An electrophoretic analysis of the histones of the house cricket

The histone complement of the house cricket, an insect, was analyzed by electrophoresis on polyacrylamide gels. Five fractions separated from each other on gels containing 6.25 m urea; their subfractions were resolved in long runs on gels 25 cm long. On comparison with the corresponding fractions in vertebrates, only the F1 fraction of the cricket seems notably different. Its mobility is much lower than that of vertebrate F1's. Further, in contrast to vertebrate F1, which consistently shows some electrophoretic heterogeneity, the F1 from nondividing tissue of cricket appears homogeneous. F1 subfractions were found in testis histone, however, and presumably reflect phosphorylation of F1, as expected of a dividing tissue. The other fractions all display as much heterogeneity as seen in vertebrates, or more. Four subfractions were visible in the F3 fraction, three in F2a1, two in F2a2, and two in F2b. The heterogenity of F2b observed in cricket is of particular interest since F2b subfractions have not been detected in studies of any other organisms. F2b's electrophoretic heterogeneity implies that it, like the other histones, is subject to modification which entails neutralization of basic groups.     

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.     

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.     

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.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

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 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.     

Individual histone messenger RNAs: Identification by template activity

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that coelectrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein synthesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.     

Segregation of histone fractions from purified rat pancreas nuclei by isoelectric focusing.

The present report describes the purification of nuclei from rat pancreas and the use of isoelectric focusing for the segregation of histone fractions, a heterogeneous mixture with respect to charge. This procedure employs gels of 5% polyacrylamide as support matrices, in 6.25 M urea, and spans a pH range of 7.5-10.8. It was, therefore, found adequate for study of the microheterogeneity of histones. The banding pattern of histone fractions in pancreas development using electrofocusing is presented.     

An evolutionary comparison of plant histones.

Histones were extracted from chromatin of the following: a moss (Polytrichum juniperinum); the primitive vascular plants Psilotum nudum and Equisetum arvense; a fern (Polypodium vulgare); the gymnosperms fir (Abies concolor), yew (Taxus canadensis) and Gingko biloba; the dicotyledonous angiosperms tobacco (Nicotiana tabacum) and maple (Acer saccharinum); and the monocotyledonous angiosperms corn (Zea mays) and lily (Lilium longiflorum). The histones were subjected to polyacrylamide gel electrophoresis and compared to standard histones of pea (Pisum sativum) and cow (Bos taurus). All species have histones of the exact electrophoretic mobility of histones F2a1 and F3 of cow and pea. All species have histones of low electrophoretic mobility assumed to be F1 histones. None of the plant histones displayed electrophoretic mobility between F3 and F2a1 while animal histone fractions F2b and F2a2 do migrate to this position. No animal histone fraction was found to migrate between F3 and F1 while a major plant fraction, designated "F2b-like" was found to migrate to this position in all plant species studied except for the moss and Psilotum. A band of similar mobility was strikingly absent from the histones of these two species.     

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.     

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.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

Electrophoretic study of plant histones: Comparison with vertebrate histones

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.     

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.     

Variability of sperm specific histones in sea urchins

The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0--8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.     

Separation and quantitative analysis of rat testis histones by one-dimensional gel electrophoresis

A procedure is presented for direct separation and quantitative analysis of histones of rat testis by use of one-dimensional cylindrical gel electrophoresis in acid/urea/polyacrylamide gels at two concentrations of urea. After separation and staining with amido black the histone fractions are determined by extraction of bound dye and spectrophotometric analysis. This method provides a rapid and accurate procedure for determination of microgram amounts of the histone subfractions which are unique to the testis as well as those which are found in somatic cells, and it is particularly useful for determination of molar proportions of the histones.     

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.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei

Summary Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts.Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days.The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei.

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.