Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

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.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Histones of spermatogenous cells in the house cricket

Histones were isolated from testis of the house cricket and analyzed by electrophoresis on polyacrylamide gels containing urea and acetic acid. Testes of two different nymphal instars and of adults were examined. The testes contained gonial and meiotic stages in the younger nymphs analyzed, and these same stages plus early spermatids in the older nymphs. At both nymphal instars, testis histone displayed the same five major fractions that are found in somatic nuclei of the cricket; the only unusual feature noted in nymph testis was a high abundance of phosphorylated F1. Adult testis has the same histone fractions as nymph testis and has two new fractions in addition. SDS electrophoresis also shows the presence of two more histones in adult testis than in nymph testis. — The unusual testis histones appear to accumulate during the nuclear elongation stages of spermiogenesis. The occurrence of these stages in adult testis is correlated with the presence of the extra histones. Nuclei of adult testis were separated into fractions enriched for early, mid, and late stages by velocity sedimentation at unit gravity. The unusual histones predominate in the fractions enriched for late spermiogenic stages. Both of the new histones appear to occur in the same stages of spermiogenesis, and display linked accumulation. Eventually they make up at least seventy percent of the histone complement.     

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

The histones of rat testis

Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X₁, X₂, and X₃) were observed. Fraction X₁ had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X₁ were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X₁ had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels.Fraction X₂ migrated between histone fractions F1 and F3 on electrophoresis while X₃ migrated between fractions F2b and F3. Fraction X₃ was isolated with F2b during fractionation by the Johns procedure. Fraction X₂ has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X₂ can be seen in electrophoretic patterns of rat liver histones.The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X₁ and F1, or X₃ and F2b are not attributable to phosphorylation.     

Fractionation of whole histone by partition chromatography on Sephadex G-25.

Whole histone from calf thymus was fractionated by partition chromatography on the basis of distribution between an aqueous phase immobilized on Sephadex G-25 beads and mobile organic phases containing various concentrations of trichloroacetic acid. The chromatography was carried out by stepwise elution with five upper phases from water and butanol-2 solvent systems containing 4 M urea and 0.1-1.5% trichloroacetic acid, and finally water. Of the six peaks obtained, two (peaks 1 and 2) contained arginine-rich histones. Although these peaks were still heterogeneous electrophoretically, the band corresponding to F2al was observed only in the electrophoretic pattern of peak 1 and the main fraction in peak 2 was F3. A histone fraction having nearly equimolar amounts of arginine and lysine was obtained from peak 3. Its amino acid composition was similar to that of F2a2. Slightly lysine-rich histone obtained from peak 4 showed an amino acid composition typical of F2b. Peak 5 contained a histone fraction with a ratio of lysine/arginine of 6.14, showing a single band on gel-electrophoresis. Very lysine-rich histone (F1) was obtained from peak 6, and the electrophoretic pattern of this fraction showed a single band.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

Changes in nuclear content of protein conjugate histone H2A-ubiquitin during rooster spermatogenesis

Electrophoretic analysis of acid-soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A-ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Characterization of histones from plutei larvae of the sea urchin Tetrapygus niger

• 1.1. Histones were isolated from plutei larvae of the sea urchin Tetrapygus niger and analysed electrophoretically. Individual histones were purified and their amino acid compositions were determined.
• 2.2. The electrophoretic analysis revealed that larval histones are microheterogeneous; H1 exhibits four subforms, the nucleosomal core histones H2A, H2B and H3 were resolved into three subforms each and H4 had two subforms.
• 3.3. The comparisons of the amino acid compositions of plutei larvae histones with data from the literature of homonimus late variants isolated from gastrulas of other sea urchin species, indicate that late histone variants are conserved proteins with a very slight degree of species specificity and with general features of classical histones.

     

The histones of rainbow trout erythrocytes include an erythrocyte-specific histone.

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erythrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation. To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to extablish identities of corresponding histone fractions, An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

The stepwise removal of histones from chicken erythrocyte nucleoprotein

1. A fractionation of chicken erythrocyte histones was achieved simultaneously with their extraction from saline-washed nuclei by stepwise titrations to progressively lower pH values. 2. Different acids and dilute buffer solutions of comparable pH behaved similarly in stepwise extractions of histones. 3. The histone preparations so obtained were characterized by their amino acid composition and behaviour on zone electrophoresis in starch gels. 4. The fractionation by titration was quite sharp at appropriate pH ranges, and the histone fraction that is apparently unique to avian erythrocytes was obtained without contamination by other histone fractions. 5. Histones prepared by stepwise titration were fractionated further by cation-exchange and exclusion chromatography. The chromatographic behaviour and amino acid composition of the components permitted comparison with histones prepared by other methods. 6. Histone fraction IIb was resolved into its subfractions IIb(1) and IIb(2) by exclusion chromatography on Bio-Gel P-60. 7. Histone fractions III and IV, previously reported to be absent from chicken erythrocyte nuclei, were found in extracts made at pH1.     

Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis

We have identified and purified TH3, a germ cell-specific histone. It has been characterized by amino acid analysis, tryptic peptide mapping, labeling with cystine, and by electrophoretic mobility as a variant of H3. On fully reduced Triton/acid/urea gels its mobility is retarded more than that of the somatic variants H3.2 and H3.3, but less than that of H3.1; it migrates between the H2As and H1s. Germinal cells from adult and sexually immature testes were purified by centrifugal elutriation followed by Percoll density gradient separation in order to study the distribution and synthesis of TH3. TH3 is found in significant levels in spermatogonia and in similar or slightly higher amounts in spermatocytes and round spermatids. The synthesis of TH3 takes place in the spermatogonia but not in spermatocytes, in contrast to the other testis-specific histones, TH2A, H1t, and TH2B. Therefore, TH3 may have a different role in spermatogenesis than do the other testis-specific histone variants.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.