Transcription of specific genes in isolated nuclei by exogenous RNA polymerases

Sklar, Virgil E. F.; Roeder, Robert G.

doi:10.1016/0092-8674(77)90028-9

Cited by 48 publications

(14 citation statements)

References 27 publications

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“…There is an extensive literature on more than 10 discrete species of small nuclear RNAs in the size range of 90-300 nucleotides, only a few of which however are NP (25)(26)(27)(28)(29)(30)(31). Although the lifetime of the large RNAs is on the order of 10-30 min, some of the small RNA species have half-lives of about 1 day and others show no detectable turnover for several days in exponentially growing tissue culture cells (reviewed in ref.…”

Section: Discussionmentioning

confidence: 99%

Definition of subclasses of nucleoplasmic RNA

Tamm

1977

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

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RNA synthesis in isolated HeLa cell nuclei prepared from cells pretreated with 5,6-dichloro-I-P-D-ribofuranosylbenzimidazole (DRB) is inhibited in a time-dependent manner. After 40-min pretreatment of cells with 60B M DRB in the presence of actinomycin D (0.04 ,g/ml), the rate of RNA synthesis in isolated nuclei, measured by[3HJUTP incorporation, is decreased by 63%. The DRB-resistant one-third of heterogeneous nuclear is distributed over the entire size range of heterogeneous nuclear RNA with some enrichment in the 18S range, as was observed earlier by pulse-labeling whole cells. 5,6-Dichloro-1-f-D-ribofuranosylbenzimidazole (DRB) has a selective and reversible action on nuclear RNA synthesis in mammalian cells by inhibiting two-thirds of heterogeneous nuclear RNA (hnRNA) (1, 2) and >95% of mRNA (2) through an apparent block in chain initiation (3,4 Milwaukee, WI. The phosphate-buffered saline (PBS) was 0.13 M NaCl/2.7 mM KCl/0.82 mM Na2HPO4/1.5 mM KH2PO4/ 0.91 mM CaC12/0.5 mM MgCl2; the pH was 7.3. The lysis buffer was 6 mM KCI/5 mM MgCl2/0.01 M Tris, pH 7.4; to the buffer was added dithiothreitol to a concentration of 1 mM prior to use. High salt buffer was 0.5 M NaCl, 0.05 M MgCl2, and 0.01 M Tris. Sodium dodecyl sulfate buffer (NaDodSO4 buffer) was 0.5% NaDodSO4/0.1 M NaCl/1 mM EDTA/0.01 M Tris, pH 7.4. Dialysis buffer was 0.01 M Tris, pH 7.4/0.1 M NaCl/0.5% NaDodSO4.The incubation mix for RNA synthesis in isolated nuclei contained 6 mM KCI, 1.6 mM MnCl2, 0.1 M (NH4)2SO4, 40,M each of ATP, CTP, and GTP, 4 ,uM UTP, 2 ,uM [3H]UTP (20)(21)(22)(23)(24)(25) ,gCi per 500-Sl sample), and 1 mM dithiothreitol in 50 mM Tris/25% glycerol, pH 7.6. Isolation of Cell Nuclei and Nucleoplasmic and Nucleolar Fractions. The procedures used have been described (1, 6). Pertinent details are given in figure legends.Pulse-Labeling Techniques. Isolated HeLa cell nuclei were labeled with [3H]UTP for 0-30 min at 26°, and 25-,il aliquots were placed on Whatman 3 MM filters that were processed in batches. The acid-precipitable counts were expressed per total sample after subtraction of 0-min counts. RNA was extracted with phenol/chloroform (1). In most experiments, actinomycin D (0.04 ,ug/ml) was used to suppress ribosomal RNA'synthesis.Gel Electrophoresis. The procedure used has been described (1). The marks above the horizontal bar in each panel of Figs. 2, 3 and 4 refer, from left to right, to 28, 18, and 4S marker RNAs from HeLa cell cytoplasm. To obtain size estimates of RNA in the 4-18S range, the logarithms of the molecular weights for marker RNAs were plotted against migration rates, with molecular weights of 1,750,000, 605,000 and 25,000 for 28, 18, and 4S RNA, respectively (7). This gave a linear relaAbbreviations: DRB, 5,6-dichloro-1-#-D-ribofuranosylbenzimidazole; hnRNA, nuclear heterogeneous RNA; [3H]UTP, [3H]uridine 5'-triphosphate; PBS, phosphate-buffered saline; NaDodSO4 buffer, sodium dodecyl sulfate buffer; NP, nucleoplasmic fraction; pre-4S RNA, -4.5S precursor of tRNA.

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Section: Discussionmentioning

confidence: 99%

Definition of subclasses of nucleoplasmic RNA

Tamm

1977

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

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“…RNA synthesis has been studied in isolated nuclei [2 l] and in nuclear suspensions with added RNA polymerase III [31] and it was suggested that this enzyme catalyzes the synthesis of LMW RNA as well as 5 S and t-RNA. Using different inhibitors of RNA synthesis it has, however, also been found that A, C and D are synthesized by RNA polymerase I [32] .…”

Section: Discussionmentioning

confidence: 99%

The differential inhibitory effect of α‐amanitin on the synthesis of low molecular weight RNA components in BHK cells

1978

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“…The genes are abundant, each early oocyte containing nearly 100 000 copies in its 4C genome [ 11, and they can be isolated as a satellite from total DNA [2]. 5 S gene transcription is subject to control during normal oocyte development [3] and is carried out by a specific RNA polymerase [4], which retains its specificity in vitro [5]. The 5 S genes expressed in oocytes are different from those expressed in somatic cells h71.…”

Section: A In~~uctionmentioning

confidence: 99%

Preparation of complementary DNA to 5 S ribosomal RNA from xenopus laevis

Morris

Beebee

1979

FEBS Letters

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a In~~uctionThe 5 S rRNA genes of Xenopus provide a convenient system for study of the molecular mechanism of gene transcription and its control. The genes are abundant, each early oocyte containing nearly 100 000 copies in its 4C genome [ 11, and they can be isolated as a satellite from total DNA [2]. 5 S gene transcription is subject to control during normal oocyte development [3] and is carried out by a specific RNA polymerase [4], which retains its specificity in vitro [5]. The 5 S genes expressed in oocytes are different from those expressed in somatic cells h71.Here we describe the preparation of radioactive DNA complementary to 5 S rRNA by reverse transcription and its use as a specific molecular probe for 5 S RNA sequences. We suggest potential uses for this cDNA in studies of 5 S RNA synthesis and accumulation during oogenesis in vivo and of 5 S gene transcription in vitro by RNA polymerase III. It may also be useful for studies with other animal species since it hybridizes to chicken 5 S RNA. Materials and methods ~~e~aZsXenopus laevis were obtained from Xenopus Ltd.,

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Transcription of specific genes in isolated nuclei by exogenous RNA polymerases

Cited by 48 publications

References 27 publications

Definition of subclasses of nucleoplasmic RNA

Definition of subclasses of nucleoplasmic RNA

The differential inhibitory effect of α‐amanitin on the synthesis of low molecular weight RNA components in BHK cells

Preparation of complementary DNA to 5 S ribosomal RNA from xenopus laevis

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