Increase In Template Activity Research Articles

Role of RNA and protein synthesis in stimulated germination of zoospores of the pathogenic fungus Phytophthora palmivora

The role of RNA and protein synthesis in differentiating spores of the fungus Phytophthora palmivora was investigated using inhibitors, precursor assimilation, and in vitro translation of directly extracted RNA. Distinct differences in the capacity to take up small molecules, including exogenous precursors of protein and RNA synthesis, were detected between the different stages of differentiation. Zoospores and cysts of this fungus were impermeable to amino acids, glucose, and inorganic phosphate. Uptake of these metabolites was observed only after emergence of the germ tube. By contrast, inhibitors of protein and RNA synthesis penetrated these cells. In the presence of either cycloheximide or actinomycin D, the cells remained as cysts and did not germinate. Although encystment was not dependent on de novo transcription or translation, an increase in the pool of polyadenylated RNA was detected in the cells during this stage. This polyadenylated RNA showed a rapid and sustained increase in template activity, as measured by cell-free translation assays. The increase commenced very early in the cyst stage, immediately following pectin stimulation, and was attributed to the transcription of new mRNA. This transcription during encystment, which was also shown to be inhibited by actinomycin D, appears to be essential for germination in this species of fungus.

Low molecular weight peptidic fraction in the chromatin from normal and cancer cells: control of transcription.

DNA isolated from cell nuclei by intensive deproteinization with chloroform/isoamyl alcohol and phenol extractons contains a low molecular weight peptidic fraction in a quantity of about 20 micrograms/mg DNA. These peptides were characterized by chromatography on CM-Sephadex, Sephadex G-25, high performance liquid chromatography on microBondapak C18 and amino acid composition. The peptides control transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase and stabilize the structure of double stranded DNA, while increasing its melting point. Their level is markedly decreased (by about 40%) in DNA prepared from tumor cells as compared to normal cell DNA. Transcriptional studies showed only a slightly increased template activity of DNA extracted at pH 9.5 versus DNA extracted at pH 6.0 for DNA preparations from tumor cells. However, there was a marked increase in template activity for DNA preparations treated at pH 9.5 from normal cells--232%, 124%, 97% and 78% for rat liver, mouse liver, mouse thymus and fibroblast L-929 cells, respectively. Also there was no difference in the melting point between these two preparations of DNA from tumor cells; normal cell DNA preparations showed increased melting point of preparations treated at pH 6.5. The data obtained indicate that the loss of low molecular weight peptides from tumor DNA during carcinogenesis is responsible for uncontrolled gene expression observed in cancer.

Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies with whole nuclei.

Immature female Sprague-Dawley rats (19-21 days old 50 gm) were used to systematically study DNA-dependent RNA polymerase activity in nuclei from the uterus after administration of 1 mcg estradiol dissolved in .1 ml of 9% NaC1 solution. Controls received only the saline solution. Experimental conditions included an excess of nucleoside 5-triphosphate and short-time assays (6 minutes) at low temperature to obtain the maximal velocity of the reaction and to minimize RNase activity. Nucleotide incorporation was measured in the absence (low ionic strength medium) or the presence (high ionic strength medium) of .25 M ammonium sulfate under various divalent cation concentrations either in the presence or absence of alpha-amanitin. The alpha-amanitin-resistant RNA polymerase activity of nuclei was identical when measured under low ionic strength conditions with either Mg or Mn and under high ionic strength in the presence of Mn (all at 4 mM concentrations). At high ionic strength alpha-amanitin-sensitive activity is about 10 times greater than activity measured at low ionic strength Mn. In all the experiments initiation of new RNA chains was negligible. Estradiol administration leads to: a 50% increase in alpha-amanitin-resistant activity within 1-2 hours under all experimental conditions; a 100% increase by 2 hours in alpha-amanitin-sensitive activity under low ionic strength conditions; but no change in alpha-amanitin-sensitive activity at 6 hours under high ionic strength conditions. Since enzyme activity measured under high ionic strength conditions mainly reflects the number of enzyme molecules engaged in transcription these results suggest that estradiol leads to an early increase in number of alpha-amanitin-resistant molecules engaged in the process of transcription while the number of alpha-amanitin-sensitive molecules remains constant. The increase in alpha-amanitin-sensitive activity obtained under low ionic strength conditions can be interpreted as either an increase in template activity or an activation of the molecules already engaged in the process of transcription or both.(AUTHORS MODIFIED)

Relationship between cell proliferation, chromatin template activity and accumulation of nuclear proteins

Trypsinization of confluent monolayers of WI-38 cells causes an extensive loss of nuclear proteins. The loss of nuclear proteins is restored only several hours after the cells have been replated at a lower density in 10% serum. When trypsinized fibroblasts are replated at a lower density in 10% serum, there is also a sustained progressive increase in the template activity of isolated nuclei, eventually leading to DNA synthesis and cell division. If 0.3% serum is used instead of 10%, there is a modest increase in nuclear template activity, but not accumulation of nuclear proteins and no DNA synthesis cr cell division.

