C. Von Holt

Fractionation of chicken erythrocyte whole histone into the six main components by gel exclusion chromatography.

Abstract A two-step procedure for the fractionation of chicken erythrocyte whole histone is described. Gel exclusion chromatography on Biogel P-60 in 0.02 M HCl, 0.05 M NaCl separates whole histone into four homogeneous fractions; F 1, F 2C, F 2a2 and F 2a1 and a mixed fraction containing F 2b and F 3. Chromatography of the F 2b-F 3 mixture on Sephadex G-100 at pH 5.1 yields pure F 2b and F 3 histones.

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.

It has often been stated that histone F2al (IV) and F3 (III) are evolutionarily extremely stable proteins [ l-3,5]. This was based on the observation that these histones had a virtually identical electrophoretic mobility regardless of the source [ 1,2] . The tryptic fingerprint maps of the two proteins from calf and pea were shown to be very similar [S] and in the case of histone F2al from pea and calf the primary structures had been established and found to differ by only two amino acid residues out of a total of lb2 residues [6]. 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. In order to establish the evolutionary relationships of histone F3 over a more extended range in the biological world we isolated this histone from a number of organisms separated widely on the evolutionary scale, namely from a shark, an echinoderm, a mollusc and a plant. 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].

Sequence of the cysteine-containing portion of histone F2al from the sea urchin Parechinus angulosus

Histone F2al has been shown to be evolutionarily a very stable protein showing only two conservative changes in organisms as widely separated on the evolutionary scale as calf and pea [ 11. The F2al from echinoderms, in contrast to both calf and pea, has been shown to contain cysteine [2]. We have isolated F2al from the sea urchin and placed in sequence 52 of the 102 amino acids. Cysteine was found to replace threonine at position 73.

Partial amino acid sequence of histone H1 from sperm of the sea urchin, Parechinus angulosus

In contrast to the conserved sequences of histones H4 and H3 [1], H1 histone has been reported to vary considerably in its structure, even within the organisms from which it has been isolated [2 -4] . In continuation of our studies of sea urchin sperm histones, histone H1 has been purified and peptides prepared by cyanogen bromide and N-bromosuccinimide cleavage. These peptides have been aligned and partially sequenced. The carboxyl half of the molecule is intensely basic while the central region contains most of the hydrophobic residues. The amino terminal region is also basic and contains many of the proline residues. The distribution is therefore very similar to that found in other H1 histones that have been partially sequenced [1-3] . In this paper sea urchin H1 is compared to rabbit thymus histone HI Fraction-3 [3], trout histone H1 [1,4] and chicken histone H5 [5,6] in terms of the amino acid distribution and partial sequences.

Proteolytic degradation of histones and site of cleavage in histone F2a1 and F3

Histones are susceptible to proteolytic degradation during their isolation [ 1,2]. This degradation can be minimized by protease inhibitors like diisopropylphosphofluoridate and by performing all operations at low temperatures [ 11. Subsequently it has been shown that a trypsin-like protease is closely associated with isolated chromatin [2-41. This enzyme is coextracted with histones in dilute acid and may autolyse such a preparation near neutral pH [2,5]. The enzyme has its optimal activity at pH 8 with nucleoprotein or free histones as substrate [2,3]. The disc electrophoretic pattern of the histone fraction isolated from degraded nucleoprotein reveals that only a limited number of fragments are formed [ 1,2], and that the 5 histone groups show different susceptibility to the proteolysis [2]. Although the enzyme has been partially purified [3], its specificity has not yet been determined. We now wish to report the purification and characterization of two histone degradation products isolated from cycad pollen nucleoprotein. The results of the determination of the N-terminal sequence of these fragments have allowed us to identify them as breakdown products of histone F3 and F2al respectively. A number of interesting features of the cleavage sites became apparent. Possible biological implications are briefly discussed.

The occurrence of histone H3 and H4 in yeast.

