Template Capacity Research Articles

Conditions that bicontinuous microemulsions must fulfill to be used as template for electrodeposition of nanostructures

The conditions to obtain metal micro/nanostructures by templated electrodeposition using bicontinuous microemulsions have been proposed. For this, different microemulsions have been tested to discover, analyze and generalize the conditions that must fulfill in order to act as a template for metal electrodeposition. Bicontinuous microemulsions were selected to provide enough conductivity to permit the process at significant velocity. Using the cobalt deposition process from a single bath in the aqueous component of the microemulsion as a test, the relation between the nature of the microemulsions and the template capacity in the electrodeposition process has been deduced. Depending on the characteristics of each selected microemulsion, either continuous or nanostructured deposits (porous films or particles of 20–50nm) were obtained. The conditions for which the microemulsions act as templates in the electrodeposition are: (1) immiscibility of oil and aqueous solution components, (2) low dielectric constant of the oil to favor the non-dissolution of the electroactive species, (3) the use of a surfactant that avoids the solubilization of electroactive species. Also, the possibility of using an electrodeposition method to characterize the structure of some microemulsions has been demonstrated.

Changes in ribonucleic acid polymerase activities in gonadotropin-treated Leydig cells: an estradiol-mediated process.

The effects of desensitizing doses of hCG on the activities of Leydig cell DNA-dependent RNA polymerases I, II, and III were investigated after optimization of the enzyme assay. Individual activities were obtained by taking advantage of their different sensitivities to alpha-amanitin. RNA polymerase III was a minor component of the alpha-amanitin-resistant activity at 0.10 micrograms/ml, and was therefore measured together with RNA polymerase I. In adult rats treated with 10 micrograms hCG, sc, RNA polymerases II and I plus III activities of Leydig cells rose within 45 min to 180 +/- 6% and 162 +/- 2% of the control value, and then decreased to control values at 60 min. The initial stimulation of polymerase activities was coincident with maximal increases in testosterone and estradiol levels in plasma. A second and more sustained increase in polymerases II and I plus III activities occurred between 12 and 24 h (212 +/- 12% and 180 +/- 10% of the control value) and was maintained until 48 h after hCG injection. The hCG-induced rise in polymerase activities was due to activation of the enzymes, since chromatin template capacity was unaltered. In animals treated with the antiestrogen tamoxifen, stimulation of RNA polymerase activity by hCG was completely inhibited. Also, hCG did not stimulate polymerase activity in animals treated with aminoglutethimide, which blocks steroid synthesis from cholesterol, or in those treated with adrostatriendione, which inhibits aromatization of testosterone leading to estradiol. Increases in RNA polymerase activities were also achieved by the administration of lower doses of hCG (0.1 and 1 micrograms) and the administration of estradiol (2 micrograms), resembling the pattern seen with 10 micrograms hCG. These studies have indicated that the hCG-induced RNA polymerase activation in the Leydig cell is mediated through nuclear actions of estradiol, since stimulation of the enzymes was prevented by tamoxifen and inhibition of steroid biosynthesis, and was induced by estradiol administration.

Alterations in myocardial RNA synthesis and RNA polymerase activity during normal growth.

The effect of normal growth (hypertrophy) on myocardial nuclear activity was investigated using male Wistar rats at 21, 50, and 100 days of age. Cardiac mass increased sevenfold during this age range. The concentration of RNA (mg X g-1) was the highest at 21 days and decreased 48% by 50 days of age and 68% after 100 days of development. RNA synthesis, corrected for alterations in the specific activity of the cytoplasmic nucleotide pool, was the highest at 21 days of age. After 50 days of growth, uridine incorporation was decreased fivefold. With continual growth (100 days), RNA synthesis was still reduced compared with the 21-day animals. RNA polymerase activity in myocyte nuclei showed little change in activity from 21 to 100 days of age. However, in the nonmyocyte fraction, RNA polymerase decreased threefold after 50 days of development. Collectively, these data suggest that the large decrease in myocardial RNA synthesis cannot be accounted for by a change in nuclear RNA polymerase activity and that an alteration in chromatin template capacity may be involved during this form of cardiac growth.

Errors in DNA synthesis induced by aflatoxin B1 modification of poly(dC-dG)

Aflatoxin B1 reacts with poly(dC-dG) to form guanine adducts and apurinic sites. Such modification strongly inhibits the template capacity of the polynucleotide and leads to an increase in non-complementary nucleotide incorporation. The template inhibition and induced errors are reduced by mild heat depurination which removes all 7-substituted modifications.

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 μg/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Preferential stimulation by aurintricarboxylic acid of DNA-dependent RNA polymerase II activity in isolated larval nuclei of Artemia salina.

