Fractionation of cricket testis nuclei on gradients of colloidal silica for study of basic protein changes during spermiogenesis

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

Hormonal control of meiosis reinitation in starfish oocytes. New evidence for the absence of efficient intracellular receptors for l-methyladenine recognition.

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Soluble acidic complexes containing histones H3 and H4 in nuclei of Xenopus laevis oocytes

Oocyte nuclei of Xenopus laevis contain nucleosomal-core histones in large amounts and in a soluble, non-chromatin-bound form. Supernatant fractions (100,000 X g) from isolated nuclei are enriched in complexes containing histones H3 and H4, which are of distinct size (5.6S by sucrose gradient centrifugation, approximate molecular weight of 270,000 by gel filtration) and negatively charged (isoelectric at pH 4.4). These complexes bind to DEAE-Sephacel and can be separated from nucleoplasmin. In diverse fractionation experiments, histones H3 and H4 have been found to comigrate with a pair of polypeptides of molecular weight 110,000 that represent the most acidic major protein present in these nuclei. After enrichment by gel filtration, ion exchange chromatography and electrophoresis, this pair of acidic polypeptides has been the only nonhistone protein detected in the histone-complex fraction. We suggest that in the oocyte nucleus, large proportions of the soluble histones H3 and H4 are not contained in complexes of all four nucleosomal-core histones but are differentially associated with specific, very acidic proteins into distinct 5.6S complexes.     

A new method for oligo(ADP-ribose) fractionation according to chain length

A new method was developed to separate mono- and oligo-(ADP-ribose) with chain lengths below 11 ADP-ribose units by size difference of one ADP-ribose residue. The separation was performed on a DEAE-cellulose column by elution with a NaCl gradient (0–0.3 $\text{M}$) in the presence of 7 $\text{M}$ urea at pH 7.6. Using this method, the chain length distribution of oligo(ADP-ribose) molecules attached to histones by incubation of isolated nuclei with radioactive NAD was determined. The average chain length estimated from this distribution coincided exactly with the value obtained by the phosphodiesterase digestion method, suggesting that the oligomers were synthesized directly on histones and not elongated from pre-existing ADP-ribose.     

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.     

Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro

In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100–200 nm; N-1) and partly decondensed (diameter of fibrils ～30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4–7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin structure, histone-chromatin binding strength, and concentration of DM.     

Histones from nucleated erythrocytes of the marine invertebrate Sipunculus nudus

Total and lysine-rich histones were extracted from purified sipunculid erythrocyte nuclei with 0.25 N HCl and 0.74 N perchloric acid, respectively. The histones were fractionated and purified by gel filtration and ion exchange chromatography. Sipunculid PCA extract shows three lysine-rich histones, one of which may be a fraction specific to erythrocytes. The histones H₃ and H₄ of this marine invertebrate are very similar to their vertebrate homologues. Whereas no fraction H_2B has been detected in our investigation, the fraction H_2A has an unusual chromatographic behaviour if compared to its vertebrate homologue. These features may be the reflection of a peculiar distribution of histones in the chromatin of sipunculid erythrocytes.     

Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

ISOLATION AND ELECTROPHORETIC IDENTIFICATION OF HISTONES OF RAT BRAIN AND LIVER

Abstract—

• 1 Relatively pure brain nuclei were prepared on a discontinuous sucrose gradient which gave yields of between 40 and 60 per cent.
• 2 An extraction procedure has been developed for the isolation of total histones from brain and liver nuclei. This procedure is also applicable to the isolation of histones directly from whole brain and liver.
• 3 The electrophoretic pattern of histones prepared from brain and liver by the above procedure was similar to that of calf thymus histones.

     

H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA.

The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones.     

Nucleosomal organization of a part of chromatin in mollusc sperm nuclei with a mixed basic protein composition

The structural organization of mature sperm chromatin from three representatives of theMytilidae family has been studied. The acid-soluble proteins in these species nuclei are primarily sperm-specific (approximately 80%) with the remainder being core histones. Previously, we have shown that the mature sperm nuclei of these molluscs are compact, dense structures formed by interaction of the spermspecific proteins with DNA (1). Here we show that: a) although the histones are minor chromatin protein fraction, they still organize a part (20–25%) of the total DNA into nucleosomes; b) one of the sperm-specific proteins, different from somatic H1 or H5 histones participates in the formation of the beaded structures.     

Immunocytochemical localization of calmodulin in PC12 cells and its possible interaction with histones

The subcellular localization of calmodulin, a multi-functional calcium-binding regulatory protein, was examined immunocytochemically in undifferentiated PC12 rat pheochromocytoma cells and cells differentiated with nerve growth factor (NGF) and dibutyryl cyclic AMP. In undifferentiated PC12 cells, diffuse immunostaining for calmodulin was observed in the cytoplasm, and weak, patch-like staining was found in the nucleus. In differentiated cells, intense immunostaining for calmodulin was observed in the cytoplasm, while nuclear immunostaining was still evident. Immunoreactivity for calmodulin was also observed along newly-formed neuritic processes, with strong staining in varicosity-like structures and growth cones. Using double-label immunochemistry, the relative intensity of immunostaining for calmodulin among the nuclei was found to correlate with the relative intensity of immunostaining for histones in the same nuclei. A comparison of a profile of ¹²⁵I-calmodulin binding in the nuclear fraction from PC12 cells to that of immunoblotting for histones in the same fraction indicated that some of the histones are calmodulin-binding proteins in PC 12 cells. These results show that the level and subcellular distribution of calmodulin are altered during the course of nerve cell differentiation and suggest the possibility that histones may function as major nuclear binding proteins for calmodulin.     

The isolation of nuclei and basic nucleoproteins from the cellular slime mold Dictyostelium discoideum.

A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X-100. Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum. The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F1. After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.     

Chromatin fractionation according to the strength of its matrix bond

Extraction in low salt concentration followed by centrifugation allows rat liver nuclear chromatin to be divided into two fractions: the supernatant chromatin and matrix chromatin. The former fraction contains about 60-70% of initial DNA and about 15% of initial protein along with all five histones, and an insignificant amount of non-histone proteins. RNA synthesis in the matrix chromatin fraction is 2-3 times more intense than that in the original nuclei. The data on gradient centrifugation do not suggest the elongation of RNA molecules synthesized in the matrix fraction. The results obtained as compared with the literature data suggest that the matrix chromatin fraction is enriched with active genes.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

Purification of nuclei from a flagellate protozoan, Trypanosoma brucei

A simple and rapid technique is described for the isolation of nuclei from the flagellate protozoan Trypanosoma brucei. Cells were disrupted by nitrogen cavitation in the presence of hexylene glycol which enhances nuclear stability. Isolated nuclei were separated from nuclei trapped with cytoskeletal fragments and flagellae by Percoll density gradient centrifugation. Centrifugation in a medium with increased sucrose concentration greatly improved the separation of free nuclei from those trapped in cell debris. The isolation procedure resulted in the recovery of 34% of the nuclei present in the whole cell suspension. Electron microscopic and chemical analysis indicated that the recovered nuclei were in good condition and were highly purified.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.