Complete Amino-Acid Sequence of Calf-Thymus Histone III

Abstract

Calf-thymus histone III is a single polypeptide chain of 135 residues (combined molecular weight of 15,324) with alanine at both the amino and carboxyl ends. The N12-terminal region (Residues 1-53) of histone III is strongly basic (net charge of +18) and contains lysines-14 and -23, which are e-N-acetylated in a fraction of the molecules, as well as lysines-9 and -27, which are partially e-N-methylated. The COOH-terminal region (Residues 54-135) is only slightly basic (net charge of +4), contains most of the hydrophobic residues, and has a 29-residue sequence that lacks a basic residue. The two cysteines are in the nonbasic region at positions 96 and 110. A few sequence similarities of calf-thymus histone III with other histones have been noted.Histones are basic proteins complexed with DNA in the nuclei of all eukaryotic organisms. Although the functions of histones have not been precisely delineated, it seems likely that they play an important role in the regulation of transcription and replication, in addition to serving as structural components of chromosomes (1).With the availability of improved methods for the separation of histones from each other and from other nuclear components, structural studies were begun on several histones from various organisms. The sequence of calf-thymus histone IV (f2al, GAR histone), one of the two arginine-rich histones, was completed first (2, 3), and this was soon followed by the sequence of pea-seedling histone IV (4). Histone IV shows a remarkable preservation of sequence throughout the evolution of eukaryotes and, in addition, has a nonrandom distribution of residues and specific sites of e-N-acetylation and e-Nmethylation (4). Some of these findings were independently substantiated in another laboratory (5), and the sequence studies have now been extended to include Novikoff hepatoma (6), rat (7), and porcine (8) histones IV.Although partial sequences of other histones have been reported (1, 9-13), the only other complete sequence reported to date is that of calf-thymus histone HUb2 (f2b) (14,15). We now present the complete amino-acid sequence of calf-thymus histone III (f3), the other arginine-rich histone, and the only histone that contains cysteinyl residues. A preliminary account of some of these studjes has been given (16). METHODSCalf-thymus histone III was prepared by method 2 of Johns (17) and further purified by column chromatography on BioRex 70 (18). The S-carboxymethyl derivative was prepared by reduction and alkylation with iodoacetate (19), and the oxidized derivative was prepared by oxidation with performic acid (20). Tryptic, chymotryptic, and cyanogen bromide peptides were obtained from the derivatized proteins by procedures similar to those described for histone IV (3, 21). Larger peptides were further hydrolyzed with trypsin, chymotrypsin, pepsin, or thermolysin to give smaller fragments. The procedures for sequential Edman degradation, hydrolysis with carboxypeptidases or aminopeptidases, and all other methods have been described (3,19,21)....

Crevices containing cysteine in the tertiary structure of calf thymus histone F3

“…Since the primary structure of F3 is known [3], suggestions may be made about the localization of a deep crevice. The cysteinyl residues are separated by 13 residues in a nonbasic region of 29 residues, but preferentially Cys-96 is present in a region of a high potential for helix formation (residues 94-106).…”

Comparison of the N‐terminal amino acid sequences of histone F3 from a mammal, a bird, a shark, an echinoderm, a mollusc and a plant.

“…3). Lys-36 in cycad is not methylated to a detectable extent which also seems to be the case in calf [7]. In contrast to the other histones Lys-5 in cycad is also partly methylated.…”

“…The terminal lysine (Lys-18) in the second repeat unit was never found to be methylated, which may be due to the preceding Pro which causes drastic alteration in the secondary structure in this region. Additionally equivalent Lys residues in the repeat unit are acetylated in calf thymus [7] and possibly also in the other organisms.…”

“…The assumption of evolutionary constancy of F3 throughout the biological world is mainly based on electrophoretic studies [ 1,2] . Only recently the sequences of histone F3 isolated from calf [7], chicken [8,9] and carp [4] have been established. The latter two were found to be identical but differed in their sequence from the calf histone with respect to residue Ser 96 which is replaced by Cys in calf.…”

“…Their electrophoretic mobility, amino acid composition and the N-terminal 40-50 amino acid sequence were compared to those of histone F3 from calf [7] and chicken [8,9].…”