Effects of prolonged quiescence on neuclei and chromatin of WI-38 fibroblasts.

DNA synthesis and cell division are markedly reduced in confluent mono-layers of WI-38 diploid fibroblasts, but resume again if the depleted medium is replaced by fresh medium containing 10% fetal calf serum. If the cells are kept quiescent for prolonged periods of time after confluence (1 or 2 weeks), the fraction of cells that can be stimulated to proliferate by fresh serum decreases and the length of the prereplicative phase increases. The template activity of isolated nuclei decreases with increasing time of quiescence, and parallel changes occur in chromatin as evidenced by circular dichroism spectra and capacity to bind the intercalating dye, ethidium bromide. When WI-38 cells are stimulated to proliferate after prolonged quiescence, the increase in template activity of nuclei is delayed by several hours in comparison to cells stimulated after short periods of quiescence. Two distinct steps, both requiring serum, can be identified in the prereplicative phase of cells stimulated to proliferative after prolonged quiescence. We interpret the results as indicating that, during prolonged quiescence, WI-38 fibroblasts go into a deeper GO state from which they can be rescued only after prolonged stimulation. In this respect, prolonged quiescence may bear some resemblance to the process of aging.

Increased intestinal chromatin template activity. Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes.

1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase. The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol. This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney. The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours. Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo. To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes. After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone. This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol. The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3. It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.

Interactions between twisted DNAs and enzymes: The effects of superhelical turns

Covalently closed bacteriophage PM2 DNA samples containing varying numbers of superhelical turns were used as substrates for the holo and core enzymes of Escherichia coli RNA polymerase, E. coli DNA polymerase I, pancreatic DNAase, and two single strand-specific endonucleases from Neurospora crassa and Mung bean. The results indicate that the superhelical turns influence the properties of DNAs as enzyme substrates in two major ways: (1) if the interaction between a DNA and an enzyme involves the unwinding or winding of the DNA helix, the interaction is strongly affected by the degree of superhelicity of the DNA, since the higher free energy of a twisted DNA favors the reduction of superhelical turns. For phage PM2 DNAs with negative superhelical density, − σ 0 less than about 0.04, this factor is the dominant one. In this range of superhelical density the sensitivity of the DNAs toward endonucleolytic attack by the single strand-specific nucleates increases only slightly with increasing − σ 0. The magnitude of transcription by E. coli RNA polymerase holoenzyme, which is known to unwind the DNA helix slightly, increases rapidly with increasing − σ 0. A similar increase in template activity with increasing − σ 0 has been observed with the core enzyme, and is interpreted as evidence that the productive binding of the core polymerase also requires an unwinding of the DNA helix. (2) When − σ 0 is greater than about 0.04, it appears that the DNA is very tightly wound. Such tight twisting might reduce the binding and/or hinder the movement of certain macro-molecules along the helix. Thus, in this range of superhelix density, the template activity appears to decrease with increasing − σ 0. While the sensitivity of the DNA toward endonucleolytic scission by pancreatic DNAase is independent of σ 0, the sensitivity toward endonucleolytic scission by the single strand-specific nucleases increases markedly with increasing − σ 0 in this range. This is interpreted as resulting from large distortions of the double helix at the sharp bends at which the DNA doubles on itself because of the tight twisting. Such distorted regions become favored sites for the single strand-specific nucleases. For the DNA samples with − σ 0 as high as 0.06, there is no evidence that there is a significant fraction of bases in the single-stranded form. In the presence of oligo-nucleotides as initiators, no significant amount of DNA synthesis on twisted PM2 templates is detected with E. coli DNA polymerase I. While the highly twisted DNA with − σ 0 = 0.06 is nicked by the Mung bean enzyme about 75-fold faster than untwisted PM2 DNA, the rate is still only a few tenths of 1% of that with a single-stranded DNA as the substrate.

Changes in the number of binding sites for ribonucleic acid polymerase in chromatin of WI-38 fibroblasts stimulated to proliferate.

1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [(3)H]UTP into RNA in the absence of reinitiation, and incorporation of [gamma-(32)P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.

Hormonal influences on histones and template activity in the rat uterus.

