Claus von Holt

More histone structures

Since the elucidation of the amino acid sequence of the prototype histones H3, H4, H2A and H2B, a considerable amount of information on the occurrence of variants of these structures has become available. (Bibliographic references to details of the histone primary structures are given in fig.l-3; for review see [341.) It appears that the histones H3 and H4, over a wide range of evolution, vary only slightly whereas the histones H2A and H2B undergo more extensive evolutionary changes (fig.1 -3). The variations of the structures can be classified into two types. The first group comprises simple point mutations or deletions of one or several residues. The second type of structural variations consists in extensive modifications of major areas of the molecules through reiteration, insertion, deletion and point mutations. Such changes have led, in some variants, to the establishment of new domains in the structure. Whereas the former type of structure variation occurs in all four histones, the second group of more extensive variations up to now has been identified only in the histones of the H2A and H2B type. The first group of alterations does not change the general structure of the molecules drastically. Such changes occur infrequently in the histones H3 and H4. In histone H3, four positions are variable. In the histone H4 only three variable positions have been identified. In both these histones there appears to be no regional preference for the conservative point mutations. The mutations involving cysteine in position 96 of

A new procedure for the isolation and fractionation of histones

1. Introduction The usual method for the isolation of histones, in- volving the extraction of histones from chromatin with dilute sulphuric or hydrochloric acid, may re- sult in the denaturation of various chromosomal com- ponents due to the extreme pH conditions. The ability of histones to regenerate the super-coil configuration of native nucleohistone is sometimes reduced [ 1,2 ] by the acid extraction technique. Evidence has been pre- sented [3] that histones to some extend bind to DNA during acid treatment and form part of the so-called non-histones. The present investigation was under- taken to develop a procedure for the isolation and fractionation of histones under mild pH conditions. Nucleoproteins dissociated at high ionic strength can be separated into protein and DNA by ultracentri- fugation [4-91 or gel filtration [8-lo] , but we have found these procedures unsuitable for the large scale isolation of histones. The method for histone isola- tion presented here is based on the finding of Mirsky and Ris [ 1 l] that protamine added in vitro to nucleo- protein directly displaces histones. A procedure which avoids extreme pH conditions is described for the large scale isolation and partial fractionation of calf thymus histones. Histones, vir- tually completely displaced from deoxyribonucleo- protein by protamine, and isolated by exclusion chro- matography, appear to be almost identical on disc electrophoresis to histones prepared by acid extrac- tion of deoxyribonucleoprotein. Histones are frac- tionated by gel filtration and ammonium sulphate precipitation into lysine-rich histones (Fl), arginine- rich histones (F3 + F2al), and slightly lysine-rich histones F2a2 and F2b.The usual method for the isolation of histones, involving the extraction of histones from chromatin with dilute sulphuric or hydrochloric acid, may result in the denaturation of various chromosomal components due to the extreme pH conditions. The ability of histones to regenerate the super-coil configuration of native nucleohistone is sometimes reduced [ 1,2 ] by the acid extraction technique. Evidence has been presented [3] that histones to some extend bind to DNA during acid treatment and form part of the so-called non-histones. The present investigation was undertaken to develop a procedure for the isolation and fractionation of histones under mild pH conditions. Nucleoproteins dissociated at high ionic strength can be separated into protein and DNA by ultracentrifugation [4-91 or gel filtration [8-lo] , but we have found these procedures unsuitable for the large scale isolation of histones. The method for histone isolation presented here is based on the finding of Mirsky and Ris [ 1 l] that protamine added in vitro to nucleoprotein directly displaces histones. A procedure which avoids extreme pH conditions is described for the large scale isolation and partial fractionation of calf thymus histones. Histones, virtually completely displaced from deoxyribonucleoprotein by protamine, and isolated by exclusion chromatography, appear to be almost identical on disc electrophoresis to histones prepared by acid extraction of deoxyribonucleoprotein. Histones are fractionated by gel filtration and ammonium sulphate precipitation into lysine-rich histones (Fl), argininerich histones (F3 + F2al), and slightly lysine-rich histones F2a2 and F2b. North-Holland PI1 blishing Company Amsterdam 2. Materials and methods

Plant histone 2 from wheat germ, a family of histone H2a variants. Partial amino acid sequences.

1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.

