No abstract

32P-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated into 15 phosphopeptides by high performance liquid chromatography. These phosphopeptides were grouped into five classes having different cell cycle phosphorylation kinetics: 1) peptides reaching a maximum phosphorylation rate in S and then declining in G 2 and M, 2) peptides reaching a maximum phosphorylation rate in G 2 and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throughout S and G 2 and reaching a maximum in M, 4) one peptide that was phosphorylated only in M, and 5) peptides that had low levels of phosphorylation that remained constant throughout the cell cycle. Amino acid analysis and sequencing demonstrated that the mitotic specific H1 phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is phosphorylated on both the Ser and Thr, does not contain the consensus sequence (S/T)PXZ (where X is any amino acid and Z is a basic amino acid). This sequence is thought to be required by the p34 cdc2 /cyclin B kinase that has maximum phosphorylating activity in mitosis. These data indicate that this kinase either does not have an obligatory requirement for the consensus sequence in vivo as generally believed or that it is not the enzyme responsible for the mitotic specific H1 phosphorylation.For over 27 years, the phosphorylation of histone H1 has been thought to play a role in controlling the cell cycle (Ord and Stocken, 1968). To examine this possibility our laboratory used synchronized CHO 1 cells to determine the cell cycle kinetics of histone phosphorylation during cell proliferation (see review by Gurley et al. (1978a)). In those studies it was found that histone H2A and H4 phosphorylations were cell cycle-independent and probably not involved in cell cycle control, while histone H1 and H3 phosphorylations were cell cycle-dependent and, therefore, more likely to have a role in cell cycle control.

Background Simultaneous measurement of cellular DNA and RNA content provides information for determination of the functional status of cells and, clinically, for the diagnosis and grading assessment of various tumors. Most current flow cytometric methods are based on resolving the fluorescence emission spectra of dyes that bind preferentially to either type of nucleic acid. However, several monochromatic nucleic acid–binding fluorochromes display resolvable differences in fluorescence lifetime when bound to DNA or RNA. The differences in the lifetime of one fluorescent probe provide an alternate means to distinguish the binding of one probe to these cellular macromolecules and to simultaneously measure their cellular contents. Methods Three nucleic acid intercalating dyes, propidium iodide, ethidium bromide, and ethidium homodimer 1, were selected to study differences in fluorescence lifetimes when bound to cellular DNA and RNA. Fixed HL‐60 cells were treated with specific nucleases to initially determine the lifetime values of each dye when bound to the cellular DNA, RNA, or both. The lifetime values were then used as the signatures to resolve the cellular DNA and RNA contents in untreated cells. Results All three dyes showed fluorescence lifetime differences when bound to RNase‐treated, DNase‐treated, or untreated cells. With these lifetime values, the fluorescence emissions from DNA, RNA, or DNA/RNA were resolved from untreated cells with the use of phase‐sensitive detection. The lifetime differences resulting from the binding to either type of nucleic acid depended on the dye, the staining concentration, and the analysis condition. Conclusions The lifetimes of the nucleic acid–binding fluorochromes were altered when binding to different macromolecules under different conditions. Phase‐sensitive flow cytometry provided a unique means for simultaneous discrimination and quantification of subcellular macromolecules with one fluorescent probe. The data demonstrated the capabilities for resolving relative cellular DNA and RNA contents based on fluorescence lifetime. Cytometry Part A 52A:46–55, 2003. Published 2003 Wiley‐Liss, Inc.

In mammalian cells, up to six serines and threonines in histone H1 are phosphorylated in vivo in a cell cycle dependent manner that has long been linked with chromatin condensation. Growth-associated H1 kinases, now known as cyclin-dependent kinases (CDKs), are thought to be the enzymes responsible for this process. This paper describes the phosphorylation of histone H1 by four different purified CDKs. The four CDKs phosphorylate only the cell cycle specific phosphorylation sites of H1, indicating that they belong to the kinase class responsible for growth-related H1 phosphorylation in vivo. All four CDKs phosphorylate all of the interphase and mitotic-specific H1 sites. In addition to the (S/T)PXK consensus phosphorylation sites, these four CDKs also phosphorylate a mitotic-specific in vivo H1 phosphorylation site that lacks this sequence. There is no site selectivity among the growth-related phosphorylation sites by any of the four CDKs because all four CDKs phosphorylate all relevant sites. The results imply that the cell cycle dependent H1 phosphorylations observed in vivo must involve differential accessibility of H1 sites at different stages of the cell cycle.