Uterine chromatin or nuclei was extracted from ovariectomized rats which had been either untreated (U), treated with estradiol (E) or with estradiol plus progesterone (EP). Their abilities to incorporate 3H-UMP into RNA were compared following preincubation at 0, 37 and 37 C in the presence of trypsin. All incorporations after treatment at 0 C were relatively low. Preincubation at 37 C caused little increase with the material from the U- or E-rats. However, the EP rat chromatin or nuclei responded with an impressive increase in template activity. Conversely, further increase by preincubation with trypsin at 37 C was least for the EP rat. Uterine histone profiles were examined. Preincubation at 37 C produced little change in the uterine acrylamide gel histone profiles of the U- or E-rat. However, extensive degradation of the lysine-rich histones, coupled with other changes were observed in the EP rat uterine histone profiles. Pregnant rat (17–19 days gestation) uterine nuclei behaved like the EP rat with r...

Non-histone proteins and gene activation in regenerating rat liver

Chromatin of 6 h regenerating rat liver exhibits a 2-fold increase in template activity over normal liver chromatin in promoting RNA synthesis in vitro. The RNAs transcribed from the 6 h regenerating liver chromatin also contain new RNA species not detectable in normal transcript. Reconstitution of chromatin from its constituents of normal and 6 h regenerating rat liver shows that the non-histone proteins, but not DNA and histones, determine the specific transcription of chromatin, and are responsible for the derepressed genetic state of the regenerating liver.

Age-dependent changes in the structure and function of mammalian chromatin—I. Variations in chromatin template activity

The in vitro template activity of chromatin isolated from submandibular gland increases threefold as rats age from two to twelve months. The increased capacity for DNA-primed RNA synthesis cannot be attributed to differences in nuclease or protease activity, and there are no variations in the ability of young and older submandibular gland DNA to transcribe RNA under these conditions. The histone content of young and older submandibular gland chromatin is similar. However, these proteins are less tenaciously bound to the DNA with age; consistent with the age-dependent increase in template activity. In contrast, there is an age-dependent decrease in the quantity of nonhistone chromosomal proteins associated with submandibular gland chromatin.

Effect of homologous RNA polymerase on the increase in chromatin template activity of stimulated fibroblasts

Confluent, non-dividing monolayers of WI-38 human diploid fibroblasts can be stimulated to synthesize DNA and divide by a change of medium and serum. Within one hour of stimulation there is an increase in the template activity for RNA synthesis of chromatin. This change in chromatin template activity is seen using either a heterologous E. coli or a homologous HeLa cell RNA polymerase. This strengthens the conclusion that genome activation is one of the earliest changes in the stimulated cells.

Composition, template properties and thermostability of liver chromatin from rats of various age at deproteinization by NaCl solutions

The composition, template activity in RNA-polymerase systems of RNA-synthesis and thermal stability of rat liver chromatin in animals of various age (1, 3, 12 and 30 months) were investigated after deproteinization with NaCl solutions of growing concentrations. Dissociation changes of arginine-rich histones were seen in 30 months old rats, testifying to a strengthening of those protiens' bonds with DNA. With age, there is also an increase in the thermostability of the chromatin proteinic fraction containing residual histones. The drop in template activity of old (30 months) rat liver chromatin in the RNA-polymerase system of RNA synthesis is associated predominatingly with changes in the protein fraction which dissociates at the interval of 1·25-2·0 M NaCl, where a strengthening of histones-DNA bonding is observed, as well as a decrease in the acid proteins. The increase in template activity of old rat liver chromatin in proportion to NaCl concentration increase proceeds much slower than in the chromatin of young (1 month old) rats.

Gene activation in WI-38 fibroblasts stimulated to proliferate: requirement for protein synthesis.

Confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by fresh medium containing 30% fetal-calf serum. Up to 80% of the cells are stimulated to divide, with a peak of DNA synthesis between 15 and 21 hr. 1 hr after the change of medium there is a 70% rise in chromatin template activity. Cycloheximide inhibited the increase in chromatin template activity. A requirement for RNA synthesis was investigated by incubating stimulated and unstimulated cells with 10 mug/ml of actinomycin D. In spite of a 95% inhibition of RNA synthesis in whole cells, purified chromatin from stimulated cells showed the usual increase in template activity. These experiments implicate a requirement for protein synthesis in template activation, and imply that the synthesis of this (or these) protein(s) is independent of RNA synthesis and regulated by a purely translational mechanism.

Diurnal rhythmicity in template activity of mouse liver chromatin

A study was initiated to define the basal state of gene expression in the mouse liver prior to an investigation of responses during stress. It was found that: 1. 1. The ability of isolated mouse liver chromatin to be transcribed in vitro is increased when the chromatin is taken during hours of darkness rather than during the day. It is suggested that this night-time increase in template activity is related to the rhythmic pattern in other phenomena of gene expression in the rodent liver. 2. 2. The magnitude of the periodic increase in template activity is diminished in the adrenalectomized animal, suggesting the involvement of glucocorticoid secretion. 3. 3. The extent of the nocturnal rise in activity is not affected by starvation of the mice. A simple hepatic response to periodic ingestion of the diet appears to be an unlikely source of rhythmicity.

