Template Activity Of Chromatin Research Articles

Effect of chronic morphine treatment on brain chromatin template activities in mice

Chromatins have been isolated from both placebo- and chronic morphine-treated mice. The specific activity of chromatin-directed UTP incorporation was measured. Evidence is presented to show that the chromatin template activity isolated from tolerant animals is increased. The increase may be due to the alteration of non-histone protein in chromatin. This is a narcotic-specific phenomenon, since the morphine-induced increase can be blocked by naloxone.

Structure of transcriptionally active chromatin.

Rat-liver chromatin has bee fractionated into transcriptionally active and inactive regions [Gottesfeld et al. (1974) Proc. Nat. Acad. Sci. USA 71, 2193-2197] and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by tne endonuclease DNase II. The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nuclease-resistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition.

Uterine chromatin template activity during the early stages of pregnancy in the rat

1. The template activity of chromatin prepared from rat uterine nuclei during dioestrus, oestrus and the first 7 days of pregnancy has been examined. 2. The DNA, RNA, histone and non-histone protein contents of uterine chromatin remained constant during early pregnancy. 3. The rate of RNA synthesis on Day 1 uterine chromatin was 8.61 ± 0.59 ( mean ± S.E. ) pmol of UMP incorporated/mg DNA per 10 min. When compared with DNA prepared from rat liver nuclei, 13.20 ± 0.27% ( mean ± S.E. ) of the Day 1 chromatin DNA was available for transcription by Escherichia coli RNA polymerase. 4. Uterine chromatin from rats in early dioestrus had significantly less template activity than during oestrus. 5. Chromatin prepared from whole uterus on Day 5 and from implantation sites on Days 6 and 7 of pregnancy had a significantly higher template activity than chromatin obtained from uteri on Day 1. Chromatin from inter-implantation tissue on Day 6 had a lower template activity than that from uteri on Day 1. 6. RNA · DNA hybridisation of RNA transcribed from chromatin obtained on Days 2, 5 and 7 of pregnancy showed that RNA transcribed from Day 5 chromatin contained species not present (or present in very small amounts) in RNA transcribed by chromatin from uteri on Day 2 and from implantation tissue on Day 7 of pregnancy. 7. The results are discussed in relation to the cellular changes occurring in the stroma immediately before implantation and it is postulated that the appearance of a new species of RNA on Day 5 is related to the preparation of the stromal cells for decidualisation.

Effect of mustine (HN2) and its monofunctional analogue (HN1) on template activity of chromatin and DNA in an RNA-polymerase system in vitro

Mustine inhibits the template activity of chromatin and DNA in an RNA-polymerase system in vitro much more strongly than its monofunctional analogue. Chromatin is more sensitive to the action of mustine than is deproteinized DNA. However, the template activity of DNA is reduced to a greater degree by the action of the monofunctional analogue of mustine than is that of chromatin. The mechanism of inhibition of the template activity of chromatin by mustine is probably connected with the ability of the compound to form DNA-protein cross-linkages in the chromatin structure.

Effect of adenosine 3′,5′-cyclic monophosphate on the RNA and DNA synthesis and cell proliferation of rat hepatocytes in primary culture: A radioautographic study

The effect of a 24-h treatment with various doses (from 1.5-10-minus 8 to 3.0-10-minus 3 M) of adenosine 3',5'-cyclic monophospahte (cAMP) on morphometric parameters, [5--3H]uridine radioactivity concentration (URC), [methyl--3H]thymidine [Me--3H]-Tr) labelling index per hour (L.I./h) and per cent mitotic index (M.I.%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographic methods. In such cells cAMP was found to induce: (1) a reduction of the apparent surface area (ASA) of total nucleoli, karyoplasm and cytoplasm; (2) significant increases in URC of all the subcellular compartments at all the dosages employed (only cAMP at 1.5-10-minus 8 M did not change karyoplasmic and cytoplasmic URC values); (3) marked increments in [Me--3H]Tdr L.I./h and M.I.% from the lowest dose up to 1.5-10-minus 4 M; at higher doses the L.I./h and M.I.% were less stimulated or approached control values. In cultured rat hepatocytes, adenosine-5'-phosphate (5'-AMP) (1.5-10-minus 4 M per 24 h) increased the karyoplasmic and total cell ASA, the lone total nucleolar URC and both the L.I./h and M.I.%. However, these metabolic effects were significantly less intense than those elicited by isomolar cAMP. Theophylline (Theo) (5.5-10-minus 5 M per 24 h) reduced the in vitro rat hepatocyte total nucleolar ASA but affected neither other morphometric nor any of the URC values. The same dose of Theo plus cAMP (1.5-10-minus M) had no morphometric effect but significantly increased the URC values of all primary rat hepatocyte compartments. Actinomycin D (DAct) (0.1 mug/ml per 24 h) plus cAMP (1.5-10-minus 4 M) decreased the cultured rat hepatocyte total nucleolar ASA but enlarged that of karyoplasm and cytoplasm and, further, markedly curtailed all the compartmental URC values. These data support the hypothesis that cAMP amplified the template activity of the liver chromatin and accelerates the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.

