Nucleotide sequence of the 5′-terminal coding region for pre-rRNA and mature 17S rRNA in<i>Tetrahymena thermophila</i>rDNA

Engberg, Jan; Din, Nanni; Saiga, Hidetoshi; Higashinakagawa, Toru

doi:10.1093/nar/12.2.959

Cited by 22 publications

(15 citation statements)

References 42 publications

(38 reference statements)

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Section: (3)mentioning

confidence: 96%

“…In agreement with this model, we have shown previously that the active (and inactive) avian alpha-globin genes alpha-pi and alpha-D are replicated in the transcriptional direction (19). The organization of the simian virus 40 and polyomaviral genomes (33) and the extrachromosomal rDNA genes of Tetrahymena thermophila and T. pyriformis (4,10,29,35) are also consistent with this proposal; however, the alternating convergent and divergent transcriptional polarities of the closely interspersed chicken histone genes (8) and the antiparallel arrangement of the nested Drosophila melanogaster GART-PCP gene pair (17) suggest that this model may not be generally applicable in its simplest form.To investigate this question further, we used an in vitro runoff replication assay (IVR) to analyze the direction of replication through the active and inactive histone H5 (2) genes of chicken embryonic erythrocytes and fibroblasts and the MSB-1 lymphoblastoid cell line. When nuclei are incubated in vitro in the presence of bromodeoxyuridine (BrdUrd) triphosphate (BrdUTP), the endogenous DNA polymerases extend replication forks previously initiated in the intact cells, leading to the preferential density labeling of origin-distal DNA segments (19).…”

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confidence: 96%

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Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Trempe¹,

Lindstrom²,

Leffak³

1988

Mol. Cell. Biol.

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We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.The replication of eucaryotic chromosomes is under both spatial and temporal controls, as evidenced by the firing of local clusters of replicons according to developmentally regulated programs and the replication of particular chromosome domains during similar intervals of the S phase in successive cell cycles (reviewed in reference 16). That the genomic location of an immunoglobulin gene sequence can influence its schedule of replication has been shown by Calza et al. (3).A relationship between transcriptional activity and the timing of replication has been inferred based on the observation that genes are generally replicated earlier in the S phase in cells where they are transcribed than in cells where they are inactive (7, 13). In the case of the Xenopus laevis kidney cell line TrXo, translocation of oocyte-type 5S rDNA genes from their normal telomeric location to a pericentromeric location is correlated with both a shift from late to early S-phase replication and the accumulation of oocyte 5S transcripts (11, 15), supporting the suggestion that the earlier replication of somatic versus oocyte 5S genes in normal cells may contribute to the selective transcription of the somatic genes (14,38).A structural link between replication and transcription is suggested by the observations that under normal cell growth conditions the Hi histones and octamer cores of bulk chromatin are conservatively replicated (23-25, 34, 36), whereas the core histone octamers of transcribed chromatin and the HMG 14/17 nonhistone pair preferentially associated with transcribed...

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Section: (3)mentioning

confidence: 96%

mentioning

confidence: 96%

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Trempe¹,

Lindstrom²,

Leffak³

1988

Mol. Cell. Biol.

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“…1. The transcription start site for the pre-rRNA is located 1.9 kbp from the palindromic center (14). The 5Ј nontranscribed spacer (5Ј NTS) contains a number of repeats that belong to three different classes: types I, II, and III.…”

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confidence: 99%

Regulatory Sequences for the Amplification and Replication of the Ribosomal DNA Minichromosome in Tetrahymena thermophila

Blomberg

Randolph

Yao

et al. 1997

Molecular and Cellular Biology

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We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (ϳ900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription. It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

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“…5A) (24). A short transcript which maps to the 5Ј external transcribed spacer (ETS) just upstream of the 17S gene has been described previously and was termed the IF (10,23,24). It was proposed that the IF is a product of premature transcription termination at the PolI promoter, because it does not correspond to any known processing product of the 35S rRNA (23).…”

Section: Resultsmentioning

confidence: 99%

“…The initiator fragment was first reported as an approximately 230-nt RNA which hybridized to a probe derived from ETS sequences upstream of the 17S rRNA gene (10,24). We prepared RNA from log-phase C3 wild-type and C3-rmm3 homozygous cells and ran these on an acrylamide gel.…”

Section: Resultsmentioning

confidence: 99%

A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

Gallagher

Blackburn

1998

Molecular and Cellular Biology

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In the ciliated protozoan Tetrahymena thermophila the ribosomal DNA (rDNA) minichromosome replicates partially under cell cycle control and is also subject to a copy number control mechanism. The relationship between rDNA replication and rRNA gene transcription was investigated by the analysis of replication, transcription, and DNA-protein interactions in a mutant rDNA, the rmm3 rDNA. The rmm3 (for rDNA maturation or maintenance mutant 3) rDNA contains a single-base deletion in the rRNA promoter region, in a phylogenetically conserved sequence element that is repeated in the replication origin region of the rDNA minichromosome. The multicopy rmm3 rDNA minichromosome has a maintenance defect in the presence of a competing rDNA allele in heterozygous cells. No difference in the level of rRNA transcription was found between wild-type and rmm3 strains. However, rmm3 rDNA replicating intermediates exhibited an enhanced pause in the region of the replication origin, roughly 750 bp upstream from the rmm3 mutation. In footprinting of isolated nuclei, the rmm3 rDNA lacked the wild-type dimethyl sulfate (DMS) footprint in the promoter region adjacent to the base change. In addition, a DMS footprint in the origin region was lost in the rmm3 rDNA minichromosome. This is the first reported correlation in this system between an rDNA minichromosome maintenance defect and an altered footprint in the origin region. Our results suggest that a promoter region mutation can affect replication without detectably affecting transcription. We propose a model in which interactions between promoter and origin region complexes facilitate replication and maintenance of the Tetrahymena rDNA minichromosome.

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Nucleotide sequence of the 5′-terminal coding region for pre-rRNA and mature 17S rRNA inTetrahymena thermophilarDNA

Cited by 22 publications

References 42 publications

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Regulatory Sequences for the Amplification and Replication of the Ribosomal DNA Minichromosome in Tetrahymena thermophila

A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

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