Histone Synthesis Research Articles

The synthesis and isolation of DNA complementary to histone mRNA from mouse embryos

A 9S poly(A) RNA preparation isolated from mouse embryos was shown to stimulate the synthesis of histones in an ascites cell free extract. This RNA preparation was used for the synthesis of a highly labelled cDNA probe complementary to histone mRNA. Hybridisation of this cDNA probe to rRNA showed that 52% of the cDNA consisted of sequences complementary to rRNA. The histone mRNA specific cDNA was purified by hybridising the impure cDNA to rRNA followed by removal of the single-stranded histone cDNA by hydroxylapatite chromatography.

Meiotic synthesis of testis histones in the rat.

The distribution and synthesis of the testis-specific variants of histones H1 and H2B, TH1 and TH2B, respectively, and of the somatic histones were studied in rat testis cells. Rat testis cells were labeled in vivo with intratesticular injections of [3H]lysine. The cells and nuclei were then separated into different developmental classes by velocity sedimentation and the histones were analyzed. TH1 and TH2B, as well as the somatic histones, were present in spermatocytes and round spermatids, but none of them were detectable in elongated spermatids. The synthesis and nuclear accumulation of TH1 and TH2B took place throughout pachytene, as well as in earlier stages, but not in the round spermatids. In addition, there was synthesis during pachytene of a histone that migrates electrophoretically with H2A. However, somatic histone synthesis, with the possible exception of H2A and H2B, was not detectable at the pachytene stage. In vivo treatment of rats with hydroxyurea reduced DNA synthesis in the testis to 1% of control values and significantly reduced the synthesis of H3, H2B, H2A, and H4, with the greatest effect being on H3 and H4. However, the hydroxyurea treatment did not significantly decrease the synthesis of TH1, H1, or TH2B. These results prove that the synthesis of several histones during the meiotic prophase is not dependent upon concurrent S-phase DNA synthesis.

Histone H5 messenger RNA is polyadenylated.

In most known systems, histone mRNA lacks the poly(A) sequence at the 3' end of the molecule typical of most mRNAs. Furthermore, the synthesis of histones, unlike that of most proteins, is tightly coupled to DNA synthesis. Nevertheless, histone synthesis occurs in amphibian oocytes in the absence of DNA synthesis. Moreover, it has recently been found that in amphibian oocytes most of the histone mRNA is polyadenylated, and the polyadenylate is probably removed during maturation of the oocyte. Histone H5, an H1-like tissue-specific histone occurring only in nucleated erythrocytes, is also atypical in that it is synthesised in the absence of DNA synthesis during maturation of the red blood cells. We report here that H5 mRNA is polyadenylated.

Nuclear Proteins in Programming Cell Cycles

Publisher Summary During mitosis, the bulk of the nuclear proteins are released to the cytoplasm and the chromosomes are condensed. The condensed chromosomes are not active templates for RNA synthesis. Nuclear RNA synthesis resumes when the released nuclear proteins begin returning to the reconstituting daughter nuclei. The temporal ordering of DNA replication, histone synthesis during S phase, and periodic bursts in the activity of several cellular enzymes show that progress of cells through cycle is highly programmed. This programming requires activation and inactivation of nuclear genes. The genes required for progress of cells through the cycle are activated and inactivated during the interphase part of the cell cycle. The appearance of small cytoplasmic polyribosomes containing histone mRNAs during S phase further argues in favor of activation and inactivation of genes in the interphase of the cell cycle. The continuous massive shuttling of nonhistone nuclear proteins between nucleus and cytoplasm in Amoeba proteus and other eukaryotes are implicated in the mechanisms of gene control in the interphase part of the cell cycle.

Host antigens in the centriolar region are induced in SV40-infected TC7 cells: SV40 small-T-function requirement.

We reported earlier (Kasamatsu and Nehorayan 1979) that infection of the TC7 subline of African green monkey kidney cells with SV40 stimulates the appearance of staining in the centriolar region by preimmune or anti-VP3 sera from rabbits, as observed by indirect immunofluorescence. The staining appears very early in infection, prior to the appearance of T antigen, and the frequency of strong staining increases rapidly. At 24 hours after infection with a multiplicity of infection of 10, about 80% of the cells showed positive staining. However, the frequency decreases as the infection progresses further. The stimulation of a number of cellular processes, i.e., synthesis of cellular DNA (Dulbecco et al. 1965; Hatanaka and Dulbecco 1966; Ritzi and Levine 1969), cellular RNA (Oda and Dulbecco 1968), mitochondrial DNA (Levine 1971), certain enzymes (Kit 1967), and histones (Shimono and Kaplan 1969; Hancock and Weil 1969; Winocour and Robbins 1970; Rovera et al. 1972),...

