Tetrahymena Histone H1. Isolation and Amino Acid Sequence Lacking the Central Hydrophobic Domain Conserved in Other H1 Histones1

Abstract

The complete amino acid sequence of a single H1 histone of the protozoan Tetrahymena pyriformis was determined, following previous determinations of the sequences of histones H2B, H2A, H3, and H4. Only a single H1 species was obtained by fractionation of a 0.5 M HClO4-soluble fraction from the whole histone extract and further purification. This starting material for sequencing contained 1.1 mol/mol phosphate and showed a single electrophoretic band after dephosphorylation. The sequence determination was performed by Edman degradation of BrCN fragments, staphylococcal protease peptides, and tryptic peptides, as well as secondary peptides from one BrCN fragment and one staphylococcal protease peptide. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that five sites of the sequence were phosphorylated to various extents (5-30%). Thus, the total sequence, consisting of 165 amino acid residues and having a molecular weight of 17,942 in the unmodified form, was completely determined. This unusually small H1 sequence differs substantially from the human spleen H1 sequence of 218 residues, having larger proportions of hydrophilic residues and smaller proportions of hydrophobic residues. Comparison of the distribution pattern of hydrophilic and hydrophobic residues, between the protozoan and human sequences, showed that the protozoan sequence lacks the central hydrophobic domain that is conserved in the known vertebrate and other H1 histones. The implications for the function of H1 are discussed from the evolutionary viewpoint.

Identification and Mutation of Phosphorylation Sites in a Linker Histone

“…Taken together, these data suggest that the five sites identified by microsequencing represent the complete set of macronuclear H1 phosphorylation sites utilized by T. (35) is shown aligned with the protein sequence determined for T. pyriformis macronuclear H1 (lower) (34). Gaps introduced to maximize homology are indicated (Ϫ), and identical residues are noted (*).…”

“…Phosphorylation at Thr-53 was detected during sequencing of the peptides spanning residues 51-56 (phosphopeptide 9) and residues 51-60 (phosphopeptide 5). These data are summarized and compared with findings for T. pyriformis (34) in Fig. 3.…”

Nucleosome Positioning Is Independent of Histone H1in Vivo

“…Prior to this study, that difference might have been ascribed to the very different structures of the micronuclear and macronuclear linker histones (13,14,30). Although histone H1 apparently does not determine nucleosome positioning in the macronucleus, we cannot eliminate the possibility that the unusual structure of micronuclear linker histones may contribute to the more compact nucleosome spacing in the micronucleus.…”

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.

“…The derived sequence of the MLH gene clearly indicates that ,B and y have compositional similarities to Hi proteins, while 8 more closely resembles HMGs. We expected that the conserved globular core found in the Hls of mitotically dividing nuclei of multicellular organisms, but absent in the Hi of amitotic macronuclei (32,62), might be found in one or more of the micronuclear linker proteins. However, no compelling similarity to the core was found.…”

“…Macronuclei contain an Hi-type histone whose solubility properties, extreme lysine richness, and cell growth/division-associated phosphorylation are typical of that class of histone. However, the derived (62) and actual (32) protein sequences of Tetrahymena macronuclear Hi indicate that it is unusual, inasmuch as it lacks some sequence elements and structural features associated with the conserved central globular core of Hi from higher organisms. Because Hi has been postulated to play an important role in higher-order chromatin structure (see reference 59 for a review), and macronuclei do not undergo mitotic chromosome condensation, it was suggested that the central globular core of Hi might be involved in-this process (62).…”