The Complete Amino‐Acid Sequence of Histone H2B₍₃₎ from Sperm of the Sea Urchin Parechinus angulosus

Abstract

The primary structure of a third H2B histone isolated from sperm of the sea urchin Parechinus angulosus has been determined. H2B(3, consists of a polypeptide chain of the following 148 amino acid residue5 : Pro-Arg-Ser-Pro- A third protein has now been identified in the complement of H2B histones of animals of the same sea urchin species. The amino acid sequence of this histone H2B(3) Parerhinus is reported. MATERIALS AND METHODS Purification of HistonesHistone H2B(3) was purified by selective extraction [3], gel exclusion chromatography on Biogel P60, and ion-exchange chromatography on carboxymethyl-cellulose (CM-cellulose) as previously described for the other histone H2B variants. The yields from 100 g wet packed sperm were: 0.7 g (histone H1) in the perchloric acid extract, 1.7 g (histones H4, H3, H2A) in the 80% ethanol/0.25 M HC1 extract and 1.0 g (histone H2B, H1 and non-histone proteins) in the 0.25 M HCl extract; about 0.5 g of H2B mixture (Fig. 1) was obtained from the 0.25 M HCl extract after Biogel P60 fractionation. The yield of histone H2B was 14.7% of the total histones extracted. The yield of histone H2B variants from a typical CMcellulose run is shown in Fig. 2 B.

The Primary Structure of Yeast Histone H3

“…Uncleaved histone H3 and the fragments generated by various cleavage methods were subjected to automated sequential Edman degradation [12] using a Beckman sequenator with programmes described previously [13]. The phenylthiohydantoin derivatives of the amino acids were identified by high-pressure liquid chromatography using a methanol gradient and an isocratic run as described previously [14].…”

“…Polyacrylamide gel electrophoresis [I 51, amino acid analysis and dansylation [16] were performed as described previously [13].…”

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

“…All phenylthiohydantoins were identified by highpressure liquid chromatography [7]. Further details on the modification of peptides and the identification of residues are given in the legends to Table 3 (in Miniprint).…”

“…In addition (Table 3-1 in Miniprint) 6.5 mg of uncleaved protein was treated for 2 min with freshly prepared water-free methanolic-HC1 (5 % w/v) to esterify free carboxyl groups, the reagent removed in a stream of nitrogen and the esterified protein incubated in 0.5 ml heptafluorobutyric acid for 40 h at 55 *C. Excess reagent was removed from the protein by vacuum applied to the spinning cup of the sequencer. The protein program [7] was then initiated with a manual 15-min phenylisothiocyanate coupling during the first cycle.…”

“…Peptide TH-11-5 (Table 3-2 in Miniprint) from the N-terminal region was incubated in heptafluorobutyric acid without prior esterification for 33 h at 55 "C. The acid was then evaporated in the spinning cup of the sequencer. The peptide program [7] was subsequently initiated with a manual 5-min phenylisothiocyanate coupling during the first cycle, and the peptide was sequenced for 11 steps. In addition (Table 3-1 in Miniprint) 6.5 mg of uncleaved protein was treated for 2 min with freshly prepared water-free methanolic-HC1 (5 % w/v) to esterify free carboxyl groups, the reagent removed in a stream of nitrogen and the esterified protein incubated in 0.5 ml heptafluorobutyric acid for 40 h at 55 *C. Excess reagent was removed from the protein by vacuum applied to the spinning cup of the sequencer.…”