Nuclear protein kinase activities during the cell cycle of HeLa S 3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S 3 cells, a nuclear phosphoprotein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G 2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G 1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G 1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G 2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.

Fate of Newly Synthesized Histones shortly after Interruption of DNA Replication

The synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on polyacrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C]marker histones by a 15 min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hydroxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hydroxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.

Histone synthesis in the salivary glands of Rhynchosciara americana during larval development

The synthesis of histones during the usually long (5–6 days) replication cycle in the salivary glands of Rhynchosciara americana larvae was investigated. The results show a coupling between DNA synthesis and that of the bands of basic nuclear proteins studied along the entire cycle. They indicate also that the synthesis of the different histones occurs in such a way as to maintain their relative amounts constant during the replication cycle both on amplified and on normal chromosomal sites.

Effect of polyamines and Mg ++ on poly(ADP-ribose) synthesis and ADP-ribosylation of histones in wheat

Polyamines in the presence or absence of Mg++ have been shown to stimulate 2–4 fold the total synthesis of poly(ADP-ribose) in isolated wheat nuclei. The stimulation by Mg++ and polyamines is dose dependent and is an affect on actual synthesis of new chains of polymer since neither Mg++ nor polyamines increase the average chain length of the polymer synthesised or inhibit the degradation of the polymer in isolated nuclei. Mg++ and polyamine treated nuclei showed an increased ADP-ribosylation of total histones to the same extent as did total polymer synthesised. Furthermore the distribution of poly(ADP-ribose) between histone H1 and the other core histones remained the same in control, Mg++ and polyamine treated nuclei.

Synthesis of high mobility group proteins in regenerating rat liver.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.

Modulations of histone messenger RNA during the early development of Xenopus laevis

The amount of translatable histone mRNA in Xenopus oocytes, eggs, and embryos was found to be roughly similar, as judged by a cell-free translation assay. This observation provides further evidence that posttranscriptional control mechanisms bring about the 50-fold increase in the rate of histone synthesis during oocyte maturation. About three-quarters of the oocyte histone mRNA was found to be polyadenylated. However, polyadenylated histone mRNA was scarcely detectable in the egg and embryo. Our data are consistent with the speculation presented here that the disappearance of the poly(A) + histone mRNA component at the time of conversion of oocyte to egg (maturation) is due to deadenylation.

The sea urchin ( Sphaerechinus granularis) codes different H2B histones to assemble sperm and embryo chromatin

Histones from sperm and gastrula nuclei of the sea urchin Sphaerechinus granularis were analyzed by three different electrophoretic methods, stained with specific color reactions and compared with one another. The histones of the two cell types showed identical properties in none of the three analytical methods employed but the greatest differences involved the H2B type of histones. H2B from embryo chromatin had properties similar to those of the corresponding molecule from calf thymus. In the sperm chromatin no such molecule was observed but two additional ones were found that differed from embryo H2B in size, charge and specific colour reactions. The differences were such that different genes may be postulated for the synthesis of the H2B histones found in sperm and in embryo.

Histones biosynthesis and turnover in epithelial lens cells cultured in vitro

Histone synthesis was compared in epithelial lens cells during exponential growth and in the stationary phase brought by contact inhibition. Double labelling experiments with 3H-lysine and 14C-lysine show a net turnover of histone H1 independent of DNA replication. The nucleosome core histones seem to turn over also, but much more slowly than H1.

Synthesis and Modification of the Histones during the Decidual Cell Reaction in the Mouse Uterus

The decidual cell reaction has been studied as a model of cellular differentiation and proliferation. The uterine histones, analyzed by Triton-acid-urea polyacrylamide gel electrophoresis, display marked heterogeneity. DNA synthesis begins 15 h after stimulation with a concomitant synthesis of histones. Three histone fractions undergo modification approximately 3 h following stimulation; histone fractions H1, H2A and H3 display increased phosphate incorporation. An increase in histone phosphorylation is observed in all histone fractions immediately prior to the increased rate of histone synthesis. At later times, only histones H1, H2A and H3 continue to become phosphorylated. Acetate incorporation into histone H2B and H4 also increases before the increase in histone synthetic rate. These histone modifications may be involved in the differentiation of stromal cells into decidual cells.