Phosphorylation of nonhistone chromosomal proteins in dog kidney cells infected with infectious canine hepatitis virus

Infectious canine hepatitis virus infection of dog kidney cells altered the metabolism of the nonhistone chromosomal proteins. The rate of incorporation of [ 32P]phosphate into the nonhistone chromosomal proteins began to increase between 6 and 7 hr after infection, and the specific activity of the proteins labeled between 8 and 9 hr after infection was 195% of that observed in the mock-infected control. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that phosphorylation of the nonhistone chromosomal proteins was increased after infection especially in some particular components of the proteins. Alteration of the metabolism of the nonhistone chromosomal proteins correlated with an increase of RNA synthesis and template activity of chromatin in infected cells.

Effects of the carcinogen urethane on nuclear RNA polymerase activities

Intraperitoneal injection of male rats with a single dose of the carcinogen urethane inhibited the Mg 2+ -dependent RNA polymerase activity of isolated lung nuclei by about 50%. The inhibition was maximal 1 hr after the injection. Twenty hours after administration, the Mg 2+ -stimulated activity was again normal. The non-carcinogenic derivatives methylcarbamate and butylcarbamate had no effect on RNA polymerase activities of isolated lung nuclei. The inhibition of nuclear RNA synthesis by urethane was not due to increased hydrolysis of newly synthesized RNA, and the template activity of lung chromatin was not affected by the carcinogen. The activity of Mn 2+ + (NH 4 )SO 4 -dependent RNA polymerase activity of isolated lung nuclei was not affected by urethane injection. The two RNA polymerase activities of liver nuclei were not inhibited by the carcinogen. Mg 2+ -stimulated nuclear RNA synthesis in vitro was inhibited markedly by urethane at concentrations above 50 mM, whereas the Mg 2+ + (NH 4 ) 2 SO 4 -dependent activity was only slightly affected. In vitro RNA synthesis catalyzed by E. coli RNA polymerase was inhibited also by high concentrations of the carcinogen.

Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin.

Estrogen administration to chicks results in an increase in the chromatin template activity of oviduct target tissue as assayed under standard in vitro assay conditions. However, the results obtained by the simple measurement of template activity may be a complicated function of the number of available RNA polymerase initiation sites, the rate of RNA chain elongation, and the rate of reinitiation. In the present study, we have measured separately the change in both the number of chain initiations as well as the rate of RNA chain propagation under conditions in which reinitiation was eliminated. Chromatin prepared from either estrogen-treated or control oviducts both supported an RNA chain elongation rate of six nucleotides per s and a chain size of approximately 700 nucleotides. Thus, both the elongation rate and size of the average product remained relatively constant following estrogen stimulation. In contrast, within 8 hours after a single injection of estrogen to unstimulated chicks, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased to 150% in comparison to control values, and by 24 hours the level of polymerase bound to chromatin was twice that of the untreated control chick chromatin. With daily injections of estrogen, polymerase binding continued to rise. Coincident with the over-all increase in chromatin-bound polymerase was an increase in rifampicin-insensitive initiation sites and newly synthesized RNA chains. Unstimulated chick oviduct chromatin initiated 10,000 RNA chains/pg of DNA, while 24 hours of steroid treatment increased the number of initiated chains to 34,000 chains. These data demonstrate that the estrogen-induced increase in chromatin transcriptive activity was due to an increased number of polymerase binding and initiation sites on the chromatin template without a detectable change in the rate of RNA chain elongation.

