Deoxyribonucleoprotein Complex Research Articles

Studies on deoxyribonucleoprotein in leukocytes and related cells of mammals. VII. The fast green histone content of rabbit leukocytes after hypotonic treatment.

Peripheral leukocytes of rabbit were subjected to the following treatments: (1) 3 parts blood to 2 parts water; (2) 0.95% sodium citrate; (3) Ohnuki's medium. Treated and control leukocytes were then smeared, fixed in 9:1 methanol-commercial formalin and stained for histones according to Alfert and Geschwind. Cytophotometry was carried out with the Barr and Stroud integrating microdensitometer. The cells treated with water increase their dye content by 50%; this increase is less marked (30%) in the cells incubated in Ohnuki's mixture, while there is a 15% decrease in case of citrate. The spread of values "between cells" is larger than the values found with the Feulgen reaction because of factors other than chromatin compaction. (1) There are fluctuations in staining intensity in different areas of the same slide. (2) Nonhistone substrates take the dye, such as hemoglobin of erythrocytes, cytoplasm and specific granules of heterophil granulocytes. (3) Apparently, a certain amount of histones is lost when nuclei swell in hypotonic citrate. It is concluded that, although histone dye binding behaves in a manner similar to Feulgen with respect to changes in the physical state of the deoxyribonucleoprotein complex, the quantitation of fast green histone is a less reliable method for the study of these changes.

Activation of deoxyribonucleoprotein in human leucocytes stimulated by phytohemagglutinin: I. Kinetics of the binding of acridine orange to deoxyribonucleoprotein

In accordance with previous findings a pronounced and rapid increase in the fluorescence intensity of fixed human leucocytes stained with acridine orange (AO) was observed only a few minutes after stimulation with phytohemagglutinin (PHA). This increase in fluorescence intensity was ascribed to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex. The measured AO fluorescence was not caused by staining artifacts due to altered autofluorescence of leucocytes, nonspecific AO binding or changes in cell membrane permeability. On the average, the number of AO binding sites increased by a factor of 2 after PHA stimulation. In activated cells, about half of all binding sites in DNP were accessible to AO. PHA-P and PHA-M induced the same final increase in AO binding sites in DNP although the rate of increase in AO binding sites was lower when PHA-M was used. Since the same decrease in rate could be obtained by lowering the PHA-P doses, it was concluded that the difference in the activity between the two PHA fractions might be due to different contents of the stimulating factor. Leucocytes sampled from infected or recently vaccinated donors exhibited a spontaneous increase in AO binding sites in DNP, which, in most cases, could not be further increased by PHA. The increase in AO binding sites in DNP, which seems to be one prerequisite for later cell growth, is apparently due to weakened interaction between DNA and concomitant proteins. This hypothesis was supported by the finding that acetylation of amino groups in the protein moiety of the DNP complex resulted in a marked increase in AO binding sites in nonstimulated cells while no such increase was found in PHA stimulated cells having a large number of accessible groups for AO binding even without acetylation.

Studies on deoxyribonucleic acid in leukocytes and related cells of mammals. VI. The Feulgen-deoxyribonucleic acid content of rabbit leukocytes after hypotonic treatment.

Peripheral blood of rabbit was subjected to the following hypotonic treatments in addition to controls: (1) 3:2 blood or plasma-distilled water; (2) 0.2 M sucrose; (3) 0.95% sodium citrate; (4) Ohnuki's medium ( Nature (London) 208: 916. 1965). The cells were smeared, fixed in methanol-acetic-formalin and stained by means of the Feulgen reaction. Cytophotometry was carried out in the Barr and Stroud integrating microdensitometer. The results show that the cells subjected to hypotonic media increase their chromatic load, the mean values being 7-12% higher than the controls. These results reinforce previous suggestions that any change in the physical state of deoxyribonucleoprotein molecules brings concomitant changes in the reactivity of their dye-binding sites and, moreover, that this increase is correlated with (1) diffusion of the deoxyribonucleoprotein complex and (2) increase of the mean nuclear size. Therefore, under the conditions used, the more diffuse the nuclear content, the higher the apparent deoxyribonucleoprotein value.

