Effects of oestradiol, α-amanitin and ionic strength on the in vitro synthesis of RNA by uterus nuclei

Raynaud-Jammet, C.; Bieri, F.; Baulieu, Étienne-Émile

doi:10.1016/0005-2787(71)90683-6

Cited by 33 publications

(6 citation statements)

References 12 publications

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“…4 for selective action of a-amanitin on soybean RNA polymerases). The ionic strength optima and aamanitin sensitivities for RNA polymerases I and II in soybean nuclei are similar to those reported previously with animal nuclei (14,27,42). Based on this differential assay, the level of RNA polymerase II activity expressed in nuclei from untreated tissue was 50 to 60% higher than RNA polymerase I activity.…”

Section: Methodssupporting

confidence: 88%

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

Chen¹,

Lin²,

Chang³

et al. 1975

Plant Physiol.

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A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA:RNA :protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of 3H-TTP into DNA.thesis. Isolated nuclei thus are essential to the study of in vitro RNA synthesis if conditions relating to the in vivo state are to be approached. Isolated nuclei from other systems have been used to great advantage in the study of several important problems: e.g. the study of the transcription of defined RNA species (15,23,25,30,34), the intranuclear metabolism and release of RNA (26), the post-transcriptional additions of poly(A) to HnRNA (16,32,33), the mechanisms involved in DNA replication (6,9,10,22), the modification of chromatin proteins and their role in gene expression (18,28,35,39), the preparation of high mol wt DNA (1), and enzyme localization (8,29). We have pursued the isolation of nuclei from soybean hypocotyl with the objective of isolating large quantities of "intact," biologically active nuclei to be used in studies on the auxin regulation of RNA synthesis. In this report, we describe a procedure for isolating nuclei from control and 2, 4-D-treated soybean hypocotyl. The attendant RNA and DNA synthetic activities, gross morphology, and macromolecular composition are described. MATERIALS AND METHODSStudies on the mechanism of plant hormone action have received much attention in recent years. Hormones are involved in the regulation of many different physiological processes; some lines of evidence indicate that in several cases this regulation may involve the control of the transcription or translation process (17). Because it is generally difficult to isolate plant nuclei in sufficient yield for extensive biochemical work, the isolation of plant chromatin has been used routinely in the study of the influence of auxin on the transcription of RNA (11,24). Recent studies with soybean chromatin isolated by conventional methodology (13)

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Section: Methodssupporting

confidence: 88%

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

Chen¹,

Lin²,

Chang³

et al. 1975

Plant Physiol.

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“…In view of the findings of enhancement of nucleolar RNA polymerase activity by estradiol-17/~ in uterus [1,24], testosterone in prostate [18], and cortisone in liver [1,35], the aldosterone-dependent shift in renal polymerase I:II activity may represent an effect common to most if not all steroids. The increase in polymerase I activity could provide a means of increasing the capacity for protein synthesis but not involve tile directed synthesis of specific proteins.…”

Section: Discussionmentioning

confidence: 94%

Cordycepin and α-amanitin: Inhibitors of transcription as probes of aldosterone action

Chu

Edelman

1972

J. Membrain Biol.

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Summary. Evidence has been presented for the existence of distinct ]~NA polymerases (I and II) in the rat kidney similar to those described previously in rat liver and identified as nucleolar (I) and nucleoplasmic (II) in location. In adrenalectomized rats, administration of d-aldosterone (1 ~tg/100 g body wt) increased the ratio of polymerase I:II activity as defined by sensitivity to a-amanitin. The time-course of this shift correlated with the previously reported changes in urinary Na and K concentrations, in that a significant change in the I :II ratio was noted 1 to 2 hr before the usual peak change in urinary electrolyte concentrations. In the isolated urinary bladder of the toad, cordycepin inhibited incorporation of 3H-uridine into epithelial RNA--with a half time of 4.3 min-and impaired the action of aldosterone on transepithelial Na transport. These findings as well as earlier studies in the literature imply that aldosterone augments ribosomal RNA synthesis, and that ribosomal or messenger RNA synthesis or both are required for the expression of mineraloeorticoid activity.Steroidal regulation of active Na transport in anuran and mammalian target tissues is apparently mediated by stimulation of DNA-dependent synthesis of RNA and the subsequent de novo synthesis of proteins [6,7,34]. This inference is based, in part, on the finding that actinomycin D impaired mineralocorticoid action [6,10,34]. In addition, aldosterone enhanced the incorporation of precursors into total or nuclear RNA of the urinary bladder of the toad, in vitro and of the rat kidney, both in vivo and in homogenates [5,10,12,13,22,27].Roeder and Rutter [25,26] have recently separated and characterized two eukaryofic DNA-dependent RNA polymerases with distinct functions.

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“…Otherauthors have shown that hormone-stimulated RNA synthesis is of a ribosomal type, which is synthesized in the nucleolus (Liao & Lin, 1967;Jacob et al, 1969;Raynaud-Jammet et al, 1971;). The results of our study have now shown an increase of RNA polymerase activity brought about by a steroid-receptor complex in intact nucleoli and also specifically in the purified nucleolar form.…”

Section: Source Of Enzyme Templatementioning

confidence: 99%

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

Davies

Griffiths

1973

Biochemical Journal

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A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

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Effects of oestradiol, α-amanitin and ionic strength on the in vitro synthesis of RNA by uterus nuclei

Cited by 33 publications

References 12 publications

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

Cordycepin and α-amanitin: Inhibitors of transcription as probes of aldosterone action

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

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