Nils R. Ringertz

Changes in deoxyribonucleoprotein during spermiogenesis in the bull

Summary The changes in deoxyribonucleoprotein during spermiogenesis in the bull were studied by the use of quantitative cytochemical methods. o 1. Feulgen reactivity was found to decrease considerably when elongated spermatids differentiated into testicular spermatozoa although the DNA content of these cells was unchanged as judged by 2650 A absorption measurements. 2. As early as the transformation of round spermatids into elongated spermatids, a decrease in the total number of DNP-PO 4 − groups available for binding by basic dyes was found. This was reflected by the decreased ability of DNP to bind acridine orange or methyl green. This decrease was most pronounced when elongated spermatids underwent metamorphosis into testicular spermatozoa. 3. Concomitant with the decrease in Feulgen reactivity and the decrease in total number of available DNP-PO 4 − groups, a marked increase in the basicity of nuclear protein as measured by the alkaline bromphenol blue method was seen. At the same time, there was an increase in the Sakaguchi reaction denoting an increase in the amount of protein-bound arginine. These cytochemical changes in the deoxyribonucleoprotein were interpreted as indications of an alteration of the spermatid histone into a more basic arginine-rich protein late in spermiogenesis. They were further interpreted as indicative of an increase in the strength of electrostatic binding of the nuclear protein to the DNA molecule. Some biological implications of these changes were briefly discussed.

Cytochemical characterization of deoxyribonucleoprotein in individual cell nuclei: Techniques for obtaining heat denaturation curves with the aid of acridine orange microfluorimetry and ultraviolet microspectrophotometry

Abstract Simple methods for obtaining heat denaturation curves on the deoxyribonucleoprotein DNP of individual cell nuclei or intact cells are described. Cells or nuclei adhering to microscopic slides were heated in a salt solution (SSC) containing formaldehyde. After heating the slides were rapidly cooled. Both the formaldehyde and the rapid cooling served to prevent renaturation of denatured DNA. The heated preparations were analyzed for changes in single- and double-strandedness by UV microspectrophotometry and by microfluorimetry after acridine orange staining. These techniques were used to obtain melting profiles of DNP from non-stimulated and PHA stimulated lymphocytes, erythrocyte nuclei activated by cell fusion with HeLa cells, normal erythrocyte nuclei and isolated HeLa nuclei. The results obtained show that the activation of a large number of genes in both human lymphocytes and hen erythrocyte nuclei is paralleled by striking changes in the melting profiles of nuclear DNP.

“Activation” of hen erythrocyte deoxyribonucleoprotein☆

Abstract In heterokaryons formed by fusing hen erythrocytes with human HeLa cells the erythrocyte nucleus is reactivated and resumes RNA synthesis. At an early stage of this reactivation process, the deoxyribonucleoprotein (DNP) undergoes characteristic changes in its cytochemical properties. We have found that similar DNP-changes can also be induced in the nuclei of intact red cells or in the nuclei of red cell ghosts by washing and incubating in salt solutions or tissue culture media devoid of serum proteins. These changes are not induced if 0.05 % (w/v) of autologous, bovine or human serum proteins are added to these media. The DNP-changes are expressed as an increased ability of DNP to bind the basic dyes acridine orange and ethidium bromide, an altered Feulgen reactivity and a lowered stability of DNP to heat denaturation. In spite of these DNP-changes the dry mass and total UV-absorbancy of the nuclei remain unchanged by the experimental procedure. The DNP changes appear to be induced ny non-enzymatic reactions and to be highly dependent on such factors as adhesion of the cells to glass, cell-to-cell interaction, the ionic environment and the presence or absence of certain serum proteins in the medium. It is suggested that the DNP-changes reflect the existence of a “chromatin activation reaction” which is essential for transforming inactive and highly condensed DNP and chromatin into active templates for RNA synthesis. This activation reaction may be intimately coupled to changes in the cell membranes and in the ionic environment surrounding the chromatin.

Chromatin changes during erythropoiesis

Summary 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 14 C-actinomycin D. The DNP modifications observed are discussed in relation to possible mechanisms of chromosome condensation and genome repression.

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

Abstract The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 μM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [ 35 S]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.

Biochemical analysis of reactivated chick erythrocyte nuclei isolated from chick-HeLa heterokaryons.