Aurintricarboxylic acid was found to stimulate preferentially RNA polymerase II activity in isolated nuclei of Artemia salina larvae. Studies on the in vitro transcription of a homologous chromatin indicate that this dye induces changes in the chromatin so as to enhance its template capacity for free RNA polymerase. Both free RNA polymerases I and II are equally sensitive to this dye in the absence of chromatin template. RNA polymerase II, however, becomes highly resistant to aurintricarboxylic acid when it is actively transcribing the chromatin template, whereas RNA polymerase I remains extremely sensitive even in the transcription complex. Therefore, in isolated nuclei, a preferential stimulation of alpha-amanitin-sensitive, but not-resistant RNA synthesis by aurintricarboxylic acid can be observed.

Immunospecificity of nuclear antigens in chicken erythroid cells.

Specific antisera were produced against chicken reticulocyte dehistonized chromatin. The antisera reacts strongly with chicken reticulocyte chromatin, but only marginally with chicken erythrocyte chromatin. There is no reticulocyte antigen detected in chicken liver. Reticulocyte maturation is accompanied by a gradual decrease in the chromatin immunological activity and template capacity. The reduction of immunological activity is due to the change of chromatin conformation during erythrocyte maturation. Dehistonization and sonication of erythrocyte chromatin raises the erythrocyte chromatin immunological activity to levels similar to those of reticulocyte chromatin. The erythrocyte nuclear antigens are class specific, not being found in frog erythroid cell or murine Friend leukemia cell chromatins.

Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis

ts Cl mouse L cells are temperature-sensitive ( ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function.

Non-random nature of 2-acetylaminofluorene-induced alterations of DNA template capacity

Male rats were fed a diet containing 0.03% (w/w) 2-acetylaminofluorene (AAF) and their hepatic DNA was isolated and transcribed with E. coli RNA polymerase. Ingestion of the carcinogen-containing diet for 4 days substantially reduced the template capacity of the isolated DNA. This reduction in template capacity was due to an apparent decreased RNA chain size (up to 50%), with no significant changes in initiation or re-initiation of RNA synthesis. This premature termination of RNA synthesis was accompanied, in some instances, by a reduced rate of RNA chain elongation. When the rats were returned to a basal diet for 7 days following 4 days of AAF ingestion, template capacity and RNA chain size returned to control values. Fractionation of hepatic chromatin on a glycerol gradient revealed that inhibition of DNA template capacity occurs on portions exhibiting characteristics of expressed, as well as those with characteristics of repressed, segments of the genome. In contrast, the DNA isolated from a small, highly condensed chromatin fraction (15% of total chromatin-DNA) showed no significant reduction in total template capacity. Analysis of the fidelity of RNA synthesis on this DNA template was performed by determining the rate of addition of individual nucleotide triphosphates to a growing RNA chain. Large reductions in the rates of adenosine and uridine polymerization were observed while no changes in guanosine or cytidine polymerization were found. This suggests the presence of functionally significant carcinogen-induced modifications of adenine. The inhibition in the rate of adenosine and uridine polymerization was reversed when the animals were placed on a basal diet after AAF ingestion.

Studies on sex-organ development. Changes in the oestrogenic response of the chick Müllerian duct as measured by chromatin template and ribonucleic acid initiation capacity

Assays of transcription in vitro, with Escherichia coli RNA polymerase or wheat-germ RNA polymerase II, were used to characterize chromatin templates isolated from the left Müllerian duct of the chick embryo during normal development, and during development in the presence of diethylstilboestrol. Control Müllerian-duct template capacity with E. coli RNA polymerase decreased from 6.42% on day 10 to 4.34% by day 15 of development. Similar results were found with wheat-germ RNA polymerase II. In the presence of rifampicin and heparin, the prokaryotic enzyme transcribed a number-average RNA chain of 670 nucleotide residues, at an average rate of 110 nucleotide residues/min, from Müllerian-duct chromatin of all developmental stages. From day 10 to day 15 there was a 44% decrease in the number of initiation sites for E. coli RNA polymerase on Müllerian-duct chromatin. A 47% decline was observed when these chromatins were transcribed with excess RNA polymerase II in the presence of rifamycin Af/013. Signs of increasing responsiveness to oestrogen developed between days 10 and 16. Embryos exposed to maximally responsive doses of diethylstilboestrol for 2 days showed increases in Müllerian-duct chromatin template capacity, RNA-chain initiation sites, wet weight, protein and RNA. The changes seen in the oviduct of the 1-week-old chick injected for 2 days with diethylstilboestrol were defined as 100% responses. By comparison, the Müllerian duct, after exposure to diethylstilboestrol from day 10 to day 12, from day 13 to day 15 or from day 16 to day 18, showed a 15%, 39% and 72% template response respectively, and a 42%, 56% and 85% initiation-site change respectively. A similar developmental trend was observed in all parameters. It is concluded that oestrogenic responsiveness in the developing Müllerian duct increases from day 10 to nearly maximal values by day 16 of development, and that this transition is paralleled by a progressive restriction of genomic activity.

Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.

Hetero- and euchromatin of synchronous Euglena cells : I. Physical fractionation of nuclei into differentially condensed chromatin

Several attempts were made to isolate intact nuclei and fractionate condensed and extended chromatin from synchronized cells of Euglena gracilis Z. Different factors affecting the recovery and the intactness of nuclei have been tested: detergent concentration, incubation time and addition of Mn 2+ (0.13 mM) and/or spermidine (0.14 mM) as protective agents. Interphase and mitotic nuclei show preserved chromatin when Mn 2+ is included, while the combination of Mn 2+ and spermidine—and, to a lesser extent, spermidine alone—leads to mitotic nuclei with enhanced clumped chromatin. The common procedure to fractionate Euglena chromatin involves swelling of nuclei before disruption. We proved that this step induces artefactual decondensation of packed heterochromatin. Two alternative methods are compared with separate condensed and dispersed chromatin: (1) breakage of swollen nuclei and subsequent addition of divalent cations and/or spermidine with mild pressure shearing forces; (2) disruption of nuclei in a medium containing Mn 2+ as a protective agent, without swelling. Electron microscopy study indicates that the normal packed appearance of condensed chromatin is preserved, according to the second procedure, while extensive shearing is necessary. Template capacity of the extended chromatin is significantly higher in both methods. Relative amounts of condensed and dispersed chromatin during interphase and mitosis are discussed.

Developmental restrictions on transcription: Determinants of the developmental program and their role in aging

A developing plant system, the soybean hypocotyl has been used to investigate early transcription events which restrict auxin induced cellular proliferation to the appropriate developmental stage. Auxin treatment of 4-day old seedlings resulted in an early (6 hour) activation of chromatin-bound RNA polymerase activity which approached 200% of control values by 18 hours. This occurred without a detectable alteration in chromatin template capacity (assayed with exogenous RNA polymerase) and resulted in the synthesis of “induced RNA transcripts” as determined in vitro by nearest neighbor analysis. In contrast, auxin treatment of unresponsive 8-day old seedlings did not alter the chromatin-bound RNA polymerase activity. Hormonal activation did, however, result in the exposure of “induced template” regions in chromatin which could only be transcribed in vitro if exogenous RNA polymerase was included in the transcription reaction. Isoelectric focusing of the endogenous chromatin-bound and soluble RNA polymerase enzymes from succesive developmental stages revealed that the chromatin-bound enzymes at the 2-day stage were first released from the chromatin complex and could be recovered in the soluble pool (4-day stage). This was followed by a gradual disappearance of these subspecies (6-day stage) until only a limited ensemble of RNA polymerase subspecies remained bound to chromatin and free in the soluble pool (8-day stage). Similar analyses of both the bound and free enzymes at the 4 and 8-day stages following auxin treatment revealed that the 4-day soluble enzymes could be induced to rebind to the chromatin complex in a defined sequence after hormone treatment while those of the 8-day hypocotyl were unable to do so. These developmental events indicate that the select loss of certain RNA polymerase subspecies serves to restrict the hormone responsiveness of this tissue to the early developmental stages. Such restrictions could thus commit the constituent hypocotyl cells to their terminal post-mitotic phase of development.

Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA.

Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (+/-)-trans, 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzo[a]pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phage-specific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.

Changes in RNA polymerase activity in isolated mouse uterine nuclei during the decidual cell reaction

The artificially stimulated decidual cell reaction has been used as a model to study changes occurring in the uterus at the time of implantation. Activities of RNA polymerases I, II and III were measured in uterine nuclei isolated from ovariectomized non-primed mice, hormonally primed mice, and hormonally primed mice following stimulation of the decidual cell reaction. Activities of all three RNA polymerases increased following hormonal priming of ovariectomized mice. In nuclei from stimulated uterine horns, activities of RNA polymerases I and III increased 9 h after stimulation of the decidual cell reaction and remained elevated through 21 h. RNA polymerase II activity did not change following stimulation of the decidual cell reaction. These changes in RNA polymerase activities occur at the time of increased histone modifications and may result from changes in the template capacity.

