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(1976) Biochemistry 15, Yarus, M., Knowlton, R., & So11, L. (1977) in Nucleic Acid-Protein Recognition, pp 391-408, Academic Press, New York. 2722-2728. 41 46-41 54. 4256-4265. 11, 1439-1446. U.S.A. 80, 6755-6759. 11332-1 1339. 147, 1310-1311. 41 3 1-4 138. Phosphorylation and Methylation of Physarum Histone I4 1 during Mitotic Cycle+ Andrzej Jerzmanowski* and Marek Maleszewskif Department of Biochemistry, Warsaw University, 02-089 Warsaw, Zwirki i Wigury 93, Poland Received July zyxwvu 5. 1984 ABSTRACT: We have shown that the heterogeneity of the typical histone H1 from the lower eukaryote Physarum polycephalum seen in sodium dodecyl sulfate (NaDodSO,) gel electrophoresis is due principally to conformational effects produced by postsynthetic modifications rather than primary sequence variants. We have also shown by labeling with L- [methyl-3H]methionineand amino acid analysis that Physarum H1 in addition to already known phosphorylation undergoes methylation on e N H 2 groups of several of its lysines. The analysis of Physarum H 1 phosphorylation by high-resolution acetic acid-urea gel electrophoresis revealed that it could accept up to more than 20 phosphates per molecule. The mitotic cycle analysis of postsynthetic modifications of Physarum H1 showed that (a) H1 undergoes superphosphorylation in mitosis accepting from about 14 to more than 20 phosphates per molecule and (b) it is not totally dephosphorylated in the following cycle retaining from about 8 to about 16 phosphates per molecule in G2 phase. The results of this work are consistent with the interpretation that the newly synthesized H1 is de$osited on DNA in nonmethylated and nonphosphorylated forms and its methylation (which is irreversible) precedes its phosphorylation in the course of chromatin maturation. The studies of Physarum H1 bisected with chy- motrypsin indicate that most of the phosphorylation sites including the superphosphorylation in mitosis are localized in a larger COOH-terminal part of the molecule. The analysis of highly phosphorylated Physarum H 1 by high-resolution NaDodSO, gel electrophoresis showed the existence of several discrete conformational subspecies resembling in their electrophoretic appearance the true sequence variants of mammalian H 1. Histone HI plays a key role in the structural transitions of the basic 100-A nucleosomal filament into the thick chro- This work was supported by Polish Academy of Sciences Project 09.7 and by the Alexander von Humboldt Stiftung, Federal Republic of Germany. tM.M. was a graduate student at the Department of Biochemistry, Warsaw University (1983-1984). Present address: Department of Em- bryology, Warsaw University, 00-035 Warsaw, Krakowskie PrzedmieScie 26/28, Poland. 0006-2960/85/0424-2360$01.50/0 matin fiber, the 300-A solenoid (Thoma et al., 1979). On the other hand, H1 shows a considerable degree of variability. Both the sequence variants of H1 and the postsynthetically modified H1 molecules usually occur at the same time in the same chromatin (Hohmann, 1983; Cole, 1984). This together has led to the suggestion that H1 through its different sequence variants and chemically modified subspecies (mostly by phosphorylation) may specifically influence the local confor- mation of chromatin and through it the genetic activity zyxwvutsrqpo 0 1985 American Chemical Society