Changes in the cytochemical properties of erythrocyte nuclei reactivated by cell fusion.

ABSTRACT When the nucleus of a chick erythrocyte is introduced into the cytoplasm of a HeLa cell it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus is associated with an increase in volume and with changes in nuclear composition. These changes have now been studied by quantitative cytochemical techniques. During the process of reactivation the dry mass of the erythrocyte nucleus shows a marked increase which takes place largely before the replication of the DNA begins. Within 24 h of cell fusion, some erythrocyte nuclei already contain an increased amount of DNA, and 48 h after fusion many of them contain twice the normal diploid amount, thus indicating that they have replicated their DNA completely. The physical properties of the nuclear deoxyribonucleoprotein complex also change. The ability of the nuclear chromatin to bind acridine orange increases 4- to 5-fold well before the synthesis of DNA begins; and changes in the melting profile of the deoxyribonucleoprotein suggest that its structure is loosened. This view is also supported by the observation that the reactivity of the erythrocyte nuclei to the Feulgen stain is altered during the early stages of reactivation.

Interaction with heparin of deoxyribonucleoprotein complex damaged in vivo by nitrogen mustards

The treatment of mice with two alkylating agents, i.e. Tris-(2-chlorethyl) amine hydrochloride and 1,6 bis(2-chlorethyl-amino) 1,6 deoxy-D-mannit dihydrochloride, leads to profound damage to desoxyribonucleoprotein complex in thymus and spleen, manifested in a distinctly increased sensitivity of DNP to the action of heparin.

Initial changes in the deoxyribonucleoprotein complexes of kangaroo lymphocytes stimulated with phytohaemagglutinin

Early changes induced in kangaroo lymphocytes by PHA have been studied by alkaline fast green microspectrophotometry and dry mass measurement. In lymphocytes exposed to PHA, there is a 20–25 per cent decrease in stainability with alkaline fast green, an initial decrease in nuclear dry mass, and an increase in cytoplasmic dry mass. These findings support the hypothesis that PHA induces blastogenesis in lymphocytes through the removal of histone from the DNP complex.

The Isolation, Properties, and Possible Functions of Chromatin Acidic Proteins

Abstract The chromatin acidic proteins have been isolated from rat liver by dissociation of the deoxyribonucleoprotein complex. These proteins are freely soluble in buffer solutions, are heterogeneous, contain deoxyribonucleic and ribonucleic acids, and are separable into four main fractions. The isolated chromatin acidic proteins form complexes with whole histone as well as with DNA. When they are incubated in vitro with 14C-tryptophan, the isotopic label is incorporated into the protein, especially in the presence of DNA.

Post-irradiation Changes in the Deoxyribonucleoprotein Complex in Animal Tissues as revealed by the Action of Heparin

IT has been shown1–6 that “labilization” of the bond between DNA and protein occurs in certain tissues after irradiation, which manifests itself in the release of deoxyribopolynucleotides, easier deproteinization, and an increase in the solubility of the deoxyribonucleoprotein (DNP) complex in solutions of a high ionic strength. Little is known, however, of either the mechanism or the nature of these changes, or of their biological significance. In order to ascertain whether there is any change in the strength of the bond between DNA and protein after irradiation, we have investigated the interaction of heparin with the DNP complex of various tissues of normal and irradiated animals.

