Initiation of RNA Synthesis in Isolated Nuclei (original) (raw)
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Studies on ribonucleic acid synthesis in nuclei isolated from rat liver
Biochimica et biophysica acta, 1969
I. Meaningful interpretation of studies pertaining to RNA synthesis using isolated nuclei cannot be made unless the factors which modify the nature and amount of the product obtained are defined and examined in detail. This is apparent in rat liver nuclei because the level of incorporation of r3H]UMP and [3H]AMP into RNA which we found (and which is quite comparable to those of other workers), indicates that less than 0.1 oh of the DNA is transcribed. 2. Less incorporation was observed at 37' than at 25" or 30'. After 90 min, there was a 50 o/o loss of incorporated material at 37' and a 20 y. loss at 30". Nuclei preincubated in suspending medium and then assayed for RNA polymerase lost activity in direct relation to the time and temperature of preincubation. Enzyme inactivation at the higher temperature was at least partially involved because at a time when no further incorporation of label occurred, exogenous Escherichia coli RNA polymerase caused further synthesis when added to the incubation mixture. By autoradiography, it was determined that the great majority of nuclei were actively synthesizing RNA. 3. There was an inverse correlation between nuclear ribonuclease activity and [3H]UMP incorporation and the ribonuclease activity was much greater at 37". The stimulation of RNA synthesis by deoxycholate and (NH&SO, reported by other workers was shown to be partially, if not wholly due to inhibition of nuclear ribonuclease activity. 4. Nuclear nucleoside triphosphosphatase was demonstrable and was capable of degrading the triphosphate of adenosine, guanosine, cytosine, and uridine. There was no inverse correlation between the activity of this enzyme and RNA synthesis because of the presence of a nucleoside diphosphokinase activity.
Initiation by the DNA-dependent RNA polymerase
Proceedings of the National Academy of Sciences, 1966
This communication presents evidence which indicates that RNA synthesis by the DNA-dependent RNA polymerase occurs in three steps: (1) Association: DNA + Enzyme = DNA-enzyme. (2) Initiation: DNA-enzyme + purine nucleotide > [DNA-enzyme-purine1 L nucleotide I (3) Polymerization: [DNA-enzyme-purine] + NTP DNA-enzyme-oligoribonucleotide + PP1. The formation of the DNA-enzyme complex, step 1, can be inhibited by high ionic strength. When initiation occurs, a different DNA-enzyme complex is formed in the presence of purine nucleoside triphosphates which is not as easily dissociated by high ionic strength. The initiation complex can also be detected by a specially devised membrane assay. The exact nature of this complex is not established, but for its formation, a relatively high level of purine nucleotide is required. It will be shown that the association DNA-enzyme complex differs from the initiation DNAenzyme complex. The process of initiation is rate-limiting at low nucleoside triphosphate concentrations and purine nucleotides in relatively high concentrations overcome this limitation. In step 3, there is no differential effect of purine over pyrimidine nucleotides. The effect of purine nucleotides on initiation correlates with the observations of Maitra and Hurwitz' that the purine nucleoside triphosphates are the predominant 5'-terminal nucleotides found in RNA synthesized in vitro.
DNA-dependent RNA synthesis in nuclear chromatin of fixed cells
Experimental Cell Research, 1973
Fixed mouse kidney epithelial cells have been examined for their capacity to synthesize RNA with their own RNA polymerases when supplied with ribonucleoside triphosphates. The endogenous polymerase activity of chromatin in fixed cells is clearly related to changes in the size and protein content of the nucleus. Cells with small nuclei which do not incorporate $H-uridine in vivo show very little RNA polymerase activity at the ionic strength of the standard assay procedure. This activity can be enhanced by increasing the ionic strength of the assay medium. Changes in RNA polymerase activity also appear to be related to changes in the ability of chromatin to bind acridine orange (AO).
The role of nuclear proteins in RNA synthesis
Experimental Cell Research, 1972
When epithelial cells from baby mice are cultured on a solid substrate in vitro, DNA synthesis and cell division take place in a majority of the cells 24 to 48 h after inoculation. This cell proliferation is regularly preceded by a sequence of nuclear changes likely to be involved in gene activation during the process of growth-transformation. These nuclear changes, which in the present investigation were studied by quantitative cytochemical methods, are characterized by: (1) increased binding of acridine orange (AO) very soon after cell attachment, reflecting initial changes of the deoxvribonucleoprotein (DNP) complex: (2) accumulation of uroteins in the cell nucl&s; (3) dispersion of the nuclear cdromatin; (4) increased rate of 14C-uridine incorporation. The initial DNP change. reflected by the increased AO-binding, was per se not a sufficient factor for stimulation of-14C-uridine incorporation. An increasedgH-uridine incorporation was only observed when protein began to accumulate in the nucleus and when the chromatin changed from a condensed to a more dispersed state. The rate of 14C-uridine incorporation was, during the whole process of growth transformation, found to be directly proportional to the amount of protein that had accumulated in the nucleus. However, the initial DNP change was a necessary prerequisite for the subsequent accumulation of protein in the nucleus. Attachment of the cells to the solid substrate was found to be necessary in expressing this sequence of nuclear events. Furthermore, in areas on the glass slide where many cells had attached, these nuclear changes occurred more rapidly.
