Human ribosomal DNA fragments amplified in hamster cells are transcribed only by RNA polymerase II and are not silver stained

1987 ◽  
Vol 7 (3) ◽  
pp. 1289-1292
Author(s):  
V N Dhar ◽  
D A Miller ◽  
A B Kulkarni ◽  
O J Miller
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Chinese Hamster ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Methotrexate Resistance ◽  
Selection For ◽  
Polymerase I ◽  
Chromosome Regions

Cloned human rRNA gene fragments that included the promoter region were introduced into Chinese hamster dihydrofolate reductase-deficient (dhfr-) cells by cotransformation with a dhfr minigene and amplified by selection for methotrexate resistance. The human ribosomal DNA was transcribed by RNA polymerase II, not RNA polymerase I or III. The metaphase chromosome regions containing the transcriptionally active human ribosomal DNA failed to show silver staining.

scholarly journals Human ribosomal DNA fragments amplified in hamster cells are transcribed only by RNA polymerase II and are not silver stained.

1987 ◽  
Vol 7 (3) ◽  
pp. 1289-1292 ◽  
Author(s):  
V N Dhar ◽  
D A Miller ◽  
A B Kulkarni ◽  
O J Miller
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Chinese Hamster ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Methotrexate Resistance ◽  
Selection For ◽  
Polymerase I ◽  
Chromosome Regions

Cloned human rRNA gene fragments that included the promoter region were introduced into Chinese hamster dihydrofolate reductase-deficient (dhfr-) cells by cotransformation with a dhfr minigene and amplified by selection for methotrexate resistance. The human ribosomal DNA was transcribed by RNA polymerase II, not RNA polymerase I or III. The metaphase chromosome regions containing the transcriptionally active human ribosomal DNA failed to show silver staining.

scholarly journals A polymerase switch in the synthesis of rRNA in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (5) ◽  
pp. 2420-2428 ◽  
Author(s):  
H Conrad-Webb ◽  
R A Butow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Sole Source ◽  
Rna Polymerase I ◽  
Yeast Cells ◽  
Dna Repeat ◽  
Polymerase I ◽  
Respiratory Deficient

Transcription of ribosomal DNA by RNA polymerase I is believed to be the sole source of the 25S, 18S, and 5.8S rRNAs in wild-type cells of Saccharomyces cerevisiae. Here we present evidence for a switch from RNA polymerase I to RNA polymerase II in the synthesis of a substantial fraction of those rRNAs in respiratory-deficient (petite) cells. The templates for the RNA polymerase II transcripts are largely, if not exclusively, episomal copies of ribosomal DNA arising from homologous recombination events within the ribosomal DNA repeat on chromosome XII. Ribosomal DNA contains a cryptic RNA polymerase II promoter that is activated in petites; it overlaps the RNA polymerase I promoter and produces a transcript equivalent to the 35S precursor rRNA made by RNA polymerase I. Yeast cells that lack RNA polymerase I activity, because of a disruption of the RPA135 gene that encodes subunit II of the enzyme, can survive by using the RNA polymerase II promoter in ribosomal DNA to direct the synthesis of the 35S rRNA precursor. This polymerase switch could provide cells with a mechanism to synthesize rRNA independent of the controls of RNA polymerase I transcription.

scholarly journals Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin

2004 ◽  
Vol 24 (4) ◽  
pp. 1791-1798 ◽  
Author(s):  
Ralf Strohner ◽  
Attila Németh ◽  
Karl P. Nightingale ◽  
Ingrid Grummt ◽  
Peter B. Becker ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Active Role ◽  
Rna Polymerase I ◽  
In Vivo Studies ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription Terminator ◽  
Polymerase I

ABSTRACT The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.

scholarly journals Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter.

1995 ◽  
Vol 15 (6) ◽  
pp. 3372-3381 ◽  
Author(s):  
W J Pan ◽  
R C Gallagher ◽  
E H Blackburn
Keyword(s):  
Rna Polymerase ◽  
Tetrahymena Thermophila ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Wild Type ◽  
Independent Evidence ◽  
Origin Region ◽  
Origin Function ◽  
Polymerase I ◽  
Wild Type Promoter

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

scholarly journals Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.

1995 ◽  
Vol 15 (8) ◽  
pp. 4648-4656 ◽  
Author(s):  
M H Paalman ◽  
S L Henderson ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Gene Promoter ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Transcriptional Terminator ◽  
Wide Range ◽  
Polymerase I ◽  
Enhancer Sequences ◽  
Stimulation Of

We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.

Abstract A46: Wnt5a signals through DVL1 to repress ribosomal DNA transcription by RNA polymerase I

2017 ◽  
Author(s):  
Randall Dass ◽  
Aishe Sarshad ◽  
Brittany Carson ◽  
Jennifer Feenstra ◽  
Amanpreet Kaur ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Rna Polymerase I ◽  
Dna Transcription ◽  
Polymerase I

scholarly journals Feedback regulation of vitamin D metabolism by 1,25-dihydroxycholecalciferol

1977 ◽  
Vol 164 (1) ◽  
pp. 83-89 ◽  
Author(s):  
K W Colston ◽  
I M A Evans ◽  
T C Spelsberg ◽  
I MacIntyre
Keyword(s):  
Vitamin D ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Subcutaneous Administration ◽  
Feedback Regulation ◽  
Rna Polymerase I ◽  
Hydroxylase Activity ◽  
Vitamin D Metabolism ◽  
Polymerase I

Many factors influence the production of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol) by the kidney. One important factor seems to be feedback regulation by 1,25(OH)2D3 itself. Administration of 1,25(OH)2D3 to vitamin D-deficient chicks abolishes renal 25(OH)D3(25-hydroxycholecalciferol)1-hydroxylase activity and induces the appearance of 25(OH)D3 24-hydroxylase activity. It is likely that these effects are mediated via a nuclear effect, as they are prevented by pretreatment with actinomycin D and alpha-amanitin. Further, 1,25(OH)2D3 has a marked effect on gene transcription in the kidney cell, as assessed by measurement of RNA polymerase activities. RNA polymerase I and II activities are 80-90% inhibited by 12.5nmol of 1,25(OH)2D3 within 30min of subcutaneous administration, indicating an immediate and massive decrease in total gene transcription. By 4h RNA polymerase II activity has returned to control values, but RNA polymerase I activity is markedly enhanced. These results are consistent with the view that regulation of cholecalciferol metabolism in the kidney is associated with an effect of the active metabolite on the kidney nucleus.

scholarly journals Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2175-2189 ◽  
Author(s):  
Stéphanie Trumtel ◽  
Isabelle Léger-Silvestre ◽  
Pierre-Emmanuel Gleizes ◽  
Frédéric Teulières ◽  
Nicole Gas
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Functional Organization ◽  
Ultrastructural Localization ◽  
Rna Polymerase I ◽  
Wild Type ◽  
Rdna Transcription ◽  
Nucleolar Structure ◽  
Fibrillar Component ◽  
Polymerase I

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

scholarly journals Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP

2001 ◽  
Vol 21 (7) ◽  
pp. 2292-2297 ◽  
Author(s):  
Imran Siddiqi ◽  
John Keener ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Rrna Transcription ◽  
Pol I ◽  
Mutant Strains ◽  
Polymerase I

ABSTRACT Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269–5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5,RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF componentRRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

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