations with MMI-CARB reproduced the geometry of the molecules within the variations observed experimentally between the two molecules, except for a C---O bond in one of the molecules. The effect of excluding the anomeric effect from the theoretical calculations was not significant. Calculations for an intramolecularly hydrogen-bonded molecule were also carried out as a model for the molecules in a non-polar solvent.     

Transformed roots of Crepis capillaris--a [corrected] sensitive system for the evaluation of the clastogenicity of abiotic agents

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots -- 'hairy roots' -- has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.     

Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.