Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Drosophila chromatin: an immunological study.

Antibodies were prepared against chromatin, various chromosomal protein preparations and against cytoplasm from Drosophila larvae. These antibodies were used to study the distribution of antigens in chromatin and chromosomal protein preparations on double diffusion plates. Antisera from all of the mammals tested precipitated both chromatin and DNA on double diffusion plates run in water. This non-specific precipitation was removed by washing in 0.06M NaCL.     

Hydroxylapatite thermal fractionation of chromatin and DNA

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.     

Cross-linking of chromosomal non-histone proteins to DNA by UV radiation and some antitumor drugs

Antibodies reacting specifically with HeLa cell chromatin can be elicited by immunization with dehistonized HeLa chromatin preparations. The nature of these chromatin-associated antigens was investigated by cross-linking with UV irradiation or by in vitro exposure of chromatin to 1-methyl-1-nitrosourea (MNU) or 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). With the exception of 1-methyl-1-nitrosourea the described treatment of chromatin (native or dehistonized) significantly increased its immunological reactivity. Dissociation of the chromosomal proteins from DNA by concentrated salt-urea solutions essentially abolished the immunological reactivity of the residual chromatin pellets. The immunological activity was found in the supernatant protein fraction after its reconstitution with purified human placenta DNA. UV irradiation or alkylation of chromatin cross-linked the active proteins to DNA and prevented their dissociation. It is concluded that the immunologically cell-specific antigens in HeLa chromatin exist as closely associated complexes of chromosomal protein(s) with DNA.     

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.     

Coexistence of nucleosomal and various non-nucleosomal chromatin configurations in cells infected with herpes simplex virus

Coexistence of four different forms of chromatin was observed by electron microscopy in nuclear spread preparations of monkey kidney cells during late stages of infection with herpes simplex virus (HSV-1 AMG). Besides typical nucleosomal (i) chromatin, thin (3-5 nm) strands morphologically indistinguishable from protein-free DNA were frequent, without (ii) or with (iii) sparse 10-22 nm large granules different from nucleosomes. In addition, uniformly thick (mean 17 nm), heavily stained chromatin strands (iv) were seen. The non-nucleosomal character of types (iii) and (iv) chromatin was also demonstrated by their resistance to histone removal in Sarkosyl and heparin. All four forms were seen in capsid-associated HSV-DNA molecules, and various combinations of these forms occurred in adjacent regions of the same DNA molecule, including the vicinity of replication branch points. Especially frequent were regions of chromatin types (ii) or (iii) alternating with thickly coated intercepts of type (iv) chromatin, the latter often displaying "bubble"-like strand separations. The appearance of chromatin types (ii)-(iv) was dependent on viral replication. These chromatin arrays were compared with structures observed in purified HSV-DNA from these cells. Patterns of single-stranded regions were found in HSV-DNA that were similar to those observed in the thickly coated type (iv) chromatin. It is concluded that, in these nuclei, non-nucleosomal arrangements can be formed, at least on viral DNA, under conditions of continued DNA synthesis and inhibited protein synthesis, and that single-stranded DNA is packed into a characteristic thick strand of non-nucleosomal chromatin by association with a special, probably virus-coded protein.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

Phosphorylation and other properties of proteins binding single-stranded DNA (SSB-proteins) from chromatin and extrachromatin fractions of Ehrlich ascites carcinoma

A comparative study of single-stranded DNA-binding proteins (SSB-proteins) isolated from chromatin and the extrachromatin fraction of Ehrlich ascites tumour cells was carried out. No differences were found either in SDS-gel electrophoretic mobility or in the single-stranded DNA-binding capacity and stimulation of the replicative synthesis of DNA. However, chromatin SSB-proteins contained 1.4-1.5 times more phosphate than extrachromatin proteins. Both preparations could be phosphorylated in vitro by protein kinase C and cAMP-dependent protein kinase, but the chromatin proteins were phosphorylated in a lesser degree. In parallel with phosphorylation the SSB-proteins displayed a higher binding affinity for ssDNA-cellulose. Phosphorylation can thus be regarded as a means of regulation of the SSB-protein function, in particular, their interaction with chromatin DNA.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

An H1-like protein from the macronucleus of Euplotes eurystomus

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.     

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.     