Effect of 5-bromodeoxyuridine on the transcriptional properties of the genome in WI-38 human diploid fibroblasts

Growth of WI-38 diploid fibroblasts in a medium containing 5-bromodeoxyuridine (BrdU) resulted in an increased GMP and a decreased AMP incorporation into the RNA synthesised in vitro on a chromatin template. This effect was similar to that previously reported using 3T6 mouse fibroblasts-1. Substitution of thymidine by BrdU in DNA, also altered the characteristics of the DNA template itself, since the increased incorporation of guanine and decreased incorporation of adenine into RNA were evident also when purified, isolated DNA was used as template. The extent of replacement of AMP by GMP was proportional to the extent of replacement of thymidine by BrdU. Although there are variations in the base composition of RNA transcribed from BrdU-containing DNA templates, there are no significant difference in overall template activity or in the number of available chromatin binding sites for E. coli RNA polymerase. Confluent monolayers of BrdU-treated WI-38 fibroblasts are still able to respond with cell proliferation to a change of medium, as evidenced by an increased incorporation of (-3H)thymidine and an increase in chromatin template activity. The length of the prereplicative phase is similar in both BrdU-treated and untreated cells, although the magnitude of the increase of (-3H)thymidine incorporation is reduced by approximately 30% after BrdU treatment. The increase in chromatin template activity is associated with an increase in the number of chromatin binding sites for E. COLI RNA polymerase, suggesting that the presence of BrdU does not interfere with the availability of initiation sites or alter the actual rate of transcription.

Distribution of murine type B and type C viral nucleic acid sequences in template active and template inactive chromatin

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant RNA homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.

Gene activation in human diploid cells: Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins

Serum stimulation of early as well as late passages of nondividing WI-38 human diploid fibroblasts to proliferate, results in DNA synthesis beginning at 12 hours and mitosis at 20 hours. A 2-fold increase in the transcriptional activity of chromatin isolated from early and late passage WI-38 cells is evident one hour following serum stimulation. An increased synthesis and association with the genome of two defined molecular weight classes of nonhistone chromosomal proteins one hour following serum stimulation of early and late passage cells is also observed. The increased chromatin template activity and nonhistone chromosomal protein synthesis occur in early passage cells stimulated to proliferate in the presence of actinomycin D. However, when late passage WI-38 cells are stimulated in the presence of actinomycin D, increases in chromatin template activity and nonhistone chromosomal protein synthesis are not observed one hour following serum stimulation. The possibility that nonhistone chromosomal protein synthesis and activation of transcription early during the prereplicative phase of the cell cycle in early passage human diploid fibroblasts may be regulated at the translational level is discussed. Consideration is also given to the possibility that there may be an age-dependent modification in such a regulatory mechanism.

Isolation, fractionation and template activity of the continuously-condensed chromatin of Euglena gracilis

A technique for isolating whole chromatin from nuclei of the lower eukaryote Euglena gracilis is presented. This chromatin, which appears under the electron microscope as uniformly condensed fibers, can, nevertheless, be subfractionated into distinct heterochromatic and euchromatic fractions. The euchromatin, comprising about 14% of the total DNA of the nucleus, contains over 80 % of the total endogenous RNA polymerase activity measured. The K m for this enzyme is higher than that found for prokaryotes, but falls in the range found for other eukaryotes. Stability constants, calculated from cation-chromatin binding data, suggest that internal carboxyl groups of chromosomal proteins, at least, are involved in the condensation of Euglena chromatin. The relationship between Euglena chromatin and that of higher eukaryotes is discussed.

Mechanism of action of steroid hormones

Despite the diversity in the target sites and physiological actions of the steroids, an impressive body of evidence has been assembled in support of a unitary theory of the mechanism of action of these hormones in vertebrates. The steroids set in motion a train of events, as follows: (a) penetration into the target cell, (b) stereospecific binding to high affinity receptors, (c) temperature-sensitive activation of the steroid-receptor complex, (d) attachment of the active complex to chromatin, (e) induction of RNA and protein synthesis, and (f) physiological expression of the induced protein. Although the overall sequence is well-defined, our knowledge of the molecular processes involved in each of these steps is still quite incomplete. Two of the major efforts now underway to elucidate these molecular processes involve, (a) purification and characterization, in terms of structure-function relations, of the putative receptors, and (b) studies on the nature of the interaction between steroid-receptor complexes and the genome. It is now apparent that steroids induce de novo synthesis of both messenger RNA (mRNA) and ribosomal RNA (rRNA); the role of the former in directing the synthesis of specific proteins is reasonably clear but that of the latter remains to be elucidated. The mechanism of induction is also under scrutiny since the observed increases in mRNA synthesis could arise in a variety of ways, e.g. negative or positive regulation of chromatin template (gene) activity, changes in processing of heterogeneous RNA to mRNA, effects on RNA polymerase or ribonuclease activities. Although steroidal regulation of RNA and protein synthesis is a dominant pathway, the possibility of direct actions on membranes or regulatory enzymes in some circumstances can not be excluded at the present time.

