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1.
Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation. 总被引:2,自引:1,他引:1
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The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. 相似文献
2.
Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules. 总被引:6,自引:3,他引:3
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We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina. All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila. Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol. Cell. Biol. 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA. In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved. However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs. We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma. 相似文献
3.
Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element 总被引:30,自引:0,他引:30
A novel genetic scheme was used to isolate mutants altered in the formation or maintenance of amplified rDNA in the Tetrahymena macronucleus. One such mutant had a cis-acting rDNA mutation that affected the ability of mutant rDNA molecules to replicate in macronuclei in the presence of a wild-type (B strain) rDNA. The mutant rDNA was lost from these heterozygous macronuclei during vegetative cell divisions, although it was maintained normally in the homozygous or hemizygous state. In contrast, wild-type macronuclear rDNA of the C3 strain used to obtain the mutant outreplicated B strain rDNA in B/C3 heterozygote macronuclei. Sequence differences were found between wild-type B and C3 and mutant C3 rDNAs in the replication origin region, changing an upstream repeat of a highly conserved rRNA promoter element. We propose that the various rDNA alleles differentially compete for limiting amounts of trans-acting factors that bind to these enhancer-like repeats and positively regulate rDNA replication. 相似文献
4.
Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA
+ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs. 相似文献
5.
Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila. 总被引:13,自引:1,他引:12
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The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development. We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA. In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA. Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested. Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly. This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines. One of the 26S rRNA mutants failed to generate transformants, but the other did. These transformants grew normally, and produced 26S rRNA containing the inserted sequence. A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA. This study reveals that some rRNA sequences can be altered without significantly affecting cell growth. 相似文献
6.
Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia. 总被引:34,自引:19,他引:15
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Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule. 相似文献
7.
Martins C Wasko AP Oliveira C Porto-Foresti F Parise-Maltempi PP Wright JM Foresti F 《Cytogenetic and genome research》2002,98(1):78-85
In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements. 相似文献
8.
The hypothetical origin of replication for the 7.5-kb plasmid common to Chlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar of C. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7-10. The evidence presented suggests that C. trachomatis has a homologue to the Escherichia coli dnaA gene and that this homologue might be involved in replication of the C. trachomatis 7.5-kb plasmid. 相似文献
9.
Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA. 总被引:13,自引:4,他引:9
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Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA. 相似文献
10.
The ribosomal RNA genes of Tetrahymena: structure and function 总被引:7,自引:0,他引:7
J Engberg 《European journal of cell biology》1985,36(1):133-151
11.
The hypothetical origin of replication for the 7.5-kb plasmid common toChlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar ofC. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7–10. The evidence presented suggests thatC. trachomatis has a homologue to theEscherichia coli dnaA gene and that this homologue might be involved in replication of theC. trachomatis 7.5-kb plasmid. 相似文献
12.
Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae. 总被引:31,自引:8,他引:23
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Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator. 相似文献
13.
14.
Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae
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Saccharomyces cerevisiae carries approximately 150 ribosomal DNA (rDNA) copies in tandem repeats. Each repeat consists of the 35S rRNA gene, the NTS1 spacer, the 5S rRNA gene, and the NTS2 spacer. The FOB1 gene was previously shown to be required for replication fork block (RFB) activity at the RFB site in NTS1, for recombination hot spot (HOT1) activity, and for rDNA repeat expansion and contraction. We have constructed a strain in which the majority of rDNA repeats are deleted, leaving two copies of rDNA covering the 5S-NTS2-35S region and a single intact NTS1, and whose growth is supported by a helper plasmid carrying, in addition to the 5S rRNA gene, the 35S rRNA coding region fused to the GAL7 promoter. This strain carries a fob1 mutation, and an extensive expansion of chromosomal rDNA repeats was demonstrated by introducing the missing FOB1 gene by transformation. Mutational analysis using this system showed that not only the RFB site but also the adjacent approximately 400-bp region in NTS1 (together called the EXP region) are required for the FOB1-dependent repeat expansion. This approximately 400-bp DNA element is not required for the RFB activity or the HOT1 activity and therefore defines a function unique to rDNA repeat expansion (and presumably contraction) separate from HOT1 and RFB activities. 相似文献
15.
16.
New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene. 总被引:7,自引:3,他引:4
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A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination. 相似文献
17.
A novel form of extrachromosomal rDNA has been identified in conjugating Tetrahymena cells. This rDNA consists of 11 kb linear double-stranded DNA molecules, each containing a single rRNA gene copy. The DNA sequence, tandemly repeated CCCCAA (Blackburn and Gall 1978) found at the termini of extrachromosomal palindromic rDNA (the macronuclear form found in vegetatively growing cells), is also present at the corresponding terminus of the 11 kb rDNA. The other end of this molecule has an extra 0.3 kb segment of DNA covalently attached to the DNA region corresponding to the center of the palindromic rDNA. The kinetics of appearance and synthesis of the 11 kb rDNA early in macronuclear development are consistent with its being an intermediate in rDNA amplification. 相似文献
18.
Long range cooperative interactions regulate the initiation of replication in the Tetrahymena thermophila rDNA minichromosome.
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The Tetrahymena thermophila rDNA exists as a 21 kb palindromic minichromosome with two initiation sites for replication in each half palindrome. These sites localize to the imperfect, repeated 430 bp segments that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine if the D1 and D2 segments act independently or in concert to control initiation, stable DNA transformation assays were performed. Single domain derivatives of the plasmid prD1 failed to support autonomous replication in Tetrahymena. Instead, such constructs propagated exclusively by integration into endogenous rDNA minichromosomes and displayed weak origin activity as detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also transformed Tetrahymena poorly, showing similar replication defects. Hence, the D1 and D2 segments are functionally non-redundant and cooperate rather than compete to control initiation. The observed replication defect was greatly reduced in a plasmid derivative that undergoes palindrome formation in Tetrahymena, suggesting that a compensatory mechanism overcomes this replication block. Finally, using a transient replication assay, we present evidence that phylogenetically-conserved type I elements directly regulate DNA replication. Taken together, our data support a model in which cooperative interactions between dispersed elements coordinately control the initiation of DNA replication. 相似文献
19.
The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila. 总被引:9,自引:2,他引:7
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P C Yaeger E Orias W L Shaiu D D Larson E H Blackburn 《Molecular and cellular biology》1989,9(2):452-460
The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia. 相似文献
20.
In most organisms, the nuclear ribosomal RNA (rRNA) genes are highly repetitive and arranged as tandem repeats on one or more chromosomes. In Entamoeba, however, these genes are located almost exclusively on extrachromosomal circular DNA molecules with no clear evidence so far of a chromosomal copy. Such an uncommon location of rRNA genes may be a direct consequence of cellular physiology, as suggested by studies with Saccharomyces cerevisiae mutants in which the rDNA is extrachromosomal. In this review, Sudha Bhattacharya, Indrani Som and Alok Bhattacharya summarize current knowledge on the structural organization and replication of the Entamoeba rDNA plasmids. Other than the rRNAs encoded by these molecules, no protein-coding genes (including ribosomal protein genes) are found on any of them. They are unique among plasmids in that they do not initiate replication from a fixed origin but use multiple sites dispersed throughout the molecule. Further studies should establish the unique biochemical features of Entamoeba that lead to extrachromosomal rDNA. 相似文献