Conditions Favoring RNA Polymerase I Transcription in Permeabilized Cells

Masson, Claude; Bouniol, Christine; Fomproix, Nathalie; Szöllösi, Maria S.; Debey, Pascale; Hernandez‐Verdun, Danièle

doi:10.1006/excr.1996.0209

Cited by 34 publications

(27 citation statements)

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“…The product of rDNA transcription taking place during 20 min is visible as large spots regularly distributed in the interior of the nucleolus ( Figure 5). This alignment of large nucleolar RNA spots is also observed without a-amanitin ( Figure 6) using run-on transcription optimized for RNA pol I transcription (Masson et al, 1996). The number of RNA spots was evaluated per nucleolus on CLSM optical sections.…”

Section: Resultsmentioning

confidence: 57%

“…Detection of RNA Synthesis by Run-On in Permeabilized Cells. Run-on transcription was performed as previously described (Wansink et al, 1993), except that glycerol was omitted from all media (Masson et al, 1996). Briefly, cells were washed in PBS and with permeabilization buffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride).…”

Section: Labelingmentioning

confidence: 99%

“…Here, the 3-D codistribution of rDNA and rRNA transcripts on one hand and of UBF and rRNA transcripts on the other hand have been addressed to establish how the 3-D organization is correlated with transcription. RNA transcription was detected by modified RNA precursors incorporated by in situ run-on in conditions favoring transcription by RNA pol I (Masson et al, 1996). PtKj cells were chosen for this investigation because they possess two clusters of rDNA, each of them giving rise to one nucleolus during interphase.…”

Section: Introductionmentioning

confidence: 99%

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Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

Junera

Masson

Géraud

et al. 1997

MBoC

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The distribution of the ribosomal genes (rDNA) and the upstream binding factor (UBF), correlatively with their RNA transcripts, was investigated in G1, S-phase, and G2. rDNA was distributed in nucleoli, with alternate sites of clustered and dispersed genes. UBF was found associated with some but not all clustered genes and proportionally more with dispersed genes. It was distributed in several foci that were more numerous and heterogeneous in size during G2 than G1. We suggest that UBF associated with rDNA during S-phase because its nucleolar amount increased during that time and remained stable in G2. 5,6-Dichloro-1-13-D-ribofuranosylbenzimidazole treatment indicated a similar amount of UBF per transcription unit, and consequently heterogeneous size of the UBF foci can represent a variable number of transcription units per foci. Direct visualization of the transcripts demonstrated that only part of UBF is associated with active transcription and that rDNA distribution varied with transcription. We propose that in the same rDNA locus three types of configuration coexist that are correlated with gene activity: 1) clustered genes without UBF; 2) clustered genes with UBF, of which some are associated with transcription; and 3) dispersed genes with UBF and transcription. These results support the hypothesis that rDNA transcription involved several steps of regulation acting successively and locally in the same locus to promote the repressed clustered genes to become actively transcribed dispersed genes.

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Section: Resultsmentioning

confidence: 57%

Section: Labelingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

Junera

Masson

Géraud

et al. 1997

MBoC

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“…Nucleolar Run-on-Ongoing synthesis of rRNA in the nucleolus was visualized by incubating transfected COS-7 cells with bromo-UTP (21,22). 48 h after the Me 2 SO shock, cells were rinsed twice each with 1ϫ PBS and run-on buffer (20 mM Tris HCl, pH 7.4, 5 mM MgCl 2 , 0.5 mM EGTA, pH 8.0, 0.5 mM phenylmethylsulfonyl fluoride).…”

Section: Construction Of Kid-1 Mutant Proteins-mentioning

confidence: 99%

Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration of the Nucleolus

Huang

Philippin

O’Leary

et al. 1999

Journal of Biological Chemistry

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The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH 2 terminus and two Cys 2 His 2 -zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.

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“…As an alternative to the autoradiographic approach, a non-isotopic immunocytochemical method of RNA synthesis detection can also be used [4,7,19,20,27,32,39]. It [19,20,31,39].…”

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confidence: 99%

Non-isotopic detection of nucleolar transcription in pre-implantation mouse embryos

Kobema

Landa²,

Kaňka³

et al. 1998

Reprod. Nutr. Dev.

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Conditions Favoring RNA Polymerase I Transcription in Permeabilized Cells

Cited by 34 publications

References 0 publications

Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration of the Nucleolus

Non-isotopic detection of nucleolar transcription in pre-implantation mouse embryos

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