Deoxyribonucleoprotein Complex Research Articles

Protein and Messenger RNA Requirements for Superhelicity of Polyoma Virus DNA

WHEN cells productively infected by polyoma virus are exposed to brief treatment with cycloheximide1 or puromycin2, a viral DNA component is formed which sediments slightly more slowly than 20S superhelical viral DNA (form I) at neutral pH. Bourgaux and Bourgaux-Ramoisy2 have obtained evidence that this viral DNA species is essentially free of protein, in conditions in which form I DNA is extracted as a deoxyribo-nucleoprotein complex and that it consists of covalently closed circular molecules deficient in superhelical turns. I wish to report that I have confirmed that a closed circular DNA species of lower superhelix density than form I viral DNA is synthesized in infected cells treated with cycloheximide. This is shown by alkaline sedimentation and equilibrium centrifugation in CsCl-propidium diiodide (CsCl-PDI) of DNA isolated by sedimentation in neutral sucrose. A stoichiometric relationship exists between the formation of form I polyoma DNA and the synthesis of protein; the protein requirement is maintained, however, by messenger RNA (mRNA) which is stable for at least 5 h. To account for these findings, a post-transcriptional mechanism for the control of viral DNA synthesis is considered.

Nucleoprotein changes and uridine incorporation in rat thymocytes: II. Effect of treatment with anti-thymocyte serum in vitro

Treatment of rat thymus lymphocytes with antithymocyte serum and gamma globulins from such serum reduced the rate of uridine incorporation in these cells. The reduction of uridine incorporation in small lymphocytes was paralleled by a reduced capacity of nuclear deoxyribonucleoprotein to bind tritium labelled actinomycin D, suggesting that the reduced uridine incorporation is due to modifications of the nuclear deoxyribonucleoprotein complex. These modifications may be triggered by changes in the cell membrane caused by the binding of antibodies to membrane components.

Cytochemical properties of nuclear chromatin as demonstrated by the colloidal iron binding technique

The colloidal iron (CI)-binding method can be used for the study of the cytochemical properties of nuclear chromatin in differentiated human lymphocytes. Growth stimulation of lymphocytes, either with phytohemagglutinin or by cell-crowding, resulted in a rapid increase of CI-binding per unit amount of DNA which is likely to reflect a change of the deoxyribonucleoprotein (DNP) complex of importance for gene activation. A similar increase of the CI-binding capacity of the DNP-complex was found to be induced in non-proliferating cells pretreated in solutions of increased alkalinity or increased ionic strength and after blocking the amino groups in histones with acetic acid anhydride. Increased CI-binding was therefore interpreted as reflecting an increased electronegativity of the DNP complex due to a decreased interaction between DNA and histones. Evidence was also obtained that conformational changes of the DNP complex played an important role in the CI-binding reaction.

The role of nuclear proteins in RNA synthesis

When epithelial cells from baby mice are cultured on a solid substrate in vitro, DNA synthesis and cell division take place in a majority of the cells 24 to 48 h after inoculation. This cell proliferation is regularly preceded by a sequence of nuclear changes likely to be involved in gene activation during the process of growth-transformation. These nuclear changes, which in the present investigation were studied by quantitative cytochemical methods, are characterized by: (1) increased binding of acridine orange (AO) very soon after cell attachment, reflecting initial changes of the deoxyribonucleoprotein (DNP) complex; (2) accumulation of proteins in the cell nucleus; (3) dispersion of the nuclear chromatin; (4) increased rate of 14C-uridine incorporation. The initial DNP change, reflected by the increased AO-binding, was per se not a sufficient factor for stimulation of 14C-uridine incorporation. An increased 3H-uridine incorporation was only observed when protein began to accumulate in the nucleus and when the chromatin changed from a condensed to a more dispersed state. The rate of 14C-uridine incorporation was, during the whole process of growth transformation, found to be directly proportional to the amount of protein that had accumulated in the nucleus. However, the initial DNP change was a necessary prerequisite for the subsequent accumulation of protein in the nucleus. Attachment of the cells to the solid substrate was found to be necessary in expressing this sequence of nuclear events. Furthermore, in areas on the glass slide where many cells had attached, these nuclear changes occurred more rapidly.

