James Allan

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome☆

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.

Transcription of globin genes in reticulocyte chromatin

The use of exogenous RNA polymerase in studies of the transcriptional specificity of chromatin is based on two unproven assumptions: (1) That the active genes in chromatin are in a unique conformation preferentially transcribed by the polymerase; (2) That this conformation survives the process of isolation of the chromatin. The further extension to the study of the fidelity of chromatin reconstitution rests on the additional precept that the specificity of transcription of reconstituted chromatin can be measured in relation to that of ‘native’ chromatin. The data presented here undermine at least one of these assumptions. We show that there is indeed little evidence for globin gene specificity in transcription of reconstituted reticulocyte chromatin.