Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei]

The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.     

Uptake of glycine-N15 by components of cell nuclei

1. The uptake of glycine-N¹⁵ by components of cell nuclei was studied. The nuclear components were derived both from tissues with high metabolic rates-mammalian liver, kidney, and pancreas-and from cells with relatively low rates of metabolism-avian erythrocytes and echinoderm sperm. N¹⁵ uptake by nuclear components of liver, kidney, and pancreas was far more rapid than by those of erythrocytes and sperm. 2. The nuclear components of liver, kidney, and pancreas for which measurements were made were DNA, histone, and residual protein of chromatin. Uptake into DNA was low, into histone higher, and into residual protein much higher still, being comparable with that into mixed cytoplasmic protein. 3. A comparison of the uptake of N¹⁵ by the chromosomal components, histone and DNA of liver, pancreas, and kidney showed that chromosomal "activity" varies in different cells and also in the same cell depending upon its over-all activity.     

Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

The binding of [6,7-(3)H]oestradiol-17beta to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.     

Comparison of the binding of anthracycline derivatives to purified DNA and to cell nuclei

Fluorescence method was used to study the interactions of anthracyclines with purified DNA and with cell nuclei at 37 degrees C, at pH ranging from 6.8 to 8. Four anthracyclines were used; adriamycin (ADR), 4'-o-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR) and aclacinomycin (ACM). The values of pKa of deprotonation of these four drugs in the pH range 6.5-8.5 are 8.4, 7.7, 8.4 and 7.0 for ADR, THP-ADR, DNR and ACM, respectively. The overall binding constants K* of these four drugs to purified DNA was determined at various pH values. The binding constants K0 and K+ of the respectively neutral form and once protonated form of the drugs to DNA were calculated. Using cell nuclei from K562 cells, the amount of drug intercalated (CN) within the nuclei of K562 cells and the amount of free drug (CE) in the solution were determined at various pH values: measuring at the same pH values, a linear correlation occurred between K* and CN/CE.     

Uptake and translocation of griseofulvin by wheat seedlings

Summary 1. A roughly quantitative technique for studying uptake and translocation of the antibiotic griseofulvin by wheat plants has been devised. Wheat plants were grown in nutrient solutions containing griseofulvin and translocation measured by bioassay of the griseofulvin appearing in the guttation drops induced by transfer to a humid atmosphere.2. Griseofulvin was phytotoxic at concentrations of 5 µg/ml and above, the first symptoms observed being stunting and swelling of the roots.3. The concentration of griseofulvin in the guttation drops was directly related to the concentration in the nutrient solution; there was evidence of griseofulvin accumulation in the leaves, the concentration in the guttation drops being frequently higher than that in the nutrient solution.4. Atmospheric conditions favouring transpiration increased uptake and translocation of griseofulvin.5. Uptake and translocation of griseofulvin was inhibited by inclusion of respiratory enzyme inhibitors in the nutrient solution.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.     

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells

Cytoplasmic extracts, prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain an extractable factor capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not detectable in resting cells. It is nondialyzable, precipitable by 30-50% saturated ammonium sulfate, and inactivated by trypsin. It is heat sensitive, but stable to cold and lyophilization. The molecular weight of the factor is greater than 100,000. This cytoplasmic activator of nuclear DNA replication is not released from the cell, and has no effect on intact cells. This suggests that it serves as an intracellular mitogenic signal in replicating cells.     

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.     

Functional and structural characteristics of polyribosomes associated with chick embryo cell nuclei

Polyribosomes bound to the outer nuclear membrane was isolated from purified preparations of chicken embryo cell nuclei. These polyribosomes were shown to consist fractions forming unstable complexes with the nuclear membrane which can be separated from the latter by treatment with high ionic strength buffer solutions. Using sedimentation and gradient density analyses, the nuclei-bound RNP complexes were shown to be predominantly composed of 80S monosomes which take an active part in collagen polypeptide synthesis in cell-free protein-synthesizing systems. A comparison of sedimentation properties and collagen-synthesizing activity of nuclei-bound polyribosomes and cytoplasmic polyribosomes forming unstable complexes with endoplasmic membranes, it was concluded that the nuclei-bound 80S monosomes are an early step in the formation of cytoplasmic polyribosomes.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.