Further studies on the nuclear chromatin of morphologically abnormal bull spermatozoa.

are commonly found in ejaculates from bulls with certain types of reduced fertility. Intranuclear spaces and a granular appearance of the nuclear chromatin were the major abnormalities observed in a high percentage of spermatozoa from two infertile bulls (McCosker, 1969). The organization of the DNA and nuclear proteins forming the deoxyribonucleoprotein complex (DNP) in the chromatin of deformed sperm heads has received little attention. Previous studies have demonstrated that most morphologically abnormal sperm heads contain the same amounts of DNA as their normally shaped counterparts (Gledhill, 1966a). Defects in the binding of nuclear protein to DNA, which probably occur during the process of chromatin aggregation in spermateliosis, have only recently been incriminated in the formation of certain misshapen sperm heads (Gledhill, Dar\l=z%o\ynkiewicz & Ringertz, 1971). Their possible r\l=o^\lein an instance of infertility was noted earlier (Gledhill, Gledhill, Rigler & Ringertz, 1966a). The present communication reports a study which had as its objective to determine if the DNA of abnormally shaped spermatozoa has a sensitivity to thermal denaturation that differs from that of normally shaped spermatozoa. The method used allows the analysis of molecular arrangements of DNA within intact, definable sperm heads. Such information has physiological importance as it relates to current work on the maturation ofnuclear chromatin and could show mechanisms which might be involved in the differentiation of abnormal sperm heads. Nine sexually mature and normally fertile dairy and beef bulls of varying ages (H to 15 years) and breeds were used. Spermatozoa from the testis and from the caput epididymidis were obtained in a manner previously described (Gledhill, 1966b). Spermatozoa were also obtained from the left vas deferens of two of the bulls (courtesy of Dr R. P. Amann, The Pennsylvania State Univers¬ ity) at 1 and 3 days after cannulation by a modification of the method of Amann, Hokanson & Almquist, (1963) and Sexton, Amann & Flipse (1971). Ejaculated spermatozoa were collected with the aid of an artificial vagina. The criteria used for differentiating morphologically abnormal from normal

Cytochemical characterization of deoxyribonucleoprotein by UV-microspectrophotometry on heat denatured cell nuclei

Denaturation of double-stranded DNA into a single-stranded state can be studied by heating fixed cells attached to quartz slides and then determining the increase in nuclear UV-absorption at 265 nm by microspectrophotometry at room temperature. In order to prevent renaturation as the slides are cooled, formaldehyde is added to the solution in which heat denaturation is performed. The influence of formaldehyde concentration, duration of heating and ionic strength on the stability of DNA to heat denaturation has been examined. The standard method involves heating of ethanol/acetone fixed cells to temperatures between 22°C and 100°C in 0.15 M NaCl, 0.015 M sodium citrate containing 4% formaldehyde for 20 min followed by cooling to room temperature and mounting in glycerol. Different cell types examined by the cytochemical method show markedly different heat denaturation curves. These differences appear to reflect differences in DNA-protein interaction in the deoxyribonucleoprotein complexes of nuclear chromatin. DNA in nuclei which are completely repressed and which have a tightly condensed chromatin, e.g. sperm heads and hen erythrocyte nucleim show greater stability to heat denaturation than does DNA in more active cell nuclei. The thermal stability of DNA in rat thymocyte nuclei, the most active cell nuclei examined, is as low as that of calf thymus DNA in solution. Hen erythrocyte nuclei show a two-step transition from the double-stranded to the single-stranded state. The first step occurs at a temperature which is slightly lower than that of isolated DNA, whereas the second step occurs at a higher temperature. Bull spermatozoa show a single-step transition and a T m value which is higher than that for bovine DNA. In all cell types examined, 85–100% of the DNA in the fixed cell nucleus appears to be in the double-stranded form.

Cytochemical evidence of nucleoprotein changes in rat adrenal cortex following hypophysectomy or dexamethasone suppression

Changes in the characteristics of nuclear chromatin of rat adrenocortical cells were studied following withdrawal of ACTH stimulation by hypophysectomy or by dexamethasone suppression. The binding of acridine orange to individual cell nuclei was measured by microfluorimetry. Both hypophysectomy and dexamethasone suppression reduced the binding of acridine orange to nuclear chromatin to 40–70% of the controls. Simultaneously, the thermal stability of the deoxyribonucleoprotein complex increased significantly. The chromatin changes observed seem to reflect the situation in vivo, bearing relation to the functional activity of the deoxyribonucleoprotein complex.

