Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication.

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.

A cytospin technique for spreading plant metaphases suitable for immunofluorescence studies.

Recent immunofluorescence techniques enable the localization of various cellular antigens, thus providing a powerful tool for cell and molecular biology research. Serious problems occur, however, when these techniques are applied to plant material. The presence of the cellulose wall can be a barrier to reproducible penetration of antibodies into cells and it often displays a confusing autofluorescence. A novel technique to prepare mitotic chromosome spreads from root tip meristems of germinating seeds is presented. Synchronous mitotic cells arrested in metaphase are converted into protoplasts using pectin and cellulose hydrolytic enzymes, and the purified protoplasts are fixed either in a methanol-acetic acid mixture to study DNA epitopes or in a nonextracting fixative to study chromosomal proteins. The latter fixative contains Triton X-100 to lyse the protoplasts and neutral formaldehyde to fix proteins by cross-linking. The protoplasts are immediately centrifuged onto microscopic slides as commonly done for mammalian cytogenetics. Using commercially available antibodies and both epifluorescence and confocal laser scanning microscopy, we demonstrated that the acid fixed chromosome slides are suitable for detection of DNA (anti-DNA antibody) or incorporated 5-bromodeoxyuridine (anti-BrdU antibody), while the cytospun formaldehyde and Triton X-100 fixed samples are convenient for detecting histones (antihistone antibody, pan). This technique should provide a general tool to study structural and functional domains of plant chromosomes.