Showing papers on "Transcription (biology) published in 1974"
TL;DR: The data discussed herein demonstrate the great variation in target-tissue response that can occur after administration of steroid hormones, and direct quantitative evidence that sex steroids cause a net increase in the intracellular amounts of specific mRNA molecules in target tissues is provided.
Abstract: The data discussed herein demonstrate the great variation in target-tissue response that can occur after administration of steroid hormones. The female sex steroids can exert regulatory effects on the synthesis, activity, and possibly even the degradation of tissue enzymes and structural proteins. Each response, nevertheless, appears to be dependent on the synthesis of nuclear RNA. In many instances, the steroid actually promotes a qualitative change in the base composition and sequence of the RNA synthesized by the target cell, implying a specific effect on gene transcription. Most important is our direct quantitative evidence that sex steroids cause a net increase in the intracellular amounts of specific mRNA molecules in target tissues. It thus appears that we are discovering a pattern of steroid hormone action which includes (Fig. 1): (i) uptake of the hormone by the target cell and binding to a specific cytoplasmic receptor protein; (ii) transport of the steroid-receptor complex to the nucleus; (iii) binding of this "active" complex to specific "acceptor" sites on the genome (chromatin DNA and acidic protein); (iv) activation of the transcriptional apparatus resulting in the appearance of new RNA species which includes specific mRNA's; (v) transport of the hormone-induced RNA to the cytoplasm resulting in synthesis of new proteins on cytoplasmic ribosomes; and (vi) the occurrence of the specific steroid-mediated "functional response" characteristic of that particular target tissue. To elucidate fully the mechanism of steroid hormone action we must study the biochemistry of the process by which information held by the steroid hormone-receptor complex is transferred to the nuclear transcription apparatus. If our assumptions are correct, we should ultimately be able to discover how this hormone-receptor complex exerts a specific regulatory effect on nuclear RNA metabolism. Such regulation might be achieved (i) by direct effects on chromatin template leading to increased gene transcription and thus RNA synthesis; (ii) by activation of the polymerase complex itself; (iii) by inhibition of RNA breakdown; or (iv) by intranuclear processing of large precursor molecules so that smaller biologically active sequences are produced, and (v) by transport of RNA from the nucleus to the cytoplasmic sites of cellular protein synthesis.
866 citations
TL;DR: Approximately 35,000 different poly(A)-containing RNA sequences are present in HeLa cell cytoplasm and the sequences are grouped in three distinct abundance classes.
Abstract: Approximately 35,000 different poly(A)-containing RNA sequences are present in HeLa cell cytoplasm. The sequences are grouped in three distinct abundance classes.
513 citations
484 citations
TL;DR: The role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species is unequivocally demonstrated and rule out nonspecific inhibitory effects in the endogenous systems.
Abstract: Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated IIIA and IIIB). The enzymes are unaffected by low α-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for IIIA and IIIB) at higher toxin concentrations. RNA polymerase I was unaffected at all α-amanitin concentrations tested. Myeloma RNA polymerases II, IIIA, and IIIB appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic α-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases.
Isolated myeloma nuclei and nucleoli continue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by α-amanitin, but only at high concentrations; and the α-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases IIIA and IIIB. In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by α-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high α-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems.
These results unequivocally demonstrate the role of RNA polymerase III (IIIA and/or IIIB) in the synthesis of (pre) 4S RNAs and a 5S RNA species.
323 citations
TL;DR: Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation.
Abstract: Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA.
314 citations
TL;DR: A series of isonuclear cytoplasmic petite (ϱ−) mutants presenting various patterns of large deletions and retentions in the C321R — E514R region was used to study the functional organization of mitochondrial DNA.
225 citations
TL;DR: It is proposed that the transcription complex is influenced by N-product to become termination-resistant at a site in the phage genome (juggernaut model) that appears to be at or near thephage promoter.