Abstract Sendai virus-induced fusion of chick erythrocytes with human HeLa cells results in the formation of multinucleated hybrid cells (heterokaryons). In these cells the erythrocyte nuclei take up human proteins from the cytoplasm and undergo a general “reactivation” reaction resulting in transcription, and eventually replication of the chick DNA. The early events in this reactivation process were analysed by isolating reactivated chick erythrocyte nuclei from heterokaryons zero to 15 hours post-fusion, at a time when extensive decondensation of the chick chromatin occurs (and transcription of chick DNA is initiated) but very little chick DNA replication is occurring. The isolated chick nuclei were sufficiently free from contaminating HeLa nuclear material to allow an electrophoretic and chromatographic analysis of 3 H-labelled proteins accumulating in the chick nuclei. The proteins were labelled by incubating the cultures with 3 H-labelled amino acids. Radioactivity in the histone group of nuclear proteins was shown to be composed predominantly of f1 histone. Furthermore, this f1 histone could be identified as being HeLa in origin and its migration into reactivating chick erythrocyte nuclei was correlated with a loss of chick-specific f2c histone. The appearance of radioactivity in the major non-histone group of nuclear proteins was highly preferential relative to the histone group of proteins. Electrophoresis on sodium dodecyl sulphate-poly acry lamide gels showed that many subgroups of non-histone proteins present in HeLa nuclei moved into the reactivated chick erythrocyte nuclei, whereas some other human proteins did not. Furthermore, pulse-chase type experiments showed that similar patterns of labelled proteins appeared in the erythrocyte nuclei irrespective of whether label was administered only to the mammalian cell before fusion or was given to heterokaryon cultures after fusion. The data presented are discussed in relation to changes in nuclear protein metabolism influencing chromatin structure and the availability of DNA for transcription.

Nucleoprotein changes and initiation of RNA synthesis in PHA stimulated lymphocytes

Abstract 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 14 C-acetate and 3 H-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.

Actinomycin binding properties of stimulated human lymphocytes

Abstract In cultures of human lymphocytes only a small percentage of the cells is capable of binding labelled actinomycin D (3H-AMD), and of incorporating 3H-uridine into RNA. Addition of phytohaemagglutinin (PHA) to such cultures markedly increases the frequency of cells binding 3H-AMD and of cells synthesizing RNA. There is a close parallelism between the frequency of cells capable of binding 3H-AMD and the frequency of cells synthesizing RNA. For a short period of time there is also a good correlation between the rate of RNA synthesis and the amount of 3H-AMD bound by individual cells. At later stages, however, practically all blast cells show a moderate AMD-binding, but a very strong incorporation of labelled uridine. Little 3H-AMD is lost from the cells if, after the labelling, the incubation is continued in the absence of actinomycin. Attempts to remove bound 3H-AMD by postincubation with unlabelled AMD for time periods up to 4 h did not result in any marked loss of 3H-AMD from control cells and PHA stimulated cells. It is pointed out that the quantity of AMD bound to living cells probably only represents a very small fraction of the total number of potential binding sites in the DNA. This fraction appears to vary depending on the functional state of the chromatin. Kinetic studies suggest that there are at least two different types of binding: one early and rapid form of binding and a second, slow, form of binding. The PHA-stimulated and the control cells differ with respect to the rapid type of binding but not with respect to the slower form of binding. It is suggested that the rapid type of binding reflects how large a portion of the chromatin is available for transcription.

Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons.

Abstract The reactivation of the chick erythrocyte nucleus was studied after erythrocytes were induced to fuse with rat epithelial cells in the presence of Sendai virus. The chick nucleus swells, shows an increase in dry mass and protein content and resumes RNA synthesis. Nucleoplasmic antigens characteristic of the rat cell are found to migrate into the erythrocyte nucleus. The rate of uptake of these molecules, which are believed to be proteins, appears to be directly related to increases in nuclear size, 3H-uridine incorporation and RNA polymerase activity. The polymerase activity which increases during the first days after cell fusion is sensitive to α-amanitin but relatively resistant to actinomycin D. At later time points there is an increase in α-amanitin resistant polymerase activity which probably reflects the appearance of ribosomal RNA synthesis. When heterokaryons containing different proportions of rat: chick nuclei are compared, reactivation is found to proceed most rapidly in those containing a high rat: chick nuclear ratio. As the number of erythrocyte nuclei in heterokaryons increases, the rate of reactivation in the individual nuclei is progressively reduced suggesting that the erythrocyte nuclei compete with each other for macromolecules of specific importance for the activation process.

Cytochemical demonstration of histones and protamines: Mechanism and specificity of the alkaline bromphenol blue binding reaction☆

Abstract 1. 1. The alkaline dye binding reaction has been studied in model systems using various types of basic proteins and on mouse lymphocytes and testicular cells. 2. 2. The specificity of the dye binding reaction was tested in model systems; proteins and polypeptides differing from each other with respect to basicity were stained at acid (pH 2.3) and alkaline pH (pH 8.2). Basic proteins were found to be selectively stained at the alkaline pH. 3. 3. The quantitative aspects of the staining method were investigated in model systems. In precipitation experiments, the stoichiometric relation between the number of basic groups and the number of dye molecules in the precipitate was determined. The proportionality between the amount of basic polypeptide and the amount of dye bound to it was studied at both the macrochemical and the microchemical level. In the latter case, this was done in a model system of microdroplets using microspectrophotometry and microinterferometry. In these model systems a linear relationship was found to exist between the amount of dye and the quantity of basic protein present. 4. 4. Various factors which might influence the amount of dye bound to basic proteins were investigated. Acid macromolecules were found to reduce the dye binding capacity of the basic proteins. The fixation method, the mode of DNA extraction and the staining conditions were also found to influence the staining results. 5. 5. The amount of dye bound to a cell at an alkaline pH reflects the quantity of basic protein present but is also dependent on qualitative properties of the basic proteins present.