Differential effects of cyclic adenosine-3',5'-monophosphate on phosphorylation of rat liver nuclear acidic proteins

The effects of cyclic AMP on the phosphorylation of different acidic proteins of rat liver nuclei were examined in vivo and in vitro. N⁶,O²′-dibutyryl cyclic AMP selectively stimulated in vivo phosphorylation of specific nuclear proteins more than twofold within 15 min after injection. Cyclic AMP caused only a small stimulation of phosphorylation of acidic proteins in isolated nuclei but the stimulation was selective for specific proteins. When isolated nuclear acidic proteins were incubated with a soluble cyclic AMP-dependent protein kinase, the cyclic nucleotide stimulated total phosphorylation about 1.7-fold. These results support the view that the regulatory effects of cyclic AMP may involve phosphorylation of acidic proteins associated with DNA in the chromatin.     

Effect of nickel(II) on DNA-protein interactions

Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using³H-thymidine and³⁵S-methionine, respectively. Nuclei were exposed to NiCl₂ in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37°C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (³H) retained on each filter was normalized to protein (³⁵S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl₂ resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl₂ inhibits specific DNA-protein interactions.     

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts: Fate of Single-stranded Bacteriophage fd DNA

Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using ³H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.     

RADIOAUTOGRAPHIC LOCALIZATION OF DEOXYTHYMIDINE TRIPHOSPHATE IN TRADESCANTIA POLLEN GRAINS DURING DNA SYNTHESIS

Tradescantia pollen grains, isolated during the period of DNA synthesis in the generative cell, accumulate deoxythymidine triphosphate (dTTP)-³H after incubation with thymidine-³H in the presence of millimolar deoxyadenosine. Most of this dTTP-³H was found to resist extraction by the fixative, cold ethanol-acetic acid, and its location was investigated by radioautography with thin, dry emulsion. Substantial binding of dTTP-³H occurred as an artifact; but when nuclei were isolated from the fixed pollen grains by sonication, it was found that they were differentially labeled: generative nuclei contained dTTP-³H, vegetative nuclei did not. This observation is discussed and is interpreted as evidence supporting the idea that thymidine is phosphorylated only in the generative cell of the pollen grain.     

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [³H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg²⁺ and was stimulated by monovalent ions such as K⁺. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1^st to 4^th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.     

Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity

A large sedimentation device was developed that allows separation of 5 × 10⁸ rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%.The in vivo binding of the liver carcinogen [³H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6–5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3–3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene.DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6–2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.     

The DNA content of sperm and hemocyte nuclei of the silkworm,Bombyx mori L.

To estimate the size of the haploid genome of the silkworm, Bombyx mori (Lepidoptera), amounts of Feulgen-DNA staining in individual nuclei of primary spermatocytes, spermatids, maturing sperm, and larval or pupal hemocytes were determined with an integrating microdensitometer and compared with the Feulgen-DNA levels found for chicken erythrocyte nuclei, or the sperm and erythrocyte nuclei of Xenopus laevis that were included with each Bombyx preparation as empirical reference standards of 2.5, 3.15, and 6.3×10^?12 g DNA per cell, respectively. Under these conditions, the haploid male genome of B. mori was estimated as 0.52±0.01 (S.E.)×10^?12 g DNA, corresponding to a molecular weight of roughly 3.1×10¹¹ daltons. From similar measurements of Feulgen-stained hemocyte nuclei, approximately 1.0±0.05 (S.E.)×10^?12 g DNA was estimated for the diploid or 2C male genome of Bombyx. These values compare favorably with estimates of genome size based upon analysis of the kinetics of reassociation of DNA isolated from B. mori and provide an independent basis for assessing the degree of polyploidy achieved by the giant nuclei in the posterior silk gland prior to its secretion of fibroin at the end of the fifth larval instar.     

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of ³H-TTP into DNA.     

Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 Å and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [³H]dexamethasone in vivo or in vitro (reconstitution experiments with [³H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30–36 Å and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 × g homogenate supernatant to low ionic strenght during preparation of cytosol resulted in conversion of the 61 Å to a 36 Å complex very similar to the intranuclear form of dexamethasone receptor. 61 → 36 Å complex-verting activity was present in both the 100 × g ?10 000 × g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 Å dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 Å. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 Å and 36 Å dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 Å complex revealed that this form had been converted to the 30–36 Å complex.Further digestion of teh 61 and 36 Å [³H]dexamethasone-receptor complexes with hypotonic extract of the 1000 × g ?10 000 × g sediment of liver homogenate or with trypsin resulted in formation of a third complex with a Stokes radius of 19 Å and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 Å dexamethasone-receptor complexes were calculated as 102 000, 46 00 and 19 000, respectively, and the frictional ratios of the molecules as 1. 84, 1. 38 amd 1.00, respectively.It is concluded that the nuclear 30–36 Å dexamethasone-receptor complex is formed from the cytosol 61 Å complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [³H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [³H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

Migration of newly-synthesized RNA during mitosis. IV. Content of labelled RNA in nuclei before and after mitosis in Amoeba proteus

Amoeba proteus were incubated with ³H-uracil for 3 h. Thereupon RNA synthesis was blocked by actinomycin D and the population separated into dividing and non-dividing cells. Nuclei were isolated from cells of both groups and their RNA radioactivity was measured by means of autoradiography. The amount of label in the nuclei of non-dividing (interphase) cells was found to be equal to the sum of labels in both nuclei of daughter cells shortly after division. It is concluded that labelled RNA leaves the nucleus at the onset of mitosis and returns to the nuclei of daughter cells immediately after its termination.     

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI¹, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI¹ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI¹ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI¹ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites of 17β-estradiol

The interactions in vitro between [³H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing ³H-labeled steroid-protein complexes or with serum protein-bound [³H]estradiol. Nuclei preferentially accumulated ³H-labelea steroids from liver cytosol protein-steroid complexes relative to [³H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[³H]estradiol and the ³H-labeled water-soluble metabolite were retained in vitro by nuclei.     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.