Abstract: The induction of λ prophage provokes the constitutive expression of the adjacent gal operon in E. coli. This “escape synthesis” can result from transcription that initiates at a phage promoter and extends into the gal operon. The effect requires the product of the λ gene N. N-mediated transcription not only fails to terminate at the prophage-bacterial junction and at the ends of bacterial operons, but ignores termination signals caused by polar insertions or ochre mutations within gal. Suppression of polarity by N-function is a cis-effect; only transcription initiated at the phage promoter is influenced. We propose that the transcription complex is influenced by N-product to become termination-resistant at a site in the phage genome (juggernaut model). This site appears to be at or near the phage promoter.
182 citations
TL;DR: The enzymatic transcription of DNA from the 70S RNA of Rous sarcoma virus is initiated on the 3' terminus of a molecule of 4S RNA which is hydrogen bonded to the viral genome, and this primer served as the principal primer for initiation of DNA synthesis by both detergent-disrupted virions of RSV and purified RNA-directed DNA polymerase with RSV 70SRNA as template.
Abstract: The enzymatic transcription of DNA from the 70S RNA of Rous sarcoma virus (RSV) is initiated on the 3′ terminus of a molecule of 4S RNA which is hydrogen bonded to the viral genome. We labeled this primer with radioactive deoxynucleotides, and demonstrated that its release from 70S RNA by thermal denaturation was accompanied by a reduction in the template activity of the viral RNA. Two-dimensional electrophoresis in polyacrylamide gels separated the 4S RNAs associated with the 70S RNA of RSV into approximately eight fractions, each of which appeared to contain a discrete species of tRNA. The RNA in one of these fractions served as the principal primer for initiation of DNA synthesis by both detergent-disrupted virions of RSV and purified RNA-directed DNA polymerase with RSV 70S RNA as template.
168 citations
TL;DR: DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2.0 to demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA.
Abstract: DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of α-amanitin (50% inhibition at 0.02 μg/ml), RNA polymerase III was completely sensitive to high concentrations of α-amanitin (50% inhibition at 20 μg/ml) and RNA polymerase I was totally resistant to concentrations of α-amanitin less than or equal to 200 μg/ml. RNA synthesis by the endogenous RNA polymerase activities in nuclei isolated from infected cells was completely sensitive to α-amanitin, thus suggesting that RNA polymerase I is not involved in viral DNA transcription even though it is present in these cells. The α-amanitin inhibition curve was biphasic and showed inflection points at about 0.02 and 20 μg/ml, suggesting the participation of both RNA polymerases II and III in the synthesis of RNA in these nuclei. Furthermore, at least a large fraction of the synthesis of the nuclear precursors to viral mRNA, monitored by hybridization to viral DNA, showed the same sensitivity to α-amanitin as did RNA polymerase II; and the synthesis of both viral 5.5S RNA and (presumably cellular) 5S RNA in the isolated nuclei exhibited the same sensitivity to α-amanitin as did purified RNA polymerase III. Thus, these data provide strong supporting evidence for previous studies which suggested the involvement of an RNA polymerase II in transcription of the adenovirus genome and demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA.
167 citations
TL;DR: It is concluded that transcription initiated at p L by E. coli RNA polymerase is so modified by interaction with N protein that even distant nonsense codons are no longer read as a cause of polarity.
152 citations
TL;DR: The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs, which supports the hypothesis that each of the 35S RNA subunits of M- Mu LV 60- 70S RNA has a different base sequence.
Abstract: The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.
TL;DR: In mouse cell lines derived from mammary adenocarcinomnas, the synthetic steroid dexamnethasone stimulates production of murine mammary tumor virus and provides a possible tissue culture source of this virus and a model system for studying the mechanism of action of corticosteroids and the regulation of transcription of integrated viral DNA.
Abstract: In mouse cell lines derived from mammary adenocarcinomnas, the synthetic steroid dexamnethasone stimulates production of murine mammary tumor virus. Viral RNA and antigens are increased as much as 20-fold, and culture fluid supernatants from steroid-treated cells contain type B particles with reverse transcriptase. These cells provide a possible tissue culture source of this virus and a model system for studying the mechanism of action of corticosteroids and the regulation of transcription of integrated viral DNA.
TL;DR: The subunits α, β and β′, the constituents of core portion of RNA polymerase, were found to be produced coordinately during steady-state growth at different rates within the range examined and to be metabolically as stable as the bulk of protein.
