DNA methylation patterns of Melandrium album chromosomes.

Melandrium album (syn. Silene latifolia) belongs to dioecious plant species possessing heteromorphic sex chromosomes, X and Y. Our previous experiments using in situ nick translation and replication kinetics analysis indicated structural and functional differences between the two X chromosomes in homogametic female cells. Here we show DNA methylation patterns of M. album root tip chromosomes using the indirect immunofluorescence approach with a monoclonal antibody raised against 5-methylcytosine (5-mC). In male cells, a more intensive 5-mC labelling on the shorter arm of the only X chromosome was observed in comparison with the longer X arm. A global hypermethylation of the male Y chromosome was not found, which indicates its prevalent euchromatic character. In female cells, the specific 5-mC pattern of the X chromosome was found on a single X chromosome, whereas the other X displayed an overall higher level of 5-mC labelling. Application of a hypomethylating drug, 5-azacytidine (5-azaC), during seed germination led to a deletion of any specific differences in the 5-mC distribution between the two X chromosomes. Confocal laser scanning microscopy analysis of DNA methylation in interphase nuclei showed hypermethylated domains that were efficiently decondensed and hypomethylated by 5-azaC treatment. The presented data show reproducible differences in the DNA methylation patterns between the two X chromosomes in M. album female somatic cells, which indicate their distinct transcriptional activities as a possible consequence of the negative dosage compensation of X-linked genes.

Ribosomal RNA genes of Phaseolus coccineus. IV. Structure and comparative-analysis of the intergenic spacer: possible involvement of some nucleotides in the transcription control of coding sequences. Sequence of the intergenic spacer of ribosomal genes.

The sequence of the intergenic spacer (IGS) of Phaseolus coccineus is determined. The IGS contains three distinct regions: Region A, constant in length; Region B, heterogeneous in length among genes, including two very similar segments 162 and 177 bp long, repeated two and nine times respectively in the investigated clone; Region C, constant in length, comprising five islands. The putative promoters and the sites of termination, processing and methylation are detected by a comparison with other plant systems.

Distribution of 5-methylcytosine-rich regions in the metaphase chromosomes of Vicia faba.

The DNA methylation pattern of Vicia faba metaphase chromosomes was examined with a specific monoclonal antibody. 5-methylcytosine (5-mC) residues are present in different chromosomal sites, and are particularly abundant in telomeric and/or subtelomeric regions and in certain intercalary bands. Chromosomal localization of methylated regions enables a better knowledge of the lengthwise differentiation of this chromosome complement. Our results also indicate that there may be differences in monoclonal antibody binding between corresponding regions of homologous chromosomes in V. faba. This behaviour is detectable in specific regions with different frequencies. The data support results previously obtained for Allium cepa metaphase chromosomes using the same monoclonal antibody.

Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine.

1. The specificity of a monoclonal IgG1 raised against a 5-methylcytidine-keyhole limpet hemocyanin conjugate was investigated by inhibition experiments with soluble competing antigens. 2. A competitive enzyme immunoassay has been set up, with the antigen immobilized on polystyrene microtitration wells. 3. The analysis of the cross-reaction profile allowed the topography of the antigen-antibody interaction to be described. 4. The binding properties of the monoclonal antibody are discussed in terms of both analytical applications and working limitations in the immunochemical study of gene methylation.