Studies on the action of the nuclear factor promoting actinomycin D-binding capacity of chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Studies on the action and chemical nature of the nuclear factor promoting degradation of chromatin DNA

Chromatin DNA of liver and kidney, obtained by the method of Dingman & Sporn, is inaccessible in 0.14 M NaCl to pancreatic DNase and cytoplasmic DNase. Under the combined action of DNase and nuclear extract (NE) (extraction with 0.14 M NaCl) on chromatin, the DNA of the latter is intensively degraded. The action of NE is tissue-specific—liver NE has almost no effect on kidney chromatin DNA degradation. The removal of protein or RNA from NE deprives it of its ability to accelerate chromatin DNA degradation by DNases. It is assumed that the active part of NE is a complex of a protein and RNA. Here, tissue specificity is determined by both components of this complex. The biological role of the nuclear factor promoting chromatin DNA degradation is not known at present.     

DNA-tropic inhibitors of nuclease action on DNA in liver nuclei

Distamycin is a potent, wide-spectrum inhibitor of the breaking of both free and intranuclear DNA with DNAse I and with own nuclear nucleases. It compares very favourably with actinomycin D, proflavin and ethidium bromide, especially in the inhibition of DNAse I action in the nuclei. This seems likely to be due to partial overlapping of the binding sites of the nuclei with chromatin proteins in contrast to distamycin that interacts with a minor furrow of DNA being blocked to a less extent by proteins. The DNA-tropic agents under test exert no qualitative effect on the kinetics of intranuclear DNA splitting by DNAse I. Carminomycin and bleomycin are the least effective inhibitors in all the systems depicted.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

7-Azidoactinomycin D: a novel probe for examining actinomycin D-DNA interactions

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

The use of intercalating dye molecules in the study of chromatin structure

This paper reviews the field of chromatin structure studied by means of dye molecules believed to intercalate within DNA. The emphasis is on dyes whose binding properties are the best characterized (ethidium bromide (EB). actinomycin D(AMD), proflavine (PF)) but studies in which less common dye molecules are used are also considered. A comparison is made between the binding of these dyes to purified DNA and to whole or partially deproteinized chromatin in order to investigate both the availability of DNA in chromatin and the localization of chromosomal proteins or the DNA backbone.     

Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster.

Specific interactions of chromatin with the nuclear envelope (NE) in early embryos of Drosophila melanogaster have been mapped and analyzed. Using fluorescence in situ hybridization, the three-dimensional positions of 42 DNA probes, primarily to chromosome 2L, have been mapped in nuclei of intact Drosophila embryos, revealing five euchromatic and two heterochromatic regions associated with the NE. These results predict that there are approximately 15 NE contacts per chromosome arm, which delimit large chromatin loops of approximately 1-2 Mb. These NE association sites do not strictly correlate with scaffold-attachment regions, heterochromatin, or binding sites of known chromatin proteins. Pairs of neighboring probes surrounding one NE association site were used to delimit the NE association site more precisely, suggesting that peripheral localization of a large stretch of chromatin is likely to result from NE association at a single discrete site. These NE interactions are not established until after telophase, by which time the nuclear envelope has reassembled around the chromosomes, and they are thus unlikely to be involved in binding of NE vesicles to chromosomes following mitosis. Analysis of positions of these probes also reveals that the interphase nucleus is strongly polarized in a Rabl configuration which, together with specific targeting to the NE or to the nuclear interior, results in each locus occupying a highly determined position within the nucleus.     

Control of in vivo protein synthesis and peroxidase formation by DNA-containing extracts of normal and crown gall tissues

Nucleic acids extracted from normal bean hypocotyl tissue (NE) and crown gall tumors (TE) affect amino acid incorporation into protein and the development of peroxidase activity when vacuum infiltrated into normal receptor tissues. TE enhances and NE inhibits both processes; NE from successively older tissues produces progressively greater inhibitions per unit of infiltrated nucleic acid. The active material has an absorption maximum at 257 nm with an A_260:280 ratio of more than 2·0. On acrylamide gel electrophoresis it shows a small DNA peak, four typical r-RNA peaks and a small low molecular weight RNA peak. Activity in such extracts is completely destroyed by hydrolysis with 0·3 N KOH or DNAase; RNAase is only slightly effective and pronase ineffective. It is deduced that the effective material contains DNA that may be complexed with RNA or other materials in the extract. Pretreatment of donor tissues with actinomycin d or 5-fluorouracil diminishes or annuls the activity of the extract. Pretreatment of receptor tissue with actinomycin d inhibits the action of TE but not of NE; pretreatment with cycloheximide prevents the action of both NE and TE.     

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.     

CHROMATIN STRUCTURE AND THE CELL DIVISION CYCLE : Actinomycin Binding in Synchronized HeLa Cells

Measurements of actinomycin-³H binding in synchronized HeLa cells reveal that the binding capacity of chromatin decreases progressively during the S phase despite a doubling of nuclear DNA content, reaches a minimal level during G₂ and mitosis, and then increases gradually throughout the subsequent G₁ interval. Since this pattern was evident in experiments with living cells, ethanol-fixed cells, and isolated nuclei, but not with purified DNA, the actinomycin binding profile may reflect changes in the degree of association between DNA and chromosomal proteins at different stages of the cell cycle.     