Histones occur in a wide variety of uniand multicellular organisms. Histone H4 is evolutionarily a very stable protein [1]. We have shown a similar conservation of the primary structure for histone H3 isolated from chicken [2], shark [3], sea urchin, a mollusc and a plant [4]. The other 3 histones HI, H2A and H2B seem to show a greater variability in their structures [5]. Whereas for multicellular organisms considerable information on the primary structures of histones is available to allow general conclusions to be drawn as to their occurrence, their identity and general evolutionary trends of their structures, this does not apply to unicellular, eucaryotic organisms. This lack of information is probably related to the difficulties experienced in the purification of the small amounts of histones present in the chromatin of these organisms the genome of which is by several orders of magnitude smaller than that of higher organisms. The occurrence of histones and their identity in fungi, green algae and protozoa is controversial [6,7]. In yeast the presence of histones H2A, H2B and H4 has been reported [8], whereas it has been found that histones H1 and H3 were absent [8]. If histones were to be essential for the structural and functional organisation of chromatin the absence from yeast of the conservative histone H3 would cast doubt on such a view. We have therefore reinvestigated the question of the occurrence and the nature of histones in yeast.

Histone H2B variants from the erythrocytes of an amphibian, a reptile and a bird.

Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.

The primary structure of histone F3 from shark erythrocytes

The amino acid sequence of chicken histone F3 (III) isolated from erythrocytes had previously been elucidated [l-3]. This had been achieved by selective chemical fragmentation and alignment of the peptides by generating not more than 3 peptides in any single cleavage. From the identity of the Nand C-terminal residues of the cleavage products and the uncleaved starting material [2,3] the peptides could be aligned. In order to further study the evolution of histone F3, we isolated it from shark erythrocytes. Sharks are believed to have evolved about 350 million years ago and their ancestors separated 500 million years ago from the main line of vertebrate evolution. They are therefore of considerable interest in this cortext. The amino acid seuqence of shark histone F3 was elucidated by the same procedure used for the chicken histone. The sequence of this protein can now be compared to that of chicken [3], calf [5] and the recently determined carp histone [6].

Partial amino acid sequence of two new arginine—serine rich histones from male gonads of the sea urchin (Parechinus angulosus)

It has frequently been postulated that histone fractions F2al and F3 are evolutionarily extremely stable proteins [l-3]. In contrast histones F2b, F2a2 and Fl show considerable variations among the organisms from which they have been isolated [4-71. The demonstration of these variations was based mainly on the differences in electrophoretic mobility without any characterization of the protein fraction, Early reports also indicated that calf thymus F2b might be heterogeneous [8-91. As part of a program to study the evolutionary relations of the variable histones among widely divergent species, the histones present in male sera urchin gonads have been fractionated and characterized. Among these histones was a new arginine-serine rich protein which on polyacrylamide electrophoresis behaved like a protein with a molecular weight of approximately 15 000 daltons and with prollne as the N-terminal amino acid. In addition, this histone behaved like F2b during Johns’ [lo] selective extraction. In this communication evidence will be given that this histone fraction contains two closely related species. The cyanogen bromide fragments (CNBr) reveal that the two histones contain an identical C-terminal section and a different but closely related N-terminal section. Sequence studies revealed that the C-terminal ammo acid sequence of the molecules is very closely related to calf thymus F2b [ 111. The N-terminal regions of both proteins consist of closely related, repetitive sequences which are only vaguely similar to the corresponding section of calf thymus

Isolation and characterization of the histones from cycad pollen.

Sequence studies of calf and pea histone F3 and F2al have revealed that these histones are evolutionarily very stable proteins [ 1,2] . It is desirable to obtain additional comparative information on histones in general. Such investigations in plants are hampered by the low nucleoprotein concentration of plant cells and the difficulty of separating the nucleoprotein complex of vegetative tissue from the non-chromatin components. Using pollen as a source of nucleoprotein should overcome some of these difficulties. The male cones of the gymnosperm Encephalartos cuffer (cycad) yielded large amounts of pollen from which we isolated nucleoprotein in high yields. We wish to report the isolation of this cycad nucleoprotein and subfractionation of its histones into various groups according to their solubility and behaviour on gel exclusion chromatography. The partial amino acid sequence of the histone F3 isolated from this source has been published in a previous communication [3].