The Relative Contributions of Newly Synthesized and Stored Messages to Hl Histone Synthesis in Interspecies Hybrid Echinoid Embryos

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.

New concepts on the interference on alkylating antitumor agents with the regulation of cell division

Low concentrations of the alkylating agent Trenmon (2,3,5-trisethyleneiminobenzoquinone) inhibit the biosynthesis of histones but do not affect the replication of DNA. The alkylating agent does not interfere with the formation of chromosomal nonhistone proteins or lysine incorporation into total cellular proteins. The uncoupling of histone and DNA syntheses represents one possible mechanism for the antitumor activity of the drug. Besides the effect of histone synthesis, the alkylating agent causes an impairment of the plasma membrane. This is expressed by an inhibition of the transport system for thymidine, aminoisobutyric acid and 3-O-methyl-glucose and furthermore by a reduced activity of the Na + K + ATPase, EC 3.6.1.3. Evidence is presented that the effects are caused by direct interaction of the alkylating agent with the cell membrane. Preliminary experiments employing chlorambucil covalently linked to polythyleneimine support the assumption that an alkylation of the cell surface is sufficient to inhibit tumor cell multiplication. At present it cannot be decided whether the uncoupling of DNA and histone syntheses or the impairment of the cell membrane is responsible for the antitumor activity.

Conservative segregation of parental histones during replication in the presence of cycloheximide

Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be "free" DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal in vivo for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.

Histones and DNA increase synchronously in neurons during early postnatal development of the rat forebrain cortex.

Cytophotometric measurements reveal that a temporally coordinate accumulation of histones and DNA occurs in rat cortex neurons between the last gestational day and the end of the third postnatal week. A rapid rise of both constituents around birth is followed by a more protracted synthesis. This leads to adult histone and DNA levels of approximately 3.3 arbitrary units as compared with 2 arbitrary units found in neuronal precursor cells at all foetal stages and in reference diploid nuclei from glia and liver. A secondary DNA increase in the fourth postnatal week, previously suspected on the basis of cytofluorometric measurements using the Schifftype stain BAO (bis-[4-aminophenyl]-1,3,4-oxadiazole), is not substantiated by the present work. This derives from the finding that alternative cytophotometric DNA measurements (ultraviolet absorption scanning) and autoradiographs of neuronal nuclei following repeated injections of [3H]thymidine into the lateral ventricles during the relevant period give no evidence of a further DNA increase after the third postnatal week. Neither does the accumulation of histones (measured by sulfaflavine cytofluorometry) continue beyond day 21. This leads us to conclude that DNA and histone syntheses cease at the end of the third week. Electrophoretic analyses of the histones show that the relative histone composition changes only slightly during neuronal development. Apart from an increase in the ratio of the histones H1o:H1 around birth no developmental alterations in histone composition are detectable.

Block of histone synthesis in isolated sea urchin cells actively synthesizing DNA

The synthesis of DNA and histones are normally coupled. Only a few exceptions have been found. Dissociated cells from sea urchin embryos stop DNA synthesis, but resume it after trypsin treatment. It was of interest, therefore, to examine whether the synthesis of histones is similarly affected. Sea urchin embryos were dissociated from swimming blastulae and the cells so obtained were divided into two batches, one of which was treated with trypsin. Normal embryos from the same culture were used as controls. It was found that, in the dissociated cells, histone synthesis ceases, but unlike DNA, its synthesis is not resumed after treatment with trypsin.

Effect of inhibition of DNA synthesis on histone synthesis and deposition.

We have reinvestigated the degree of coupling between DNA and histone synthesis in mammalian cells. In at least one cell line (HTC cells), the coupling is not nearly as tight as had previously been inferred from experiments with HeLa cells. The site of deposition of such histones which continue to be made in the presence of sufficient hydroxyurea to depress DNA synthesis almost totally has been studied. Deposition seems to be on material which absorbs at 260 nm. This material is not a part of the bulk chromatin and binds histone in a relatively tight manner. The possible role of such a material in histone synthesis and deposition is discussed.