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The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E.F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [14C]lysine and thymidine, and then for one generation in the presence of [3H]lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient centrifugation after ultraviolet irradiation. The results indicate that some of the chromatin proteins can segregate with the DNA strand synthesized in the same generation when the cells subsequently replicate. Using chromatin with a protein to DNA ratio of 2.6, in different experiments, 5-22% of the chromatin proteins were estimated to segregate with the appropriate DNA strand, while the remaining proteins were randomly distributed to daughter chromatin. The segregating proteins have not been specifically identified but they migrate in sodium dodecyl sulfate gel electrophoresis in the region where the four smaller histones migrate.

Previous studies have suggested that the phosphorylation and dephosphorylation of histone H5 play an important role in controlling the condensation of avian erythrocyte chromatin. The present work locates in the polypeptide chain the major sites at which H5 is phosphorylated in vivo. The majority of the radioactivity in 32P-labeled H5 is clustered in two regions of the molecule. Nearly 50% of the 32P is found in the amino-terminal N-bromosuccinimide (NBS) peptide (residues 1-28); the remainder is confined to three phosphopeptides arising from the C-terminal half of the molecule (residues 100-200). All phosphopeptides are found in a tryptic digest of monophosphorylated H5, indicating the phosphorylation of a given site is a random event. Automatic Edman degradation of the amino-terminal fragment shows that the radioactivity is equally divided between serines at positions 3 and 7. The C-terminal phosphorylated tryptic peptides share some features with the C-terminal phosphorylation sites in H1. If, as has been postulated, the sites of phosphorylation are in or near DNA combining regions, then H5 may have two DNA combining sites. The location of the phosphorylation sites is discussed in relation to a possible mechanism for controlling chromatin condensation.

A method has been devised for the separation of chromatin containing 5-bromodeoxyuridine (BrUdRib) in one strand (HL) of the DNA from that with BrUdRib in both strands (HH). Ultraviolet light breaks chromatin containing HH DNA into smaller fragments than chromatin containing HL DNA and the two species can be partially resolved on neutral sucrose gradients. Unfiltered ultraviolet light is not suitable since it causes considerable alteration in the electrophoretic pattern of chromatin-associated proteins. Irradiation with 313-nm light causes much less damage to the associated proteins. The ability to separate, isolate, and examine chromatin containing HL and HH DNA makes studies on the distribution of chromatin-associated proteins possible.