BIOCHEMICAL CHARACTERIZATION OF HIGHLY ACETYLATED REGIONS IN BARLEY NUCLEI

¹ Science University of Tokyo, Shinjuku-ku 162-8601, Japan

² Hokuriku National Agricultural Experiment Station, Joetsu 943-0193, Japan

³ Joetsu University of education, Joetsu 943-8512, Japan

⁴ Faculty of Horticulture, Chiba University, Mastudo 271-8510, Japan

⁵ Faculty of Engineering, Graduate School of Osaka University, Suita 565-0871, Japan

⁶ Anatomy Department, University of Birmingham Medical School, Birmingham B15 2TT, UK

Histone H4 in a nucleosome core is one of the targets of chemical modification, which is post-translational acetylation at the four defined lysine residues in the N-terminal domain of histone H4. We already observed that there were several dot-like hyperacetylated regions (HARs) in the non-fixed interphase nuclei of barley. In addition, they were located in the same area at the centromeric region. To know the biological significance of HARs, analyses of the western blotting and the immunoprecipitation with southern blotting to the nucleosome fractions using antibodies (H4Lys 5, 8, 12,16) against the acetylated histone H4 were performed. It was found that the nuclear fraction and crude histone fraction showed the different pattern of blotting bands according to the antibodies H4Lys 5, 8 and 16. HARs in the barley root nuclei could not be observed clearly after fixations by p-formaldehyde (PFA) or glutaraldehyde. The fixation with 70% ethanol for the tissue section sample also did small influence to the nucleus which showed only fluorescence graduation as in the case of 1% PFA fixation. Including these chemical sensitivities, HARs found only in non-fixed nuclei were fully investigated to understand the biochemical and physiologial meanings of H4 acetylation within centromere regions.