A distinct RNA polymerase activity, synthesizing 5.5 s, 5 s and 4 s RNA in nuclei from adenovirus 2-infected HeLa cells

Price, Richard; Penman, Sheldon

doi:10.1016/0022-2836(72)90551-7

Cited by 161 publications

(66 citation statements)

References 12 publications

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“…The direct engagement of RNA polymerase B in transcribing structural genes has been to date reported only for viral mRNAs (28)(29)(30)(31) and for the fibroin mRNA (32). Our results demonstrate that RNA polymerase B transcribes the ovalbumin gene.…”

Section: Methodsmentioning

confidence: 46%

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydrylagarose and hybridized to radioactive ovalbumin cDNA. (ii) (3HJUTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of a-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vivo RNA synthesis is retained in isolated nuclei.The synthesis of the egg white proteins in the tubular gland cell of the chicken oviduct is primarily related to the cellular accumulation of the corresponding mRNAs (1-7). During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2, 4, 5-7). Similar results have been found for the induction of ovomucoid and lysozyme mRNA (7). The steroid-mediated induction of specific mRNA molecules in the chicken oviduct may result from altered rates of synthesis and/or degradation. We therefore studied the synthesis rate of the egg white protein mRNAs. Because it was not possible to measure the rate of specific mRNA synthesis in the animals, we chose isolated oviduct nuclei as the cell-free system that might best reflect the transcriptional activity of the intact cell (8). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Section: Methodsmentioning

confidence: 46%

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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“…Heterogeneous nuclear RNA is synthesized by polymerase II, which is strongly inhibited by a-amanitin (12, 15-17) but not by a low concentration of actinomycin D (14,18). Under the same conditions, synthesis of smaller molecular weight RNA (4, S, 5 2, etc) is not inhibited by either drug and may be performed by a third polymerase (11,19).…”

Section: Resultsmentioning

confidence: 99%

“…Synthesis of tRNA, which accounts for about 20%o of total RNA of intact cells is resistant to both drugs, but in contrast to the larger molecular weight RNA, most tRNA synthesized in isolated nuclei passes out into the medium (19). Probably for the same reason virtually all incorporation into the ghost monolayers was either preribosomal or heterogeneous nuclear RNA.…”

Section: Resultsmentioning

confidence: 99%

Regulation of RNA Synthesis in Fibroblasts During Transition from Resting to Growing State

Mauck

Green

1973

Proc. Natl. Acad. Sci. U.S.A.

111

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Addition of serum, containing fibroblast growth factors, to a culture of resting 3T6 cells stimulates a transition to the growing state. Studies of ghost monolayers prepared with the aid of detergent at intervals after stimulation showed an increase in the rate of ribosomal RNA synthesis within 10 min. The rate continued to increase for many hours and reached a level 2.5-to 3.5-fold higher by the time DNA synthesis began. The increasing rate of ribosomal RNA synthesis appeared independent of an increase in the number of ribosomal genes, since it was not affected by prevention of DNA synthesis with cytosine arabinoside.In contrast to ribosomal RNA, the overall rate of transscription of heterogeneous nuclear RNA was not directly affected by serum growth factors and does not appear to be regulated during the transition from resting to growing state. It seems, instead, to be fixed in relation to the amount of template, for it increases proportionally to DNA content.The rate of RNA synthesis in mouse-fibroblast line 3T6 can be measured as the incorporation of labeled ribonucleoside triphosphates by nonviable "ghost monolayers" prepared with the aid of the detergent, NP-40 (1). Estimates of the rate of RNA synthesis under these conditions are not affected by the cell-membrane permeability barrier and the slowly equilibrating cell-nucleotide pools, which complicate interpretation of incorporation rates in whole cells (2)(3)(4)(5)(6).After stimulation of resting cultures of 3T6 with serum containing fibroblast growth factors, an increase in total RNA synthesis could be detected in ghost monolayers prepared less than 30 min later (1). The rate rose 2-fold by the time DNA synthesis began, and continued to rise for at least 20 hr. No attempt was made to distinguish effects on transcription of different classes of RNA.We describe here experiments on the rates of synthesis of ribosomal RNA (rRNA) and of heterogeneous nuclear RNA in ghost monolayers prepared during the serum-induced transition from resting to growing state. These two classes of RNA respond quite differently, indicating that their synthesis is controlled independently. An increased rate of ribosome synthesis is lprobably an essential part of the preparation for DNA synthesis, but the overall synthesis of heterogeneous nuclear RNA, the precursor of cytoplasmic mRNA (7) Assay of RNA Synthesis in Ghost Monolayers. All operations were done at 37°. The medium was removed and the monolayer was washed with 2 ml of serum-free medium. The monolayer was then treated with 1 ml of 0.5% NP-40 in assay buffer containing 0.05 M Tris HCl (pH 7.8), 5 mM MgCJ2, 6 mM KCl, 75 mM (NH4)2SO4, 0.20 M sucrose, and 1 mM freshly prepared dithiothreitol. After exactly 3 min, the NP-40 solution was removed and the ghost monolayer was washed with assay buffer. 0.80 ml of a solution of [3H]UTP (10 ,4Ci, [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] Ci/mmol), 5 mM ATP, 0.2 mM CTP, and 0.2 mM GTP in assay buffer was gently pipetted onto the ghost monolayer. The reac...

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“…Price and Penman prepared nuclei from HeLa cells infected with adenovirus that are able to initiate the synthesis of 5.5-S adenovirus RNA with GTP [13]. Meilhac and Chambon [21] who used calf thymus DNA, and Mandel and Chambon [22] who used SV40 DNA as templates, obtained the synthesis in vitro of RNA initiated with ATP and GTP by animal RNA polymerases Al and B.…”

Section: \ Imentioning

confidence: 99%

RNA Metabolism in Nuclei Isolated from HeLa Cells

Busiello

Girolamo

1975

European Journal of Biochemistry

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Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [ Y -~~P I G T P and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four ~-~~P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation mefhylates the nucleolar RNA by utilizing S-adenosyl-Lmethionine as a methyl-group donor.

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A distinct RNA polymerase activity, synthesizing 5.5 s, 5 s and 4 s RNA in nuclei from adenovirus 2-infected HeLa cells

Cited by 161 publications

References 12 publications

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Regulation of RNA Synthesis in Fibroblasts During Transition from Resting to Growing State

RNA Metabolism in Nuclei Isolated from HeLa Cells

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