Isolation and characterisation of histone transacetylases
Doctoral Thesis
1977
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University of Cape Town
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Abstract
Acetylation, one of the post-synthetic modifications of histones, weakens histone-DNA interactions and may play a regulatory role in gene control of eukaryotes. The literature available on histone acetylation as well as other post-synthetic modifications of histone has been reviewed. Histone acetylation is catalysed by an enzyme(s) which transfers acetyl groups from a donor molecule to histones. A crude histone transacetylase preparation was isolated from nuclei and the optimal conditions for the acetylation of histones were determined. This enzyme(s) was shown to be specific for histones with protamine displaced histone complex being the best substrate. Using this histone transacetylase preparation ³H-acetyl total histone was prepared in sufficient yield and with a high enough specific activity to enable sequential Edman degradation of the histone sub-fractions isolated from the total histone complex to be undertaken. Histones H3 and H4 were isolated from the acetylated total histone as they exhibited the highest degree of acetylation. Histone H4 peptides were generated by chymotryptic and tryptic digestion as the intact histone H4 polypeptide chain is blocked at its N-terminus. The Edman degradations of histone H3 and H4 showed that the acetylation sites that have been determined under in vitro conditions are the same as those undergoing acetylation in vivo. All of the acetylation was found in the N-terminal region of histones H3 and H4 with histone H4 showing a gradient of decreasing acetylation from the N- to the C-terminus, in contrast to histone H3 where the first two possible acetylation sites are acetylated to a minor degree only.
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Bibliography: pages 108-124.
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Thwaits, B. 1977. Isolation and characterisation of histone transacetylases. University of Cape Town.