Influence of cell density on the acridine orange binding to deoxyribonucleoprotein complex in leucocytes from patients with infectious mononucleosis and acute leukaemia.

The acridine orange (AO) binding to the deoxyribonucleoprotein (DNP) complex of individual normal lymphocytes, infectious mononucleosis (IM) lymphoid cells and leukaemic blast cells was determined by microfluorometry. Increased AO binding to DNP was found with increasing cell density on the slide and was similar in the different cell populations. staining Only in exceptional cases of IM, the AO binding was high irrespective of cell density. The increased AO binding was due to changes in the DNP complex, which might be induced by the release of macromolecular substances from the cells. The results show that in clinical cytological studies, the fluorescence intensities cannot be used for the discrimination between normal and malignant cells.

Chromatin changes during erythropoiesis

During erythroid maturation in the chick the nuclear chromatin undergoes marked condensation and ceases to act as a template for RNA synthesis. In an attempt to examine the molecular basis for these phenomena, the deoxyribonucleoprotein (DNP) complexes of nuclear chromatin were examined by quantitative cytochemical methods at various stages of erythropoiesis. With increasing degree of maturation and nuclear condensation, the DNP showed a progressively lower capacity to bind the basic dye acridine orange. The DNA component of the chromatin became more stable to acid hydrolysis and thermal denaturation. No change could be detected in the capacity of the chromatin to bind 14C-actinomycin D. The DNP modifications observed are discussed in relation to possible mechanisms of chromosome condensation and genome repression.

The use of cell fusion in the analysis of gene action.

When, under the influence of inactivated Sendai virus, the nucleus of a mature hen erythrocyte is introduced into the cytoplasm of a human or mouse tissue culture cell, it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus in the heterokaryon is associated with a marked increase in its volume. There is a direct relation between the volume of the nucleus and the amount of RNA that it makes. The nuclear enlargement is not the consequence of increased RNA synthesis, or of DNA synthesis: enlargement is the primary event, and the increase in RNA synthesis is determined by it. During the process of reactivation, the erythrocyte nucleus shows a five- to six-fold increase in dry mass which takes place largely before the replication of DNA begins. This increase is due to the passage of cytoplasmic proteins into the erythrocyte nucleus. The physical properties of the deoxyribonucleoprotein complex in the erythrocyte nucleus change as the nucleus enlarges. The ability of the nuclear chromatin to bind acridine orange and other intercalating dyes increases four- to fivefold; and changes in the melting profile of the deoxyribonucleoprotein indicate that its structure is loosened. It appears that, as the nucleus expands, more of the chromatin passes from a condensed to a dispersed state and more of it is transcribed. At the concentrations used to induce cell fusion, Sendai virus is haemolytic and rapidly lyses the nucleated erythrocytes. Fusion then takes place between the other cells in the combination and erythrocyte ghosts. The erythrocyte nucleus is thus introduced into the cytoplasm of the recipient cell without any appreciable contribution of erythrocyte cytoplasm. The reactivation of the hen erythrocyte nucleus is therefore achieved by signals emanating from human or mouse cytoplasm.

On the presence of histones in Euglena gracilis var. bacillaris

Euglena gracilis cells stained with alkaline Fast Green (FCF) show strong nuclear, but weak cytoplasmic reaction. The basic proteins of the deoxyribonucleoprotein complex of Euglena gracilis were extracted, and characterized by amino acid analysis and by polyacrylamide gel electrophoresis. Amino acid composition of these proteins indicates lysine-rich nature and a close similarity to those of histones from plant, protozoa and calf thymus. However, some differences with calf thymus histones, such as lower arginine content and slightly higher amounts of proline and alanine, are observed. It is concluded that the basic proteins associated with nuclear DNA of Euglena gracilis are histones.

Changes in binding between DNA and arginine residues in histone induced by cell crowding

