Phosphorylation of the lysine-rich histones throughout the cell cycle.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.     

Immunological relationships among vertebrate lysine-rich histones

1. The relationship between sequence homology and immunological cross-reaction was investigated by enzyme-linked immunosorbent assay and immunoblotting using polyclonal antisera against lysine-rich histones (LRH) of known sequence, chicken H1 and H5, trout Hl and Xenopus H1s. 2. The order of immunological relatedness was consistent with known homologies among these LRH and goose H5, but quantitative correlations reflected varied locations of antigenic determinants. 3. When immunoblotting was extended to LRH from eight more vertebrates, it was evident that avian H5, mammalian H1o and anuran H1s form a sub-class, to which turtle H1s may belong, that urodele erythrocytes contain no H5-like histone and that fish "H5" is more like H1 than the H5/H1s/H1o subclass.     

Interaction between lysine-rich histones and DNA

Using a membrane filter retention technique we have studied the interaction between DNA and lysine rich histone H5 in vitro. It is found that, depending on the ionic conditions, H5 can bind DNA in a random or cooperative manner and exhibits a preference to DNA with high molecular weight and/or high A+T content, as also observed with H1. The presence of 6 M urea in the assay mixture does not impair the selectivity of H5 to A+T rich DNA but partly affects its selectivity to DNA size. In contrast to H1, H5 does not discrimate between the superhelical and relaxed forms of circular SV40 DNA.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Predictability of sequence homologies among lysine-rich histones by immunological distance

The relationship between immunological distance (I.D.) measured by microcomplement fixation and amino acid sequence difference for lysine-rich histones was tested using antisera to lysine-rich histones of known sequence, chicken H1 and H5, goose H5, and trout H1 as well as to trout H5. The best relationship between I.D. (y) and percent sequence difference (x) for lysine-rich histones, y = 2x, applies as well to other histones of known sequence but it differs from y = 5x, reported for other proteins and often used for histones. Although deviations indicate that I.D. is a poor predictor of primary sequence differences among histones, it suggests that trout H5 is more closely related to H1 than to chicken H5.