CHARACTERIZATION OF THE 32,000 DALTON MEMBRANE PROTEIN—I. EARLY SYNTHESIS DURING PHOTOINDUCED PLASTID DEVELOPMENT OF SPIRODELA

AbstractThe kinetics of 35S methionine incorporation into soluble and membrane proteins during the transition from steady state dark growth to greening was studied in Spirodela. A sharp increase in the rate of incorporation occurred at 3 h, which was several h before major increases in chlorophyll were apparent. Part of this enhanced incorporation was due to enhanced synthesis of a 32,000 dalton membrane protein. This synthesis was paralleled by a temporal increase in in uitro template capacity for this protein and an increase in 0.5 × 106 dalton plastid messenger RNA.

Developmental restrictions on hormone modulated gene transcription. I. Effects of auxin on template capacity and chromatin-bound RNA polymerase

The auxin induced growth response of the developing soybean hypocotyl has been employed as a model system to investigate the transcriptional mechanisms which may in part determine this tissue's hormonal responsiveness during early differentiation. Thus, the molecular components present at the responsive (4 day old) developmental stage, but absent at the unresponsive (8 day) stage, are regarded here as developmental “off-switches” which may commit the older tissue to the synthesis of a limited ensemble of gene products. Such commitments are considered central to the onset of the terminal developmental stages of maturity and senescence. The initial studies have focused on the RNA synthetic capacity of chromatin isolated from control and auxin-induced 4 and 8 day old seedlings. While the young tissue displayed a 160% increase in chromatin bound RNA polymerase activity in vitro, the older plant showed only an 18% increase in this parameter following auxin treatment. Investigations with an exogenous source of RNA polymerase ( Escherichia coli) ruled out template inaccessibility to RNA polymerase as a limiting component in 8-day unresponsive chromatin. In addition, new hormone induced transcripts as determined by nearest neighbor analysis of the RNA synthesized in vitro were detected if the reaction was templated by 4-day hormone induced chromatin and its bound RNA polymerases. However, auxin specific transcripts were not observed with 8-day induced chromatin unless the reaction was supplemented with an exogenous RNA polymerase. The findings reported herein suggest that limitations on hormone controlled gene transcription may in part be determined at the RNA polymerase level, and suggest a developmentally programmed change in the specificity of the endogenous RNA polymerase enzymes.

Cellular RNA synthesis in normal and mengovirus-infected L-929 cells.

Cellular RNA synthesis was studied in mouse L-929 cells and in these cells infected with mengovirus. RNA polymerases I, II, and III were partially purified and their chromatographic properties were analyzed by DEAE-Sephadex A-25 chromatography. RNA polymerase II was purified from mouse liver and its subunit structure was compared to that of normal and virus-infected L-929 cells by two-dimensional gel electrophoresis. By these criteria, the enzymes from all three sources were identical. The RNA synthetic activities and capacities of chromatins from normal and virus-infected cells were compared under a variety of conditions. The endogenous activity in chromatin from infected cells was inhibited relative to controls but the residual activity responded normally to stimulation by ammonium sulfate, heparin, and Sarkosyl. The template capacity of the chromatins was compared with added RNA polymerase II and by a rifampicin challenge assay utilizing Escherichia coli RNA polymerase. Identical results were obtained in each case. The number of growing RNA chains and the rates of their elongations were determined. The results showed that nuclei and chromatin from infected cells have a smaller number of RNA polymerase II molecules engaged in RNA synthesis than normal cells do but that the active molecules elongate RNA chains at the same rate.

Transcription of nucleosomes from human chromatin

Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes.

Folded chromosomes of vegetative Bacillus subtilis : composition and properties

The isolation of folded DNA from Bacillus subtilis, a Gram positive bacterium is described. When the lysis was achieved with 1 M NaCl a slow sedimenting nucleoid was obtained (1600-2000 S). Conversely, when the lysis was achieved with 0.2 M NaCl a fast sedimenting nucleoid was obtained (3500-4000 S). The yield of folded DNA was between 80 to 90 % of the total lysate DNA. Both nucleoids contained the same amount of RNA, but the relative proportions of lipids and proteins were different. Folded chromosomes were prepared in the presence of spermidine: artifactual protein binding is shown to be unlikely. Electrophoresis of nucleoid proteins showed a dominant polypeptide (MW = 36,000), which remained associated with DNA after sarcosyl treatment and could be partially removed by heat mediated DNA unfolding. In vitro transcription by endogenous RNA polymerase bound to the fast sedimenting-nucleoid was rifamycin resistant; the template capacity of the fast sedimenting-nucleoid was compared with that of the completely unfolded chromosomes.