Tetrahymena Histone H2A. Isolation and Two Variant Sequences1

Abstract

The H2A histone of the protozoan T. pyriformis was isolated by Bio-Gel P-10 chromatography from the H2A + H3 fraction obtained on a large scale, as described previously (Nomoto, M. and Iwai. K.) and further purified by Sephadex G-100 chromatography. The purified H2A was shown to comprise approximately equimolar amounts of 2 variants. H2A(1) and H2A(2), differing in MW. The H2A mixture was fragmented with cyanogen bromide, yielding 1 N-terminal fragment (101 residues), 1 middle fragment (17 residues) and 2 C-terminal fragments (19 and 14 residues). The N-terminal fragment, whose N-terminal was blocked, was sequenced by overlapping its tryptic peptides with the peptides derived from the fragment with 3 proteases and the tryptic peptides from citraconylated intact H2A. One of the citraconylated tryptic peptides showed the arrangement of the N-terminal, middle and C-terminal fragments; the latter 2 fragments were directly sequenced by Edman degradation. Thus, the total sequences of H2A(1) and H2A(2) were competely determined; the 2 variants differ in the total residue number, 137 or 132 the MW in the unmodified form, 14,654 or 14,126, His or Asn at residue 40, Ser or Thr at residue 124, and the C-terminal sequence at residues 128-137 or 128-132, respectively. These sequences are compared with the known H2A sequences, and the implications for the structure and function relationship of this histone species are discussed.