The purification of cell nuclei from calf uterus by zonal centrifugation in reorienting density gradients

A method is described for isolation of substantial amounts of pure and enzymatically active nuclei from whole calf uterus. The technique involves a multistep sequential homogenization of the tissue and a zonal centrifugation of the crude nuclear preparation in a reorienting density gradient rotor. Electron and phase contrast microscopic observations show that the nuclei are intact and practically free from cytoplasmic contamination. Based on DNA recovery, the purified fraction contains 9% of the nuclei of the total tissue and more than 19% of the filtered homogenate. The pure nuclear fraction consists of 29% DNA, 7% RNA, and 64% protein, which parallels the composition of purified nuclei from other mammalian tissues.     

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.     

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma

DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×10⁶ daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×10¹⁰ daltons of DNA or 7.5×10⁷ nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×10¹⁰ daltons, thus indicating that vegetative cells of P. obtusum are diploid.     

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

Characterization of Allomyces genome

Allomyces arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (alpha) and two minor components (beta & gamma) with buoyant densities in neutral CsCl corresponding to 1.721, 1.710 and 1.702 g/cm3, respectively. The DNA obtained from purified nuclei contains alpha component only. The beta component corresponds to mitochondrial DNA. The gamma component is also extra-nuclear but has not been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25 % bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of both bulk and nuclear DNA, have a sequence complexity of approximately 7.2-10(6) daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for about 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7-10(10) daltons.     

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromatin-nuclear envelope complex from rat liver: isolation and purification

We describe a method for the isolation of a fraction of nuclear envelope (NE) from rat liver. The method includes mild treatment of pure nuclei with either endonuclease of DNase I under low ionic strength conditions in the presence of magnesium, which allows the nucleomeric organization of the chromatin (Ch) to be preserved. The NEs were purified by centrifugation in sucrose gradients followed by floatation in sucrose. No more than 3% of the Ch present in the purified Ch-NE complexes was due to the non specific adsorption of Ch to the NE. The main components of the complex (Ch and NE) retained their in situ ultrastructure. The complex consisted of 9--10% DNA, 3--4% RNA, about 63% protein and about 24% phospholipids.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Purification of 14C-labelled deoxyribonuclease II from HeLa S3 lysosomes and its use as a marker for the study of nuclear deoxyribonuclease II

Deoxyribonuclease II (DNAase II) in mammalian cells has generally been considered to be located in the lysosomes. Several recent studies have indicated that some DNAase II activity is present in purified nuclei; this, however, could have been due to some contamination of the nuclear fraction by lysosomes, or alternatively, it could have been caused by specific binding of lysosomal DNAase II to the nuclear fraction during isolation. Our previous studies have eliminated the possibility that lysosomal contamination was the cause of the presence of DNAase II in isolated nuclei. In this study I have purified (14)C-labelled lysosomal DNAase II and added it to cells during isolation of their nuclei. This study demonstrates that there is no specific binding of lysosomal DNAase II to the nuclear fraction and concludes that DNAase II activity observed in isolated nuclei represents an intrinsic activity that might be involved in nuclear DNA metabolism.     

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Summary Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).