In vitro RNA synthesis and nuclear proteins of isolated sea urchin embryo nuclei

Isolated nuclei and/or chromatin from Strongylocentrotus purpuratus gametes and early developmental stages were analyzed for their basic protein content as well as their ability to serve as templates for in vitro RNA synthesis. 1. 1. Sperm chromatin exhibited little or no template activity. Unfertilized egg nuclei directed 1.5 % of the RNA synthesized by pure sperm DNA. This level of template activity increased at 32–64 cell, blastula and gastrula stages reaching a maximum of 6 % during pluteus stage. 2. 2. Trypsin digestion of sperm chromatin and blastula nuclei caused a 6–8-fold increase in template activity. Similarly treated egg nuclei showed no increase in available template. Sonication of egg and blastula nuclei resulted only in a slight increase in template activity. This was less pronounced for egg nuclei. Sonicated and trypsinized blastula nuclei showed an increased template activity over controls. Similar treatment of egg nuclei resulted in no release of genetic restriction. 3. 3. Typical histone patterns could not be electrophoretically demonstrated from extracted nuclear protein prior to blastula stage. 4. 4. The above results indicate a lack of histones during early sea urchin development although there is substantial template restriction.

Influence of trypsin on in vitro RNA synthesis by disrupted nuclei of rat and human uteri in different hormonal states.

Ovariectomized rats were either untreated, treated with estradiol, or treated with estradiol plus progesterone for 3 days. Nuclear preparations of the uteri were incubated with 3H-UTP, ATP, CTP and GTP and excess RNA polymerase with and without preincubation with trypsin. The study focused on the enhancement of template activity mediated by trypsin. Five experiments were performed. In incubations of nuclei from estradiol-treated rats the increase in template activity mediated by trypsin ranged from 327 to 651 %. The results with the untreated rats were not statistically different from those with the estradiol-treated rats. On the other hand, the enhancement of template activity with the nuclei obtained from rats treated with estradiol plus progesterone was far less, ranging from 34 to 127%. When the rats were treated with progesterone alone (2 experiments), the average increase was 316%. Nuclear preparations of human endometrium were studied in the same way. In 17 studies of proliferative endometrium, the average trypsin-mediated increase was 369%, whereas with secretory endometrium (13 cases) the average was 143%.

Template activity of chromatins isolated from regenerating rat liver.

1. The priming (template) activities for RNA synthesis of normal and regenerating rat liver chromatins were investigated using E. coli RNA polymerase [EC 2. 7. 7. 6] and isolated chromatins. A slightly higher template activity was observed in regenerating liver chromatin but no detectable difference in the ratio of histone to DNA compared to that of normal liver chromatin. 2. The template activities of normal and regenerating liver chromatins were compared after removal of different extents of histone with citric acidNaCl. The difference in the template activities of normal and regenerating liver chromatins were still apparent up to a stage of selective removal of lysine-rich histones, but disappeared when most of the histones were removed. 3. Removal of histones with dilute HC1 caused some structural damage of the chromatins resulting in change in the template activity and melting profile. 4. The addition of histones after their removals from chromatins inhibited template activity slightly in contrast to the dramatic increase in template activity seen on removal of histones from chromatins. 5. The regulatory roles of histones in the synthesis of are discussed on the basis of the results.

The effect of salt extraction and heat-denaturation on the behavior of rat liver chromatin

Rat liver chromatin which has been extracted with NaCl at concentrations up to 0.9 M shows a biphasic melting profile and a coincident increase in template activity for RNA synthesis. Heat denaturation of chromatin which has not been subjected to salt extraction shows an enhanced template activity compared to that of the chromatin in its native state. Chromatin extracted with 0.6 M NaCl exhibits a decreased template activity after heat denaturation. The protein content and ultraviolet absorbance characteristics of rat liver chromatin have also been determined in relation to NaCl extraction.

On the mechanism of hormone action. VIII. Further studies on the effect of cortisol on isolated rat-liver nuclei

The incubation of isolated rat-liver nuclei in vitro in the presence of cortisol led to an increase in template activity of the nuclear RNA, measured in a cell-free amino acid-incorporating system. This increase in template activity of nuclear RNA is evident after 15 min of incubation. Nuclei from adrenalectomized rats respond to lower concentrations of cortisol than nuclei from normal rats. Very high concentrations of cortisol have an inhibitory effect on template activity. The absence of any effect of testosterone, progesterone, androstenedione, pregnenolone and 17α-hydroxyprogesterone point to the specificity of the effect of cortisol.