Wilhelm bernhard workshop on the cell nucleus

NUCLEAR PROTEINS, MATRIX AND BODIES

DEVELOPMENTAL CHANGES IN THE DISTRIBUTION OF TIGHT DNA-PROTEIN COMPLEXES ALONG BARLEY CHROMOSOMES REVEALED BY MAPPED MOLECULAR MARKERS

¹Sjakste T., ²Röder M.

¹Institute of Biology, Salaspils, Latvia; ²Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany
Rearrangements in DNA packaging as in DNA interactions with functional protein groups reflect the switches in gene expression during plant development. PCR-amplification of mapped microsatellite markers and RFLP-STS markers was successfully used to label DNA fragments in chromatin profiling experiments. Three methods of fractionation of DNA complexes with functional protein groups were used: isolation of the nuclear matrix, isolation of DNA complexes with tightly bound proteins (TBPs), and nucleoprotein-celite (NPC) chromatography. The presence of specific DNA fragments in various fractions obtained by the different isolation procedures was monitored by PCR-amplification of the 22 and 24 markers previously mapped along barley chromosomes 1H and 7H. In young leaves amplification of all markers was found in the nuclear matrix. Five markers on 1H and six markers on 7H were specific for nuclear matrix fraction in young leaves and for insoluble chromatin fraction in senescent leaves. Probably, these markers are located closely to origins of replication in young leaves. In old leaves these DNA attachment points are degraded. Specific patterns of TBPs were revealed in all types of tissues studied. The first leaf development was followed by a release of most parts of the chromosomes from the complexes with TBPs. On the contrary, senescence of the leaf was followed by an increase in DNA sites involved in interactions with TBPs. The distribution of TBPs appeared to be organ specific. In seeds the PCR-amplification of most markers was evenly distributed between all fractions of NPC-chromatography, some of them between the chromatin and tight DNA-NM complexes. During germination and leaf development the pattern changed and some markers became highly specific for the DNA-NM fraction. All methods revealed the tissue specific developmental reorganization of tight DNA protein complexes along the chromosomes. Apparently, different methods detected interactions of different protein groups with DNA.