ATYPICAL CHANGES OF DEOXYRIBONUCLEOPROTEIN DURING SPERMIOGENESIS ASSOCIATED WITH A CASE OF INFERTILITY IN THE BULL

we reported that, normally during spermiogenesis in the bull, several pro¬ nounced cytochemical changes occur in the deoxyribonucleoprotein (dnp) complex. Summarily, these changes are: a decrease in number of phos¬ phate groups (DNA-PO4) available for binding by cationic dyes, an increase in total basicity of nuclear protein, an increase in protein-bound arginine and a reduction in stainability of dna by the Feulgen reaction. These changes

Metabolic integrity of deoxyribonucleohistones

To study the relative turnover rates of histones and DNA in various tissues, rats were simultaneously injected intraperitoneally with [5-125I]iodo-2′-deoxy-uridine to label the DNA and L[U-14C]lysine to label the histones. At various times after administration, animals were killed and lysine-rich and other histones as well as DNA from various tissues isolated. After determining the specific activities (cts/min/mg) of isolated materials, turnover rates were calculated from the first-order decay curves plotted by the method of least squares. The ratios t½ (days) lysine-rich histones/t½ DNA as well as the ratios t½ “other histones”/t½ DNA were found to be close to unity for various rapidly growing rat tissues and tumors. Long-term experiments with liver showed that neither ratio deviated largely from unity. These data indicate that histones, or at least the bulk of histones, and DNA turn over at approximately the same rates, which would appear to be evidence for metabolic integrity of the deoxyribonucleo-protein complex.

The increased sensitivity of the deoxyribonucleoprotein complex of irradiated tissues to the action of heparin.

The increased sensitivity of the deoxyribonucleoprotein complex of irradiated tissues to the action of heparin.

Properties of a Deoxyribonucleoprotein Complex Derived from Bacillus subtilis*

Properties of a Deoxyribonucleoprotein Complex Derived from Bacillus subtilis^*

Nuclear Proteins of Neoplastic Cells

Publisher Summary Proteins and enzymes of the nucleus are important for the synthetic reactions involved in neoplastic cells and to the aberrations of growth that characterize these cells. It has been difficult to isolate and purify the proteins of the nucleus but in recent years, the enzymatic activities of some of these proteins have been elucidated. In studies on the nuclear proteins, it is important that the nuclei be isolated in such a manner that the nuclear proteins remain in the nucleus, and the proteins of the cytoplasm do not enter the nucleus or contaminate the preparation. The high rate of biosynthesis of both histones and acidic nuclear proteins in the growing tissues and neoplastic cells are associated with rapid biosynthesis of DNA. However, even in resting cells, there is biosynthesis of all nuclear proteins. Because the possibility exists that the individual substructures of the nucleus of tumor cells contain proteins important both to their structural and functional characteristics, it is essential to consider the kinds of structures from which the nuclear proteins may originate and the methodology for their isolation. The criteria generally used to evaluate the purity of nuclei are that the nuclei should be anatomically identical with those of the whole cell, the contents of the nuclei as they exist in the cell should all be present in the isolated product, and the isolated nuclei should not contain cytoplasmic constituents. The chapter describes the techniques for isolation of nuclei and isolation of nuclear components. Further, the enzymes of the nucleus, the acidic nuclear proteins, nuclear globulins, the nuclear ribonucleoproteins, acidic proteins of the deoxyribonucleoprotein complex, and the histones have also been discussed in the chapter.

Thymus deoxyribonucleoprotein I. Preparation and thermal denaturation

Preparations of undissociated deoxyribonucleoprotein having a reproducible composition were obtained from calf-thymus glands essentially by extraction with water. Spectrophotometric titration and other ultraviolet-absorption measurements showed that this deoxyribonucleoprotein was denatured by heat and by alkali in both 0.001 M and 1.0 M sodium chloride in a manner similar to DNA. This similarity confirms that the double-helical structure of DNA exists within the original undissociated deoxyribonucleoprotein complex in solutions.

Thymus deoxyribonucleoprotein I. Preparation and thermal denaturation

Preparations of undissociated deoxyribonucleoprotein having a reproducible composition were obtained from calf-thymus glands essentially by extraction with water. Spectrophotometric titration and other ultraviolet-absorption measurements showed that this deoxyribonucleoprotein was denatured by heat and by alkali in both 0.001 M and 1.0 M sodium chloride in a manner similar to DNA. This similarity confirms that the double-helical structure of DNA exists within the original undissociated deoxyribonucleoprotein complex in solutions.