A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid.
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Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded. (+info)
Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.
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To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription. (+info)
Differentiation of population of peripheral blood lymphocytes into cells bearing sheep erythrocyte receptors in vitro by human thymic extract.
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A small population of human marrow cells has been shown to be differentiated in vitro by thymic extract into cells bearing T-lymphocyte (thymus-derived lymphocyte) characteristics. By a similar method, the differentiation of human peripheral blood lymphocytes has been studied. A discontinuous gradient of bovine serum albumin was used to isolate lymphocytes into four layers and cells from layers I and III demonstrated the greatest potential for differentiation by human thymic extract. Appearance of T-lymphocyte characteristics was recognized by the spontaneous E-rosette technique with sheep erythrocytes. Ability of human marrow cells to be differentiated under the influence of human thymic extract was abolished by specific inhibitors of nucleic acid synthesis, however, had no inhibitory effect on the maturation of peripheral blood lymphocytes during a 2 hr incubation with human thymic extract but puromycin, an inhibitor of protein synthesis, abolished this differentiative step in cells of layer I. It is suggested from these studies that many of the cells in peripheral blood that are differentiable by thymic extract are at a stage of maturation more advanced than those in human marrow that are also differentiable by thymic extract. (+info)
Stimulation by aldosterone of the sodium efflux in barnacle muscle fibres: effects of RNA inhibitors and spironolactone.
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1. Single muscle fibres from the barnacle Balanus nubilus have been studied to provide information about the mode of action of aldosterone on Na transport in a symmetric cell. 2. Neither internal nor external application of aldosterone modified the course of the Na efflux. However, fibres pre-exposed overnight to a dose of aldosterone showed a biphasic response to external aldosterone. The first phase was prompt in onset and consisted of a falling rate constant for Na efflux becoming a constant. This has been interpreted as indicating that aldosterone acts by releasing sequestered or bound Na. The second phase was delayed in onset with the average latent period being 68 min. It consisted of a step-up in the rate of Na efflux, followed by a transitory rise in the efflux rate constant. This second phase was dose-dependent, the minimum concentration being 10(-9) M. 3. Internal application of aldosterone in a concentration as low as 10(-10) M promptly stopped the rate constant for Na efflux from further falling but failed to cause delayed stimulation. 4. The response of the Na efflux into Li-ASW following external application of aldosterone was bi-phasic. The magnitude of the delayed stimulation was comparable to that found in controls. 5. No correlation between latent period or size of the internal Na bound fraction, and the magnitude of the delayed stimulation could be established. 6. Internal application of actinomycin-D, alpha-amanitin or cordycepin shortly before application of external aldosterone caused complete abolition of the delayed stimulation. 7. Overnight pre-exposure of the barnacle to actinomycin-D caused complete abolition of the falling rate constant for Na efflux, as well as the delayed stimulation caused by external aldosterone. 8. Internal application of spironolactone SC-14266 shortly before external application of aldosterone caused complete abolition of the biphasic response to the steroid. 9. It is concluded that (i) barnacle fibres can be made sensitive to aldosterone, (ii) the biphasic effects on the Na efflux depend on prior induction of RNA, (iii) the first effect caused by internal or external aldosterone involves mobilization of sequestered Na, (iv) the results obtained with spironolactone are in keeping with the current view that aldosterone interacts with a receptor system before causing de-repression. 10. The implications of the finding that aldosterone releases sequestered Na are briefly touched upon. (+info)
In vitro differentiation of human marrow cells into T lymphocytes by thymic extracts using the rosette technique.
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The manner by which human and calf thymic extracts induce precursor cells of human marrow to differentiate in vitro into T lymphocytes has been studied using as a T-cell marker the spontaneous rosette formation technique of human T lymphocytes with sheep erythrocytes (E rosette). These findings confirm previous observations made in the study of the same process using a different T-cell marker, specific antigenicity recognizable by a heterologous anti-human T-cell serum in a microcytotoxicity test. The number of cells revealing evidence of differentiation demonstrated by the E rosette formation technique is smaller than that obtained with the anti-human T-cell serum, indicating perhaps that a different stage of maturation of T lymphocytes is recognized by the antiserum from the one detected by spontaneous rosette formation. Based on the effects of specific inhibitors of nucleic acids and protein synthesis, it can be concluded that these thymic extracts ultimately act by influences exerted in the cell nucleus and that RNA and protein synthesis are required for the differentiation of precursor cells into T lymphocytes induced by thymic extracts. In addition, continued protein synthesis appears to be required for maintenance of receptors for sheep erythrocytes on the cell surface. (+info)
Conditional expression of RNA polymerase II in mammalian cells. Deletion of the carboxyl-terminal domain of the large subunit affects early steps in transcription.
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The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA. (+info)
Transcription termination by RNA polymerase III in fission yeast. A genetic and biochemically tractable model system.
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In order for RNA polymerase (pol) III to produce a sufficient quantity of RNAs of appropriate structure, initiation, termination, and reinitiation must be accurate and efficient. Termination-associated factors have been shown to facilitate reinitiation and regulate transcription in some species. Suppressor tRNA genes that differ in the dT(n) termination signal were examined for function in Schizosaccharomyces pombe. We also developed an S. pombe extract that is active for tRNA transcription that is described here for the first time. The ability of this tRNA gene to be transcribed in extracts from different species allowed us to compare termination in three model systems. Although human pol III terminates efficiently at 4 dTs and S. pombe at 5 dTs, Saccharomyces cerevisiae pol III requires 6 dTs to direct comparable but lower termination efficiency and also appears qualitatively distinct. Interestingly, this pattern of sensitivity to a minimal dT(n) termination signal was found to correlate with the sensitivity to alpha-amanitin, as S. pombe was intermediate between human and S. cerevisiae pols III. The results establish that the pols III of S. cerevisiae, S. pombe, and human exhibit distinctive properties and that termination occurs in S. pombe in a manner that is functionally more similar to human than is S. cerevisiae. (+info)
Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development.
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Transforming growth factor beta (TGFbeta) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFbeta receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFbeta signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFbeta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2(delta)exon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2(delta)exon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2(delta)exon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGF(delta) superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho-Smad2/Smad2(delta)exon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2(delta)exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo. (+info)