Earlier reported alterations of the cytochemical properties of nuclear chromatin correlated with growth stimulation were further investigated with respect to changes in the DNA histone interaction. The alkaline bromphenol blue (BPB) binding reaction was quantitated by microspectrophotometry after DNA extraction either with trichloroacetic acid (TCA) or picric acid (PA). This method was used to differentiate between DNA bound arginine and lysine residues in histone. The BPB binding after TCA hydrolysis reflects both arginine and lysine residues in histone bound to DNA, while the BPB binding after PA hydrolysis mainly reflects the lysine residues. The accessibility of the phosphate groups in the deoxyribonucleoprotein (DNP) complex to basic dyes was studied by acridine orange (AO) microspectrofluorimetry. Gradual changes in the DNP complex of human leukocytes on slide cultures can be induced by cell crowding. This biological system was used in the present work to study the interaction between DNA and histone. The results showed that a gradual increase in AO binding to phosphate groups in DNA was accompanied by a gradual decrease in BPB binding to histone after TCA hydrolysis. After PA hydrolysis, however, the BPB binding to histone remained unchanged. These data indicate that changes in the binding between arginine residues in histone and phosphate groups in DNA play an important role in the earlier reported changes in the cytochemical properties of the DNP complex. This indication was further supported by microspectrophotometric measurements of the PA binding capacity of the cell, which also reflects the arginine residues in histone bound to DNA. The observed changes in binding between arginine residues and phosphate groups in DNP may be of importance for the regulation of the genome function and for the growth activity of the cell.

UV-irradiation induced changes in the uptake of acridine orange in living mouse fibroblasts in vitro

Earlier microspectrofluorimetric investigations showed that the capacity of nuclear chromatin in binding acridine orange (AO) was correlated to the functional state of the cells. The variation in the degree of AO binding was shown to reflect structural changes in the deoxyribonucleoprotein (DNP) complex which might be of importance for the regulation of genome activity. These studies have exclusively been performed on cells after fixation. The purpose of the present work was to evaluate the possibility of characterizing the nucleoprotein complex in individual living cells with the AO technique. The UV irradiation used for excitation of fluorescence was then found to strongly influence the uptake of AO in living mouse fibroblasts and was studied in more detail. An increased cellular uptake of AO was found during continuous irradiation. This seemed to be a direct irradiation effect rather than a photodynamic effect of AO. Most of the intracellular AO was bound to nucleoproteins. The augmented AO binding might be due to increased permeability of cell membranes induced by irradiation and/or to labilization of the nucleoprotein complex resulting in liberation of AO binding sites.

Structural Changes in the Deoxyribonucleoprotein (DNP) Complex of Leukocytes from Patients with Infectious Mononucleosis

Abstract Quantitative microfluorimetric measurements on individual acridine orange (AO) stained cells showed that leukocytes from patients with infectious mononucleosis (IM) exhibited considerably greater AO binding to deoxyribonucleoprotein (DNP) than leukocytes from healthy persons. The higher binding of AO could not be explained by an increase in the amount of DNP in IM cells but by an increased number of binding sites in DNP accessible to AO. This was the result of structural changes in the DNP complex of the IM cells, possibly due to weakened bonds between the DNA and protein moieties of DNP. The binding of AO to IM leukocytes was highest in the early phase of the disease and usually normalized after recovery. However, an increased AO binding of leukocytes from patients after recovery was frequently observed when these cells were exposed to plasma from patients in the acute phase of IM.

Rubella infection of synovial cells and the resistance of cells derived from patients with rheumatoid arthritis.

The mechanism of growth stimulation in allogeneic lymphocytes mixed in vitro was studied at the cell level by means of cytophotometric techniques. A pronounced increase in fluorescence intensity of fixed and acridine orange (AO) stained lymphocytes was observed as soon as after 1-3 hr in mixed culture. No increase in the amount of DNA took place during this time. The higher fluorescence intensity was due to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex, most probably as a result of weakened bonds between the DNA and the protein moiety in the DNP complex. Similar DNP changes have been found in other systems of growth stimulation and may be one prerequisite for later induction of cellular synthetic processes. Increased AO binding only occurred when the lymphocyte donors were incompatible at the major histocompatibility locus (HL-A); there was no change in AO binding in cases of HL-A identity. The AO binding reaction probably reflects a specific recognition of HL-A antigens, whereas other antigenic discrepancies between the individuals do not seem to cause an analogous response.

Rapid lymphocytic recognition of histoincompatibility. Changes of the deoxyribonucleoprotein complex in mixed cultures of allogeneic human lymphocytes.

