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.     

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.     

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.