A simple modification converts the spinning cup protein sequencer into a vapour-phase sequencer

Spinning cup protein sequencer Vapour‐phase sequencer Protein structure

Chapter 12 Fractionation of Histones on Molecular Sieve Matrices

Publisher Summary This chapter describes the methods for fractionation of histones on molecular sieve matrices. Methods for the isolation and fractionation of histones are governed by two properties of the nuclear proteins inherent to their biological function: their strong positive charge and their tendency to aggregate. Purified nuclei facilitate the subsequent histone fractionation as a source of nucleoprotein. Histones are dissociated from DNA for the purpose of fractionation of the total complement or the purification of individual histones. A fraction containing the total complement of histones either as hydrochlorides or sulfates, if not contaminated with other proteins, neutral polysaccharides, or polyanionic polysaccharides, can be fractionated on molecular sieve matrices like Bio-gel or Sephadex. To minimize the aggregation of histones dilute HCl is introduced as a solvent for the fractionation of histones on molecular sieves. The purification of histones on molecular sieve matrices in dilute acid in the presence or absence of NaCl is successful in conjunction with the selective procedures of extraction. Gel electrophoresis is used as a sensitive and rapid method to establish the purity of histone fractions.

The complete amino acid sequence of histone F3 from chicken erythrocytes

We wish to report the complete sequences of the 136 amino acids of the only cysteine containing histone isolated from chicken erythrocytes. For the first time, to our knowledge, all the positions of the amino acids, in the course of establishing the primary structure of a protein, have been assigned without the use of overlapping sequences. To achieve this, specific chemical cleavages rather than enzymatic degradations were chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the Nand C-terminal amino acid in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradations of automatic sequencing, allowed us to perform a rapid and relatively non-laborious primary structure determination of histone F 3 .

Purification and characterization of the cysteine‐containing F3 histone from chicken erythrocyte

Knowledge of the amino acid sequence of histones will assist in the understanding of the molecular mechanisms of their function in the cell nucleus. Up to now only histones of group F3 have been shown to contain cysteine giving to this histone group particular properties with respect to protein-protein interaction. The structure of F3 histones is therefore of special interest. A new method has been developed to isolate highly purified F3 (Arg-rich) histone from chicken erythrocytes in gram amounts. The protein was characterized by its molecular weight, amino acid composition and end groups. Evidence was obtained that the microheterogeneity of this protein is due to fractional acetylation. The three CNBr-cleavage fragments were isolated and their relative sequence in the protein determined. Amino acid analysis of these fragments revealed clustering of acidic and basic amino acids.

The partial amino acid sequences of the two H2B histones from sperm of the sea urchin Psammechinus miliaris

Two new histone H2B variants have been isolated from sperm cells of the sea urchin Psammechinus miliaris. They have been designated sperm histone H2B(1) Psammechinus and sperm histone H2B(2) Psammechinus. Both histones are highly homologous to the previously described sperm histones from Parechinus angulosus (Strickland et al. (1977) Eur. J. Biochem. 77, 263--275 and 277--286). The amino acid sequences of the Ps. miliaris sperm histones, though highly homologous, are not identical to the amino acid sequence derived from the codon sequence of a histone H2B gene, characterized from the same organism by Birnstiel et al. ((1977) Nature 266, 603--607).

Formaldehyde and glutaraldehyde in the fixation of chromatin for electron microscopy

Glutaraldehyde and formaldehyde have been used to fix chromatin core particles for electron microscopy. Glutaraldehyde crosslinks protein only, whereas formaldehyde crosslinks protein and DNA. This is confirmed by the observation that the detergents sodium dodecyl sulphate, Sarkosyl NL 35 and benzylalkyldimethyl ammonium chloride separate the DNA from the protein in the case of glutaraldehyde fixed core particles but not in the case of formaldehyde fixed core particles. The fixative used in the preparation must therefore be considered as a further variable when evaluating electron microscopic images of chromatin.

A partial structure of histone H1 from sperm of the sea urchin sphaerechinus granulosus

Abstract 91 amino acids from sperm H1 histone of the sea urchin Sphaerechints angulosus have been placed in sequence. 24 of these amino acids are in the amino-terminal random-coil region of histone H1, while the remaining 67 amino acids comprise the central hydrophobic globular region. The sequence is compared to that of other H1 histones from various species.