Increased susceptibility of activated rat liver chromatin to DNAse I

Rat liver chromatin activated by partial hepatectomy is more susceptible to the action of DNAse I than control chromatin isolated from intact liver. The study on the transfer of chromatin material to the acid-soluble fraction reveals a higher rate of activated chromatin degradation. Activated chromatin shows also an increased capacity for ethidium bromide (EB) binding as estimated from the isotherms of adsorption. The difference in EB binding between activated and control chromatin is abolished after DNAse I treatment. Conditions of mild digestion with DNAse I have been found under which the number of binding sites for EB per nucleotide decreases to almost the same level in activated and non-activated chromatin. The results suggest a preferential degradation of those DNA sequences in activated chromatin that are responsible for the increase in the ligand binding.     

Cytochemical properties of interphase chromatin condensed as a result of treatment with caffeine

Treatment of living cultured cells with caffeine (10 mg/ml, 2 h, 37 °C) brings about marked chromatin condensation which results in the appearance of small, distinct chromatin clumps in the majority of interphase nuclei. The changes taking place in the chromatin properties under the action of caffeine are rather similar to those observed in mitotic condensation (an increase in acridine orange and berberine binding and a sharp decrease in [³H]actinomycin D binding in situ; inhibition of [³H]uridine incorporation in vivo) and are reversible from the point of view of the criteria studied (nuclear morphology, ligand binding, [³H]uridine incorporation, culture viability). It is concluded that caffeine treatment can be regarded as a promising approach to the study of events occurring in chromatin condensation.     

An analysis of the binding of the chick oviduct progesterone-receptor to chromatin.

The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.     

Actinomycin D binding to DNA and chromatin: A colorimetric procedure suitable for the analysis of turbid preparations and for simultaneous processing of several samples

A simple, sensitive, and inexpensive method is presented for measuring the binding of actinomycin D (AMD) to either chromatin or DNA. The procedure gives results which are comparable with those obtained by the optical titration procedure, but is superior to that method in that it does not require continuous attention, can be used with turbid chromatin preparations, and allows simultaneous processing of several samples. Chromatin or DNA that has been allowed to react with AMD is subjected to sucrose density gradient centrifugation to remove unbound antibiotic. The AMD-DNA or AMD-chromatin complex is then dissociated by incubation with 5m urea, and the released AMD is extracted with ether and estimated spectrophotometrically.     

Ran binds to chromatin by two distinct mechanisms

Ran GTPase plays important roles in nucleocytoplasmic transport in interphase and in both spindle formation and nuclear envelope (NE) assembly during mitosis. The latter functions rely on the presence of high local concentrations of GTP-bound Ran near mitotic chromatin. RanGTP localization has been proposed to result from the association of Ran's GDP/GTP exchange factor, RCC1, with chromatin, but Ran is shown here to bind directly to chromatin in two modes, either dependent or independent of RCC1, and, where bound, to increase the affinity of chromatin for NE membranes. We propose that the Ran binding capacity of chromatin contributes to localized spindle and NE assembly.     

Effect of DNA on thyroid-hormone binding by specific receptor proteins from rat-liver nuclei

Influence of double-stranded native DNA on the binding of thyroid hormone, 3,5,3'-triiodo-L-thyronine, by the isolated nuclear receptors was studied and the following results were obtained. (1) The receptor-triiodothyronine complexes bound to DNA with moderate affinities. (2) DNA enhanced the hormone binding of the receptors. (3) The stimulatory DNA effect on triiodothyronine binding of the receptors was dependent on DNA concentration, showing its maximum at 30 microgram/ml. (4) The increase in triiodothyronine binding was observed not only in the initial velocity but also in the plateau level which was attained after sufficient incubation time. (5) There were two types of specific receptors in the rat liver nuclear extract. The dissociation constants and the maximal binding capacities for triiodothyronine, which were determined by Scatchard plot analysis in the presence and absence of DNA, suggested that DNA exerted its effect through increasing binding capacity on one class of the receptors and through enhancing affinity for the hormone on the other class of the receptors. (6) Among various polynucleotides examined, the double-stranded eukaryotic DNA was most effective in enhancing the hormone binding by the receptors. These results indicate that the nuclear thyroid hormone receptors interact with double-stranded DNA in a specific manner and are induced to bind more thyroid hormone. We interpret these results as suggesting that a ternary complex of triiodothyronine, the receptor and DNA is formed in the cell nucleus in vivo, probably representing an intrinsic step in the hormone action. Possible physiological significance of this effect of DNA on the receptors is discussed.     

Ran及其结合与调控蛋白在核膜装配过程中的调控作用

核膜在细胞周期中呈现高度的动态性：在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR（lamin B receptor）以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。