Rna Synthesis by Exogenous Rna Polymerase on Cytological Preparations of Chromosomes (original) (raw)
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A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments
Nucleic Acids Research, 1977
Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E.coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA result from a DNA-dependent reaction.
Proceedings of the National Academy of Sciences, 1984
Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent pre-rRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle.
Experimental Cell Research, 1987
Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.
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).
Effect of cycloheximide on RNA synthesis in Chironomus polytene chromosomes
Chromosoma, 1977
Modifications in the synthesis of salivary gland RNA were induced by treatments with 10 microgram/ml cycloheximide (CHM) on 4th instar larvae of Chironomus pallidivitattus. After 3, 6 and 24 h CHM treatment, RNA was labeled "in vitro", by incubating the salivary glands in a medium containing H3-uridine. The electrophoretical analyses corresponding to the 3 and 6 h treatment showed a stimulation of the non-ribosomal components of the newly synthesized RNA, while preribosomal RNA synthesis appeared depressed. This fact was also confirmed at cytological level, since autoradiograms made after 3 h of CHM treatment showed a reduced H3-uridine label over the nucleolus and an increase of diffuse labeling over the chromosomes. Longer treatments (24 h) causes a considerable inhibition of the synthesis of all RNA species. The role played by protein synthesis inhibition in the aforementioned effects is discussed.--Some of the morphological implications of CHM treatment, such as modifications of the nucleolar structure (nucleolar segregation) are also reported. The use of a squash technique based on glutaraldehyde fixation of the salivary glands, considerably facilitates such studies.
Ribosomal and non-ribosomal RNA synthesis in vitro
Biochimica et biophysica acta, 1975
The synthesis of total and ribosomal RNA using nucleoids of Escherichia coli as template was measured; of the total RNA synthesized by endogenous RNA polymerase which only completes chains, and added RNA polymerase which initiates new chains, 50-70 and 3-5%, repectively, was rRNA. Total RNA synthesis by added enzyme, however, was 10-20 times higher than endogenous RNA synthesis; thus rRNA was synthesized at the same rate by the endogenous and the added enzyme. We conclude that the percentage rRNA in vitro is no measure of the rate of rRNA synthesis. Furthermore, it follows that the added enzyme, like the endogenous one, is packed at the physical limit on the ribosomal cistrons. Consequently, initiation of ribosomal cistrons by added enzyme was at or near the maximal rate possible for this system in which the elongation rate is 10-20% of that in vitro. When RNA synthesis was assayed at various ratios of RNA polymerase to phenol-extracted DNA, the amount of rRNA made per DNA, which is...
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.
The RNA polymerases of Drosophila melanogaster during early development
Biochemical Genetics, 1985
The enzymatic basis for the changing rates of RNA synthesis in earlyDrosophila development has been examined through a description of the multiple forms, relative proportions, and total and specific activities of the RNA polymerases of theDrosophila embryo. Six chromatographically identifiable forms of RNA polymerase are detectable in embryos (Forms Ia, Ib, IIa, IIb, IIIa, IIIb), one of which (Form IIa) appears to be cytoplasmically localized and is present only in unfertilized eggs and pregastrular embryos, while another (Form IIb) appears only at gastrulation and remains throughout the remainder of development. The total and specific activities of the embryonic RNA polymerases are related to the patterns of embryonic RNA synthesis and to the nuclear and cellular events of holometabolous development.
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.
European Journal of Biochemistry, 1972
The role of protein factor B and C on RNA synthesis by RNA polymerase CI isolated from chromosomal non-histone proteins of coconut nuclei has been studied further, Factor B has been implicated as the initiation factor on the experimental evidences that (a) in its absence, RNA polymerase CI shows only minimal activity; (b) it can bind with RNA polymerase and the enzyme * factor B complex then binds to DNA, but factor B alone can not bind to DNA; (c) it promotes the incorporation of [ , ! ?, y-S2P,]ATP into RNA and this stimulation reaches a plateau rather quickly while the incorporation of [14C]ATP in the interior of RNA chain continues; (d) it is active with native homologous DNA as template, but not with denatured or A DNA; (e) RNA molecules synthesized in its presence are of higher sedimentation value (10-20 S) than that synthesized in its absence (4 S) ; (f) it can completely counteract the inhibitory effect of rifampicin, of RNA synthesis by rifampicin is reversible by factor B a t high concentration. RNA polymerase CI seems to be sensitive to cc-amanitin whereas RNA polymerase CII is comparatively resistant.