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Yeast histones H2A, H2B, and H3 were purified using the standard histone purification procedures of differential solubility and exclusion chromatography. Yeast histone H4 was isolated by the same methods in a fraction containing one other major protein component. The four yeast core histones were identified by their reactions with antisera against rye and (or) calf histone fractions as well as by their electrophoretic, chromatographic, and solubility properties. The immunological distances between yeast H2B and rye and calf H2B fractions are substantial, as is the rye-calf distance for H2B. The immunological distance between yeast H2A and rye H2A is also large and is similar to the rye H2A - calf H2A distance. On the other hand, the immunological distance between yeast H3 and rye and calf H3 is much greater than that between rye H3 and calf H3. These and other results indicate that yeast H3 differs appreciably from the H3 of higher eucaryotes.

The rye and calf histones were fractionated by differential solubility and exclusion chromatography. Antibodies were produced against histones H1, H2A, H2B, and H3 of both species and the extent of cross-reaction was measured by microcomplement fixation. The results allowed the identification of rye histones H2A and H2B. The near structural identity of the H3 histones was confirmed by the small immunological distance which indicated an amino acid difference of less than 2% between rye and calf. The interspecific differences found for both H2A and H2B were greater, the amino acid difference estimated from the immunological distance being between 10 and 20%. H1 gave no immunological cross-reaction, indicating a large (greater than 40%) difference in the structure of this protein in the two species. The extent of variation that a histone fraction shows between plant and animal species is probably related to its role in chromatin organization.

The Raman spectra of aqueous solutions of histones H3 and H4 from calf thymus and from rye reflect the high degree of conservation from species to species of the primary and secondary structures of these proteins. The amount of beta-sheet structure is estimated at 40 +/- 5% in H4 and at 33 +/- 5% in H3 from the intensities of the amide I and amide III bands at 1663 and 1241 cm-1, respectively, in the spectra. These values are independent of the salt concentration of the solutions, mostly likely because of the high histone concentration (approximately 3 mM) required to obtain the spectra, which results in some aggregation of the proteins. The intensity ratio of the tyrosine doublet at 852 and 826 cm-1 indicates that the four tyrosine residues in H4 are relatively exposed to the solvent or weakly bound to positively charged groups of basic amino acids, whereas in H3 at least one tyrosine is buried inside the protein and tightly bound to a carboxylate group. The results also show that the secondary structure of H3 is slightly influenced by the state of oxidation of the two cysteine residues it contains.

Laser Raman spectra of the calf thymus histones H1, H2A, and H2B in aqueous solutions are presented. The amide III band in the spectrum of the very lysine-rich histone H1 in aqueous solution appears at 1245 cm-1, which is almost at the same frequency as the corresponding vibration of the ionized form of poly(L-lysine). Upon increasing the NaCl concentration to 1 M, the frequency of the amide III vibration shifts to 1250 cm-1 as a result of the formation of a more compact disordered structure of at least the N-terminal region of the protein. Changing the pH from 3 to 5 induces the same frequency shift. The amide III regions of the Raman spectra of the slightly lysine-rich histones H2A and H2B shows two bands at 1247 and 1265 cm-1 for H2A, and at 1254 and 1265 cm-1 for H2B. These doublets are attributed to vibrations involving the backbone of at least two structurally distinct parts of the histone molecules. The low frequency component is assigned to the random-coil regions of the proteins which appear to have similar conformations for H1 and H2A. The frequency of this component also suggest that the structure of the disordered regions of H2B are more compact and less extended. These conclusions confirm the conformation predictions based on the primary structures of these proteins. The high frequency component at 1265 cm-1 is assigned to the alpha-helical and rigid disordered structures of H2A and H2B, since this band increases in intensity upon addition of NaCl. The amide I' region of the histone spectra is also presented but appears to be much less sensitive to the conformation than the amide III region. The intensity of the bands due to the single bond C-C stretching modes, as well as the intensity ratio of the tyrosine Fermi doublet at 855 and 830 cm-1, are also discussed.

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.

The selective extraction of histones from rye chromatin was studied using three different methods. Extractions with NaCl-phosphate buffers at pH5.5 gave results similar to those already obtained with other types of chromatin. Histone H1 was selectively extracted with 0.6 M NaCl-0.001 M PO4, while the selectivity of dissociation of the other fractions was reduced at higher NaCl concentrations. The use of phosphate-urea buffers at pH5.5 also revealed that the histones were dissociated at the same concentrations as were calf thymus histones. Histone H1 was extracted with 0.5 M PO4-1 M urea; H1, H2A, and H2B were extracted with 0.8 M PO4-2 M urea; and all histones were removed with 0.8 M PO4-5.3 M urea. It was, however, observed that the dissociated rye histones H2A and H2B were unstable in these buffers. This instability was maximum in the presence of 3 M urea, where both histones were absent from the extracted proteins and the residual nucleoproteins. Finally, a solution of 30% ethanol-0.35 M NaCl-6 M urea produced a rye nucleoprotein fraction containing only histone H1.

Amino acid composition and tryptic fingerprints of rye (Secal cereale) H1, H2B (PH1), and H2A(PHII) histones indicate the presence of major differences between these and the corresponding calf or rabbit fractions. In addition to variations for other amino acids, fraction H1 from rye contains twice as much arginine as the corresponding animal fraction; the plant H2B (PHI) and H2A (PHII) histones show lysine to arginine ratios greater than those of their animal counterparts. The tryptic maps of the same proteins appear to differ between plants and animals by the number and the general pattern of the peptides, as well as by the quantity and distribution of the arginine-containing peptides. Such results suggest the presence of differences in the primary structure of the calf and rye lysine-rich and moderately lysine-rich histones. Furthermore, the possibility is ruled out that each of these plant histones consists of an animal-like protein with an additional segment of 20--30 amino acid residues. On the other hand, the rye and calf arginine-rich fractions H3 and H4 show similar amino acid compositions and tryptic peptides maps.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.

Peripheral blood lymphocytes are a naturally occurring population of G0 cells which can be activated in vitro to grow and divide. Upon activation with phytohemagglutinin (PHA), they enter G1 and, after a 24-h lag, begin DNA replication (S phase). Using radioisotope labeling and gel electrophoresis of acid-soluble chromatin proteins, we investigated histone synthesis in G0, G1, and S phase cultures of human and pig lymphocytes. In G0 and G1 cultures, which have less than 0.1% S phase cells, all five histones are synthesized and are incorporated into chromatin in equimolar amounts. In G0 lymphocytes histone synthesis accounts for at least 6% of nuclear protein radioactivity, and the rate of synthesis is about 2-3% of that of S phase lymphocytes. In contrast to histone synthesis by S phase cultures, G0 and G1 histone synthesis was completely resistant to treatment with hydroxyurea.

The degree of actinomycin D binding to DNA in chromatin is dependent upon the state of repression of chromatin. Living cells bind three times more tritiated actinomycin to euchromatin than to genetically inactive heterochromatin. Extraction of histone results in a general increase in tritiated actinomycin binding and in a ratio of the uptake in heterochromatin to that in euchromatin approaching unity.