The mechanism of growth stimulation in allogeneic lymphocytes mixed in vitro was studied at the cell level by means of cytophotometric techniques. A pronounced increase in fluorescence intensity of fixed and acridine orange (AO) stained lymphocytes was observed as soon as after 1-3 hr in mixed culture. No increase in the amount of DNA took place during this time. The higher fluorescence intensity was due to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex, most probably as a result of weakened bonds between the DNA and the protein moiety in the DNP complex. Similar DNP changes have been found in other systems of growth stimulation and may be one prerequisite for later induction of cellular synthetic processes. Increased AO binding only occurred when the lymphocyte donors were incompatible at the major histocompatibility locus (HL-A); there was no change in AO binding in cases of HL-A identity. The AO binding reaction probably reflects a specific recognition of HL-A antigens, whereas other antigenic discrepancies between the individuals do not seem to cause an analogous response.

Studies on the yeast nucleus: III. Properties of a deoxyribonucleoprotein complex derived from yeast

1. A deoxyribonucleoprotein complex was isolated from Saccharomyces cerevisiae. It is composed of 36% DNA, 4% RNA and 60% protein. About 70% of the protein is acid-extractable. The complex sediments as a single band with a s°20,w of 27 S.2. The yeast deoxyribonucleoprotein shows a biphasic melting profile. About 50% of the deoxyribonucleoprotein melts at 71° which is close to the Tm of DNA (68°) and 50% melts at 83°. When protein, mostly histone, was dissociated from the deoxyribonucleoprotein by extraction with NaCl of increasing concentrations, the percentage of deoxyribonucleoprotein melting at 83° decreases gradually, while the Tm of the remainder shifts to that of DNA. After dissociation of deoxyribonucleoprotein at 1 M NaCl, the melting profile is identical to that of yeast DNA.3. The template activity for RNA synthesis of yeast deoxyribonucleoprotein was compared to that of yeast DNA using Escherichia coli RNA polymerase. The template activity is dependent on the concentration ratio of enzyme to template. A maximum template activity of 60% was observed for yeast deoxyribonucleoprotein at high concentration ratios of enzyme to template.4. It is concluded that yeast deoxyribonucleoprotein resembles the deoxyribonucleoprotein of higher organisms in several aspects.

Changes in deoxyribonucleoprotein during spermiogenesis in the bull: 3H-actinomycin D binding capacity

The kinetics of 3H-actinomycin D (AMD) binding to the nuclear deoxyribonucleoprotein (DNP) of male germ cells were studied by means of quantitative autoradiography. A progressive decrease in the ability of DNP to bind 3H-AMD occurred as the nucleus matured and condensed during spermiogenesis. The physical and chemical changes known to occur in the DNP complex at this time are thought to be responsible for this reduction in binding rate.

Nucleoprotein changes and initiation of RNA synthesis in PHA stimulated lymphocytes

Stimulation of human lymphocytes with phytohaemagglutinin in vitro induces marked changes in the properties of the nuclear deoxyribonucleoprotein complexes. These changes manifest themselves in an increased capacity to bind acridine orange, acetylation of histories and an altered affinity for actinomycin D and can be observed before RNA synthesis is initiated. An increased capacity to bind acridine orange is observed in all cells but only part of the lymphocyte population appears to acetylate histones and to show an enhanced actinomycin binding. Double emulsion autoradiography after labelling with both 14C-acetate and 3H-uridine suggests that it is those cells which acetylate histone which will also initiate RNA synthesis. It is suggested that the phenomena studied mainly reflect different stages of dissociation of the nucleoprotein complex and are part of a multistep preparatory mechanism for initiation of transcription.

Early changes in the binding between DNA and histone in human leucocytes exposed to phytohemagglutinin

Previously reported changes in the deoxyribonucleoprotein (DNP) complex of human leucocytes responding to phytohemagglutinin (PHA) were further investigated in the present work by using the alkaline bromphenol blue (BPB) binding reaction for basic proteins. The extraction of DNA preceding the BPB-binding was performed both in trichloroacetic acid (TCA) and in picric acid (PA). TCA liberates both arginine and lysine residues for the subsequent BPB-binding. PA, which was found to bind strongly to the guanidino-groups of arginine, mainly liberates the lysine residues for the following BPB-binding. By measuring the amounts of PA and BPB bound after PA-hydrolysis and BPB staining, information was obtained about the relative amounts of arginine and lysine in histone bound to DNA. In PHA-treated cells a 25% decrease in BPB-binding was observed after TCA-hydrolysis. No such decrease in BPB-binding could be demonstrated after PA-hydrolysis. However, as much as an 80% decrease was found in the PA-binding capacity. These results were interpreted as a reduced number of arginine residues in histone bound to the DNA-phosphate groups in cells exposed to PHA.