Abstract: SummaryIn an effort to elucidate the control of synthesis of the DNA-dependent RNA polymerase in Escherichia coli, intracellular amounts of the individual subunits were determined by polyacrylamide gel electrophoresis of cell lysates and of precipitates formed with specific antibody against holoenzyme I.Polyacrylamide gel electrophoresis of cell lysates in the presence of sodium dodecyl sulfate has been believed to separate the two larger subunits, β and β′, of RNA polymerase from the bulk of protein in E. coli. However, a polypeptide unrelated to the polymerase was found to migrate in the immediate vicinity of the β′ subunit, interfering with the accurate measurement of this subunit. Taking account of the presence of this peptide, designated tentatively as χ, the quantity of RNA polymerase was estimated relying only on the β subunit.Subunits content was also measured by polyacrylamide gel electrophoresis of the precipitates formed by treating cell lysates with anti-holoenzyme I serum. Since the isolated individual subunits as well as the holo- and core enzyme could be precipitated by the antibody, the present procedure permitted to determine total amounts of the subunits within cells.The subunits α, β and β′, the constituents of core portion of RNA polymerase, were found to be produced coordinately during steady-state growth at different rates within the range examined (0.22 to 1.87 generations/hr) and to be metabolically as stable as the bulk of protein. The rate of synthesis of these subunits relative to the total protein was found to be balanced with the growth rate; the differential rate of synthesis of enzyme core (αp) can be represented by the following empirical equation: αp(%) = 0.7 μ + 0.45, where μ represents growth rate (generation/hr). In contrast, the content of σ subunit was considerably small, i.e. only about one third mole equivalent to enzyme core, and was almost unaffected by the rate of growth.
TL;DR: The fact that nuclear poly (A) is not quantitatively converted to cytoplasmic poly(A) indicates that polyadenylation of a heterogeneous RNA molecule is not sufficient to ensure that it will be properly processed and transported to the cytopLasm.
TL;DR: The transcripts of native and reconstituted chromatins were indistinguishable in respect of both the total yield of the RNA and the fractional yield of globin-specific sequences.
Abstract: Chromatin was reconstituted from the purified DNA and histones of chicken erythrocytes and the nonhistone proteins of either chicken reticulocytes or chicken liver. Reconstituted chromatins, native chicken reticulocyte chromatin, and free DNA were transcribed with Escherichia coli RNA polymerase and the concentrations of globin-specific sequences in the RNA products were measured by hybridization with [3H]DNA complementary to chicken globin messenger RNA. Reticulocyte, but not liver, nonhistone proteins were shown to activate the globin genes in reconstituted erythrocyte chromatin. The transcripts of native and reconstituted chromatins were indistinguishable in respect of both the total yield of the RNA and the fractional yield of globin-specific sequences.
TL;DR: A positive factor enhancing the expression of this operon binds at the promoter, but exerts its effect downstream on a DNA region which acts as an attenuator of transcription.
Abstract: A positive factor enhancing the expression of this operon binds at the promoter, but exerts its effect downstream on a DNA region which acts as an attenuator of transcription.
TL;DR: There is no widespread elimination of globin genes in non-erythroid somatic mouse tissues, and globin cDNA is a faithful, but partial, transcript of reticulocyte 9 S RNA, which contains both o and β globin messenger RNAs.
TL;DR: The dependence of the viral transcription on the presence of SAM and the methylation of terminal nucleotide suggests that the transcription of CPV is a "methylation-coupled" reaction.
Abstract: S-adenosyl-L-methionine (SAM) activated the virus-associated RNA polymerase of cytoplasmic polyhedrosis virus in vitro. Synthesis of single-stranded viral RNA (mRNA) proceeded depending on the presence of SAM.A methyl residue of SAM was incorporated into an RNA molecule. A ribose moiety of adenylic acid in the 5'-terminal region of the nascent RNA was methylated in the very early stage of the transcription. The dependence of the viral transcription on the presence of SAM and the methylation of terminal nucleotide suggests that the transcription of CPV is a "methylation-coupled" reaction.
