Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments

Erickson, Jeanne M.; Rushford, Caroline L.; Dorney, D. J.; Wilson, Golder N.; Schmickel, Roy D.

doi:10.1016/0378-1119(81)90055-x

Cited by 223 publications

(90 citation statements)

References 28 publications

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“…1 were generated by using gel-purified fragments and standard cloning techniques (30). pES-ETS (which contains nucleotides -514 to +697 relative to the transcription initiation site) and pES-28S (which contains 596 nucleotides of 28S rRNA and 384 nucleotides of downstream sequence) were constructed as previously described (38), using clones generously provided by N. Arnheim (34) and R. Schmickel (14). To generate pEB-ETS, pES-ETS was cleaved with BstEII (which cleaves at position +75 relative to the initiation site) and HindIII, filled in with Klenow fragment, and ligated.…”

Section: Methodsmentioning

confidence: 99%

“…To generate pET-28S, pES-28S was cleaved with TthlllI (which cleaves at a position 218 nucleotides downstream of the 3' end of 28S rRNA) and HinidIll, filled in with Klenow fragment, and ligated. The 700-nucleotide SnaBIPvuII fragment of pADBB (generously provided by R. Schmickel [14] SI nuclease maps. S1 nuclease probes were 3' end labeled with the appropriate a-32P-labeled deoxynucleotide triphosphate and with Klenow fragment or T4 DNA polymerase or were 5' end labeled with [y-32P]ATP and polynucleotide kinase to a specific activity of 1 x 106 to 10 x 106 cpm/,jg, and the coding strand was purified on a strand-separating gel (30).…”

Section: Methodsmentioning

confidence: 99%

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Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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Human rRNA precursors from normal or stressed HeLa cells were studied by Si nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with[32p]PO4. Heating cells to 43°C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42°C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43°C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Parker¹,

Bond²

1989

Mol. Cell. Biol.

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“…The hybridization antisense probe (residues Ϫ43 to ϩ5 relative to transcription start) was produced by in vitro transcription with biotin-16-UTP using T7 RNA polymerase. The ITS1 region of human pre-rRNA corresponding to a XbaI-KpnI fragment of human rDNA (between 58 nt upstream from the 3Ј-end of the 18S rDNA coding region and 596 nt into ITS1; nt ϩ5469/ϩ6124) derived from pA XK (Erickson et al, 1981) was inserted in pBluescript SK(Ϫ). The hybridization antisense probe (ϩ5904/ϩ6124) was produced by in vitro transcription using T7 polymerase after linearization with BsaI.…”

Section: Hybridization Probesmentioning

confidence: 99%

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Dundr

Olson

1998

MBoC

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Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5Ј-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5Ј-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3Ј-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli. INTRODUCTIONThe nucleolus is a prominent membraneless subnuclear compartment assembled around clusters of tandemly repeated rDNA genes from which prerRNA is transcribed and subsequently folded, processed, modified, and assembled into small and large ribosomal subunits (Scheer et al., 1993;Shaw and Jordan, 1995;Maden and Hughes, 1997). A remarkable aspect of the cell cycle is the disintegration of the nucleolus during mitosis and its reassembly as the daughter cells proceed toward G 1 phase. A pivotal event in this process is the repression of RNA polymerase (pol) I-driven pre-rRNA synthesis between prophase and telophase (Prescott and Bender, 1962). At the same time, nucleolar components disperse to various subcellular locations as the maternal nucleoli diassemble (Goessens, 1984).A comprehensive understanding of the locations and fates of nucleolar components during mitosis is slowly emerging (Scheer et al., 1993;Hernandez-Verdun and Gautier, 1994). For example, much of the RNA pol I transcription machinery and DNA topoisomerase I remain associated with the nucleolus organizer regions (NORs) 1 of chromosomes between nucleolar disassembly in late prophase and reassembly in telophase (Scheer et al., 1993; Weisenberger and * Corresponding author. 1 Abbrevations used: DFC, dense fibrillar component; ETS, external transcribed spacer; GC, granular component; ITS, internal transcribed spacer; NDF, nucleolus-derived foci; NOR, nucleolus organizer region; PNB, prenucleolar body; pol, polymerase; snoRNA, small nucleolar RNA; sno-RNP, small nucleolar ribonucleoprotein.© 1998 by The American Society for Ce...

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“…A pGEM plasmid containing a 2.1-kb insert specific for human 28S rRNA (Bauman and Bentvelzen 1988;Erickson et al 1981) was labeled with digoxigenin-11 -deoxyuridine triphosphate (Boehringer-Mannheim, Mannheim, Germany) by nick translation and purified by Sephadex G50 (Pharmacia Biotech, Woerden, The Netherlands) gel filtration. The probe lengths were 100-400 bp as estimated by Southern blotting.…”

Section: Probes and Labelingmentioning

confidence: 99%

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells

Macville

Dorp

Dirks

et al. 1996

Histochem Cell Biol

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The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibro blasts (rat 9G). Experimental variables to balance penetra tion of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was ob tained using mild cross-linking fixation followed by treat ment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

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Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments

Cited by 223 publications

References 28 publications

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells

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