Chromatin as a template for RNA synthesis in vitro
Proceedings of the National Academy of Sciences, 1975
RNA transcribed in vitro from myeloblast chromatin by exogenously added RNA polymerase B predominantly consists of short chains that remain in hybrid structure with the template; the remainder of the product is free RNA of heterogeneous size. Addition of polyanions during synthesis caused an increase in the size and amount of free RNA with a concomitant decrease in the proportion of small RNA. The large molecular weight RNA is derived from the short RNA chains, which are synthesized de novo during the reaction in vitro. The effect of polyanions on the size and nature of the product may be related to structural changes induced in the template rather than to an inhibition of nuclease activity. Cells infected with RNA tumor viruses contain virus-specific information in the form of DNA in their genome (1); transcription of this (proviral) DNA is thought to be an obligatory step in viral replication. Since progeny RNA is probably synthesized by host nucleoplasmic RNA polymerase (2, 3) and viral RNA in transcript products can be detected with complementary probe DNA, these cells offer a system for studying the expression of specific genes in eukaryotes. Recently, we reported the presence of virus-specific RNA in products synthesized in vitro from myeloblast chromatin by exogenously added eukaryotic RNA polymerase B (2). This chromatin was isolated from myeloblasts of chickens infected with avian myeloblastosis virus (AMV). The proportion of virus-specific sequences in transcript products (1-2%) was approximately the same as that in isolated infected cells (4) and that synthesized in isolated myeloblast nuclei (2). Since the amount of viral RNA in the product was 102to 103-fold higher than expected from random transcription, it appeared that the RNA synthesizing system in vitro maintained some of the specificity exhibited during transcription in vivo. For this reason, we have examined some properties of chromatindirected RNA synthesis in vitro. In this communication we present studies on the rate of chromatin transcription, the structure and size of RNA products formed, and the effect of polyanions on RNA synthesis. The results of these studies are consistent with the suggestion that gene activation in eukaryotes may be caused by an unwinding of chromatin DNA (5). MATERIALS AND METHODS Cells and Enzymes. Myeloblasts from AMV infected chickens were generously supplied by Dr. J. W. Beard, Life
Journal of Molecular Biology, 1978
A mild enzymic procedure for fractionating nuclei was based on a combination of light-scattering properties and electron microscopy in order to monitor the structural integrity of rat liver nuclei and to establish the gentlest conditions possible for their disruption. Incubation of nuclei with as little as 0.1 unit of micrococcal nuclease per ml for 60 seconds at 20 to 29"C, followed by EGTA, caused their total disruption with minimal perturbation of chromatin or transcriptional characteristics. A simple two-step differential centrifugation resolved the gently disrupted nuclei into three mechanically unsheared fractions. One of these (fraction P2) consisted of aggregates of 6 to 30 covalently linked nucleosomes, each containing about 200 base-pairs of DNA, and which are here termed polynucleoeomes. This fraction represented about 10% of nuclear DNA (200 to 6000 base-pairs), whose properties corresponded to euohromatin prepared by other methods. Since there is virtually no reinitiation of RNA synthesis in vitro by isolated nuclei or subnuclear preparations, endogenous RNA polymerase activities represent the elongation of RNA chains that were initiated in vivo. When the distribution of free (inactive in the absence of exogenous template) and endogenous template-engaged RNA polymerases I(A) and II(B) was monitored, the latter as an index of transcriptional complexes that existed in the intact nucleus, mild nuclease digestion was found not to alter the autonomous transcriptional characteristics of the disrupted nuclei. Over 85% of the template-engaged RNA polymerase II was recovered with polynucleosomes. Further nuclease digestion of this fraction showed that a minimum of six nucleosomesjunit was necessary for retaining the enzyme on its template. Polynucleosomes can therefore be considered as the basic structural units of chromatin which are selectively released by extremely mild enzymic treatment of whole nuclei from the transcriptionally active compartment and are capable of continuing *in vitro the elongation of RNA chains initiated in. viva.
Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis
The Journal of cell biology, 1980
A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).
The path that RNA takes from the nucleus to the cytoplasm: a trip with some surprises
Histochemistry and cell biology, 2002
It is now clear that nuclear context is playing an essential role in gene expression. For this reason we have developed methods to study gene expression in situ. Transcription takes place in discrete foci in the nucleoplasm of mammalian cells. These sites concentrate several RNA polymerases II and factors involved in the production of mRNA. Moreover, these sites also contain the active machinery to carry out protein synthesis. Once the mRNA leaves the transcription site, it interacts with the nuclear pore complex and the mRNA is exported using the filaments of the nuclear pore complex in to the middle part of the nuclear pore complex and finally is released in the cytoplasm.
Relationship Between Rna Synthesis, Cell Division, and Morphology of Mammalian Cells
Journal of Cell Biology, 1966
Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P32 and H3-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division ...