Search

Wheat nuclei have a remarkably well defined interphase organisation, and we have made use of this to determine the relationship between interphase chromosome organisation, the positioning of specific transgenes and induced changes in DNA methylation and histone acetylation, using in situ hybridisation and confocal 3D imaging. After germinating seeds either in the presence of 5-Azacytidine (5-AC), which leads to DNA hypomethylation, or trichostatin A (TSA), which results in histone hyperacetylation, the architecture of the interphase chromosome arms changes significantly even though the overall Rabl configuration is maintained. This suggests that specific chromosome segments are remodelled by these treatments but that there is a strong link of both centromeres and telomeres to the nuclear envelope. In lines carrying multiple transgene integrations at widely separated sites, we show that the multiple transgenes, which are usually colocalised during interphase, are dispersed after 5-AC or TSA treatment and that there is an increase in transgene activity. This suggests that the colocalisation/dispersion of the transgenes may be a function of specific interphase chromosome organisation and that these lines containing multiple transgene copies may all be partially transcriptionally repressed.

The standard rye cultivar `Imperial' and a structural variant carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms)were used to investigate expression patterns of homologous rDNA loci, and the influence of chromosome structural change on their interphase organisation and relative disposition. Sequential silver staining and in situ hybridization with the rDNA probe pTa71, established a correspondence between the expression and organization patterns of rDNA domains in metaphase and interphase cells. In most cells of the cultivar Imperial, nucleolar organizer region (NOR)silver staining on metaphase chromosomes with equivalent numbers of rDNA genes revealed a size heteromorphism between homologous rDNA loci, resulting from their differential expression. NOR heteromorphism in the structural variant line was significantly reduced. The preferential activity of one NOR over its homologue was found to be random within cells and independent of parental origin. Nucleotypic modifications mediated by changes in the 1R chromosome structure include increased proximity between homologous rDNA loci in interphase, and an increase in the frequency of cells with intra-nucleolar ribosomal condensed chromatin. These results seem to indicate a `sequence recognition' process for the regulation of homologous loci.

Probes for B chromosome-specific sequences in the distal region of the long arm of the rye B have been used to investigate the interphase arrangement of the Bs in rye and in hexaploid wheat. The Lindstro¨m strain of wheat carries the rye Bs as additions. The number of in situsignals in nuclei with two, three and four Bs is often less than the maximum B number, and it seems that the Bs may be grouped together in various ways rather than being randomly dispersed throughout the nucleus. The degree of physical association is greater in rye than in the alien wheat background. The results are discussed in relation to the pairing and recombination preferences of the Bs in rye and in Lindstro¨m wheat.

Genomic imprinting of rye origin rDNA sequences in triticale is modulated by DNA methylation responsible for ontogenic expression patterns of those sequences. Considering the dynamic nature of these phenomena, we evaluated the influence of plant development on the inheritance of modified rye rDNA expression patterns. DNA hypomethylation was induced in triticale by 5-azacytidine (5AC) treatments at distinct developmental stages of M1plants, and expression patterns were analysed in M2. The activity of rye origin rRNA genes in progeny of untreated and 5AC-treated plants was evaluated by silver staining in meristematic root tip cells and in meiocytes at diplotene. In the progeny of 5AC-treated plants, a significant increase in rye rDNA expression was observed, contrasting with the residual activity in untreated plants. Significant differential effects of 5AC treatments were observed in M2plants and correlated with the M1plant developmental stage in which DNA hypomethylation was induced. Hypotheses to explain the origin of those differences are discussed here.