TL;DR: Division of the RSV-infected chicken embryo fibroblast is required for the initiation of transcription of virus RNA, but it is not necessary for the maintenance of transcription in stationary cells.
Abstract: Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) did not synthesize virus RNA until after division of the infected cells. Initiation of RNA transcription after cell division resulted in transcription of the entire viral genome and was followed shortly thereafter by appearance of progeny virus. Addition of colchicine to RSV-infected stationary chicken embryo fibroblasts resulted in inhibition of cell division and a 24-h delay in the appearance of virus RNA. RSV-infected chicken embryo fibroblasts made stationary 10 to 15 cell generations after infection continued to produce an amount of progeny virus similar to that produced in dividing RSV-infected cells. Teh virus produced from cells made stationary after virus production had begun contained RNA synthesized in stationary cells. Therefore, division of the RSV-infected chicken embryo fibroblast is required for the initiation of transcription of virus RNA, but it is not required for the maintenance of transcription of virus RNA.
TL;DR: It is possible that deinduction may proceed by way of a decrease in messenger RNA production, leading to a rapid fall in the synthesis of phosphoenolpyruvate carboxykinase.
Abstract: The rate of synthesis of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in rat liver decreased with a half-life of 30 min after fasted rats were refed either a chow diet or glucose. A requirement for both glucose and insulin to bring about this rapid deinduction was shown, as well as the ability of dibutyryl adenosine 3′:5′-cyclic monophosphate to block the decrease in enzyme synthesis. Estimates of the stability of messenger RNA of phosphoenolpyruvate carboxykinase were made by using the inhibitors actinomycin D and cordycepin to block further messenger RNA synthesis, and then measuring the decrease in specific enzyme synthesis. It is suggested that the use of actinomycin D yields an overestimation of the template stability. The results with cordycepin imply that the enzyme messenger RNA has a short half-life of approximately 1 hr. Thus, it is possible that deinduction may proceed by way of a decrease in messenger RNA production, leading to a rapid fall in the synthesis of phosphoenolpyruvate carboxykinase.
TL;DR: The correct transcription of the hut (histidine utilization) operon DNA of Salmonella typhimurium requires activation either by 3':5'-cyclic adenosine monophosphate and catabolite-activating protein or by nonadenylylated glutamine synthetase.
Abstract: The correct transcription of the hut (histidine utilization) operon DNA of Salmonella typhimurium requires activation either by 3′:5′-cyclic adenosine monophosphate and catabolite-activating protein or by nonadenylylated glutamine synthetase (EC 6.3.1.2.). Thus, glutamine synthetase plays a regulatory role distinct from its enzymatic function.
TL;DR: It is concluded that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.
Abstract: The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.
TL;DR: Evidence indicates that viral polypeptide abundances are specified at the transcriptional level and the requirement for capsid proteins in RNA replication suggests that these proteins are positive control elements for replication of paramyxovirus RNA.
Abstract: This review evaluates possible mechanisms which regulate paramyxovirus protein and RNA syntheses. Evidence indicates that viral polypeptide abundances are specified at the transcriptional level. RNA replication and transcription are competing processes utilizing an encapsidated single-stranded RNA template. The requirement for capsid proteins in RNA replication suggests that these proteins are positive control elements for replication of paramyxovirus RNA.
TL;DR: Results suggest that an α-amanitin sensitive, DNA-dependent RNA polymerase, possibly the host cell nucleoplasmic polymerase is responsible for HSV-1 RNA synthesis in the infected cell.
TL;DR: A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin, and the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized.
Abstract: A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors
TL;DR: DNA-dependent synthesis of tryptophan (trp) operon mRNA and enzymes was studied in vitro and it was concluded that read-through transcription is not repressible in vitro.
TL;DR: The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus, and these primers were purified by isolation of the covalent complex between primer and nascent DNA, differential melting of the 70SRNA, and by two-dimensional electrophoresis in polyacrylamide gels.
Abstract: The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways—by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5′ terminus and CpCpAOH at the 3′ terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (Ψp, Ψp, Cp) Gp (possibly in place of the characteristic sequence GprTpΨpCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro.