The Complete Amino‐Acid Sequence of Histone H2B₍₁₎ from Sperm of the Sea Urchin Parechinus angulosus

Abstract

Two histones have been isolated and purified from sperm cells of the sea urchin Parechinus angularus. They have been designated sperm histone H2B(1)1+,~~.~hi~~~ and sperm histone H2B~)~aveehinus on criteria of solubility and partial sequence homology with H2BCalf.H2B (1) consists of a polypeptide chain of the following 144 amino acid residues: Pro-Ser-GlnLys-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Gln-Lys-Gly Gly-Lys-Gly-Gly-Lys-Gly-Ala-Lys-Arg-Gly-Gly-Lys-Ala-Gly-Lys-Arg-Arg-Arg-Gly-Val-GlIn a previously published partial sequence serine was incorrectly assigned to position 30. A comparison of the carboxyl-terminal two thirds of H2B(1)parechinus and H2B,,lf reveals that 79 of the corresponding positions are occupied by identical amino acid residues. Much of the variability occurs in a short polar region of 20 amino acid residues. The remainder of C-terminal regions is hydrophobic and almost completely invariable.A comparison of the amino-terminal one third of H~B ( I ) P~~~,~~~~~~~ and H2B,,lc reveals that they are both highly basic with a considerable homology of the position of basic amino acids. Further, H2B(1)pavec,,inus possesses an amino terminal extension of 19 amino acids. These 19 amino acids can be recognized as a repeating pentapeptide.Five major species of histones have been recognized in calf thymus chromatin and their primary structures have been determined. These five histones have been described in other species as well and in a number of cases, the primary structures have been partially or completely determined. From these investigations it appears that the sequences of histones H3 and H4 are extremely conservative over a wide spectrum of the biological world [l], while that of HI is quite variable even between the HI histones isolated from different tissues of the same organism Ahhreviation. Dansyl, 5-dimethylaminonaphthalonesulfonyl. [2,3]. Histones H2A and H2B appear to be intermediate in variability [4-81. We have previously extracted two proteins from sperm of the sea urchin ParechinuJ angulosus that we classified by solubility criteria as H2B [9]. Partial sequence of these two proteins indicated a close homogology to calf thymus H2B at the carboxyl end. The N-terminal sequences, however, of the two proteins not only differed considerably from each other but showed little homology to calf thymus histone H2B. Nevertheless, these two sea urchin proteins were sub-classified as H2B(1) and H2B(2, on the strength of their solubility criteria and C-terminal sequence homologies. In this paper the complete amino acid sequence of sea urchin sperm histone H2B(1) is described, while the following paper presents the complete amino acid sequence of sea urchin hist one H 2B ( 2 ) .

The Primary Structure of Histone H2A from the Sperm Cell of the Sea Urchin Parechinus angulosus

“…Histone H2A was purified by selective extraction of chromatin [8] and subsequent gel exclusion chromatography on Biogel P-60 [5] of the fraction soluble in ethanolic 0.25 M HC1 (80% vjv ethanol in water) (Fig. 1).…”

“…The fractions containing the purified peptides were desalted on suitable Sephadex or Biogel columns using 10 mM HCl as the eluant and subsequently freeze-dried. Details of the purification procedures and the nomenclature of the peptides have been described previously [5]. The designations are : TH = thermolysin digestion; MT = tryptic digestion of maleylated protein or peptide; T = tryptic digestion; NP = neutral proteinase digestion; SA = Staphylococcus uureus protease digestion and NB = cleavage by N-bromosuccinimide.…”

“…by s u b t r a c t i o n * 1 mq h l s t o n e H4 was added w l t h the p e p t l d e and the p e p t l d e program Was used used to determine the sequence are given in Tables 3-1 to [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. This histone, as other H2A histones [3,21] has no free amino terminus.…”

The Histone H2B from the Sperm Cell of the Starfish Marthasterias glacialis

“…To 50 mg protein (10 mg/ml in 70"/,, wiv, formic acid) a 100-fold excess (based on methionine) of CNBr (in 70% formic acid) was added and the mixture incubated with stirring, in the dark, at room temperature [9] for 24 h. Excess CNBr was evaporated under a stream of N2, the sample diluted 1 : 10 with distilled water, and freeze-dried. Separation of peptides was accomplished on CM-cellulose (details in Table 1).…”

The reconstitution of histone H3H4 tetramer from acid extracted histones in the absence of urea

“…Methods for the isolation of histones can be divided into two groups; the first results in the isolation of the individual polypeptide chains by employing solvent solubility properties of the histones [ 11, charge differences of the individual histones in the presence of urea or guanidinium chloride [2] and size-charge and aggregation differences in dilute hydrochloric acid 131. All these methods have in common that the separation conditions are known to seriously interfere with the higher order structures of proteins.…”