Chromatin template activity during aging in WI38 cells

A reduction in the chromatin template activity of WI38 cells of different ages has been demonstrated using (1) the endogenous RNA polymerase of the host cell and (2) exogenously supplied E. coli RNA polymerase. The consistency between these two methods suggests that the reduction in chromatin template activity of these cells is age-associated and may represent changes in gene activity during in vitro aging.

The effect of urethan on cyclic events in the regenerating rat liver

The effects of urethan on cyclic events associated with cell replication in the regenerating liver of the male rat were measured in an effort to determine actions of potential significance to the hepatocarcinogenic activity of this agent. A single intraperitoneal dose of urethan (1 mg/g), injected at either 0.5, 12 or 24 hr after partial hepatectomy, caused inhibition of the incorporation of 3H-thymidine into nuclear DNA. Measurements of the effects of urethan through two cycles of DNA biosynthesis indicated that the inhibition was transient and reversible. The induction by partial hepatectomy of certain enzymes closely linked to DNA replication (i.e. ribonucleoside diphosphate reductase and thymidine kinase) were affected by urethan in a similar manner, while induced DNA polymerase activity and the activities of other enzymes not linked to DNA replication (i.e. adenine and hypoxanthine-guanine phosphoribosyltransferases) were relatively unaffected by the carcinogen. Urethan had no detectable effect on the ability of chromatin to serve as a template for RNA synthesis as measured by the incorporation of both 14C-ATP and 3H-GTP into RNA, catalyzed by E. coli RNA polymerase, when the urethan was injected during the prereplicative phase of liver regeneration. However, when the carcinogen was administered during the S phase (18 hr after partial hepatectomy) and the chromatin isolated 9–10 hr later, the template activity of chromatin was depressed and the composition of the nascent RNA, as measured by the ratio of AMP to GMP incorporated, was altered.

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot ( Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.

Accessibility of chromatin to DNA polymerase I and location of the F1 histone.

The effect of prebound poly-L-lysine upon the template activity of DNA, chromatin and F1 histone-depleted chromatin for E. coli DNA polymerase I has been investigated. Measurements have been made in the absence and presence of 0.1M NaCl. From the results we conclude that only ca 60% of the polylysine-accessible DNA, i.e. 22% of the total DNA of the chromatin, is accessible to the DNA polymerase I and that ca. 30% of the polylysine-accessible DNA, i.e. 11% of the total DNA, is associated with the F1 histone.

Primary Tissue Culture of Normal Adult Human Decapsulated Adrenal Cortex: Radioautographic Studies on the Metabolic Effects of ACTH<sub>1-2</sub><sub>4</sub>

ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-3H and 1-leucine-3H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH1-24 (3 X 10(-6) M) the incorporation of the precursors for RNA and DNA synthesis and the cell proliferation were found to be markedly stimulated. The apparent uptake of leucine-3H was instead reduced in 2-day and, less still, in 7-day treated cells. In parallel with ultrastructural studies, these data suggest that ACTH orderly activates the template activity of chromatin while eliciting the differentiation and hypertrophy of human adrenocortical cells.

Alterations in chromatin functions during aging in vitro.

Age-associated alterations in the chromatin functions of human diploid cells have been observed. These alterations include: (1) a decline in the rate of histone acetylation; (2) a reduction in the rate of RNA synthesis as measured by (a) the rate of 3H-uridine incorporated into the RNA of young and old cells; (b) comparison of the template activity of isolated chromatin from young and old cells using E. coli RNA polymerase and (c) measurement of chromatin template activity using the endogenous RNA polymerase of young and old cells. It is suggested that the nondividing state of old cells may be the result of the inability to synthesize specific RNA molecules (and perhaps specific proteins) necessary for the cell to continue through the cell cycle.

Changes in the G0 state of WI-38 fibroblasts at different times after confluence

Some events in the prereplicative phase of WI-38 human diploid fibroblasts stimulated to proliferate are found to be a function of the length of time the cells have been quiescent. At 5 days after plating, when the cells first become confluent, the prereplicative phase upon stimulation by a nutritional change is relatively short, DNA synthesis begins at 8 h after stimulation, and there is no increase in chromatin template activity. At 9 days after plating the prereplicative phase of stimulated cells is lengthened to 14 h and there is an increase in chromatin template activity within 1 h of stimulation. Finally, in 18-day cells, the prereplicative phase is lengthened even further to 20 h, and there is a lag after stimulation before the increase in chromatin template activity. It is proposed that confluent WI-38 cells initially arrest in G 1, subsequently pass into G 0, and continue to go deeper into G 0 as they remain quiescent.