Probing chromatin structure in the early phases of apoptosis

Abstract. A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time-course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light-scattering properties, and spectrophotometric determination of the protein content. An augmentation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as 1 h after irradiation. As this phenomenon is not correlated to changes in actual protein content, one can conclude that modifications of basic protein accessibility occur from the early phases of the apoptotic process. Also DNA stainability augmented with time, generating the transient appearance of a hyperdiploid peak that preceded the appearance of the hypodiploid peak typical of the late stages of the process, and that shared with the latter the same light-scattering properties. Chromatin status was further explored by staining apoptotic nuclei using DNA probes with peculiar molecular weight. Propidium iodide (PI) and ethidium bromide (EB), but not the much bulkier 7-aminoactinomycin D (7-AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to binding sites was responsible for the phenomenon. Remarkably, all dyes identified the same proportion of hypodiploid nuclei when an apoptotic nucleus shed its fragmented chromatin. Control experiments included differential interference contrast and fluorescence microscopy that showed the purity of nuclei preparations and the typical morphological apoptotic features. Finally, the simultaneous evaluation of DNA by PI and nuclear proteins by FITC in a time course study allowed a thorough assessment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolysis and probably renders it easier the final endonucleolytic step leading to DNA fragmentation and loss.     

Studies on the preparation and properties of ribonucleoprotein particles from rat liver nuclei

Ribonucleoprotein particles of 38 S were extracted from rat liver nuclei with isotonic salt buffer under concomitant sonication. The fate of the endogeneous nuclear RNAases assayed with poly(A), high molecular weight yeast RNA and rapidly labeled hnRNA was followed during the preparation of 38-S nuclear ribonucleoprotein (nRNP) particles. Essentially all the RNAase activity could be removed from the particle preparation. The effect of synthetic RNAase inhibitors on the nRNP particles was studied. Upon extraction of nuclei with 0.14 M NaCl, approximately 38% of the total nuclear radioactivity was found in the 38-S nRNP particles. By two successive extractions of the remaining chromatin with either isotonic or 0.22 and 0.3 M NaCl, an additional 25 and 9% of rapidly labeled hnRNA of 38 S particle were dissociated from chromatin, respectively. The chromatin components, DNA, nonhistone proteins, histones and RNA were determined after successive salt extractions. Particularly alterations in the nonhistone proteins and RNA were found. The protein patterns upon SDS-acrylamide gel electrophoresis of the salt-extracted chromatin preparations were compared with those of the 38-S nRNP particles. Particularly proteins in the molecular weight range of 32 000-43 000 were dissociated from chromatin after treatment with 0.22 or 0.3 M NaCl.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

Tissue Differences in Antigenic Properties of Non-Histone Protein-DNA Complexes

THAT DNA and histones are not tissue specific^1,2 implies that other components of chromatin may be responsible for the tissue specific control of eukaryotic gene expression. We now report studies of the antigenic properties of non-histone protein-DNA complexes isolated in an undissociated state from native chromatin and compare these with the properties of native chromatin. Because DNA is a very weak immunogen³, the antigenic determinants in these preparations should be principally caused by the protein components.     

Properties of single-stranded DNA-binding proteins (SSB-proteins) from chromatin and nonchromatin fractions of Ehrlich ascites tumour: phosphorylation enhances the affinity of SSB-proteins for single-stranded DNA.

To assess the possible functional role of single-strand DNA-binding (SSB) proteins in eucaryotic cell, a comparative study was made of SSB-proteins isolated from chromatin and the nonchromatin fractions of Ehrlich ascites tumour (EAT) cells. No appreciable differences between the two groups could be found either in SDS-gel electrophoretic patterns or in the ssDNA-binding capacity and stimulation of DNA replication in permeable EAT cells. However, the chromatin SSB-proteins incorporated 1.4-times more labelled phosphate in vivo; phosphate assays in the isolated chromatin and nonchromatin SSB-proteins yielded ca. 3 and 2 moles Pi/mole protein, respectively. Both preparations could be further phosphorylated in vitro with Ca-phospholipid-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase, but the non-chromatin proteins were phosphorylated to a greater degree. In parallel with phosphorylation, the SSB-proteins displayed stronger binding to ssDNA cellulose. Phosphorylation may thus be a means of regulating the functions of SSB-proteins, in particular their interaction with chromatin.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.