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At least 19 recordsLinked to original sources

PubMed1979

A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.

Animals↗

PubMed1979

Absence of cytosine methylation at C-C-G-G and G-C-G-C sites in the rDNA coding regions and intervening sequences of Drosophila and the rDNA of other insects.

Cytosine residues in C-G dinucleotides are frequently methylated in eukaryote DNA. In DNA of the dinoflagellate C. cohnii, the sequence C-MeC-G-G apparently renders Hpa II (C-C-G-G) incapable of digesting whole cell DNA in general, and rDNA in particular. Msp I, which also recognizes C-C-G-G but cleaves irrespective of methylation, degrades C. cohnii DNA and produces rDNA segments of 10.2 to 1.4 kb. We have applied this Hpa II/Msp I test to unfractionated DNA, and to rDNA and the rDNA intervening sequence of Drosophila virilis embryos and adults. There is no evidence of C-MeC-G-G sequences in either developmental stage of this species. Absence of G-MeC-G-C from coding and intervening sequences of rDNA was shown in comparisons of Hha I (G-C-G-C) cleavage patterns of unfractionated DNA and cloned (unmodified) segments of rDNA. Comparisons of Hpa II and Msp I cleavage products of DNA from the house fly, the flesh fly and a bumblebee also revealed no internal cytosine methylation in the sequence C-C-G-G. Because amounts of McC in C-G dinucleotides vary greatly among species, from apparent nonexistence to substantial proportions, no inference may yet be drawn about the role of such base modifications in DNA.

Animals↗

PubMed1977

Evidence for an excess of rDNA in the testis of Drosophila melanogaster during rDNA magnification.

Hybridization of rRNA and DNA extracted from different tissues of different genotypes have been performed. The results show that: 1) in DNA extracted from the testis of premagnified males there exists an excess of rDNA, which is consistent with the model proposed by Ritossa (1972) and by us (1973) to explain the phenomenon of magnification. 2) in DNA extracted from diploid tissues of different genotypes the percent of rDNA is directly proportional to the number of ribosomal genes. 3) in polytene cells the percent of rDNA for all genotypes so far studied is less than that in diploid cells and is not significantly dependent on the genotype. This last result is consistent with those of Spear and Gall (1973).

Animals↗

PubMed1979

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

D. melanogaster males of XYbb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb+/O females, yielding XYbb/O male progeny, and to XNO- w sn bb+ females, yielding XYbb/XNO- females. The male and female offspring were scored for the bb+ phenotype. Results show that there is a higher percentage of bb+ flies in the XYbb/O male progeny than in XYbb/XNO- female progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters. These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.

Animals↗

PubMed1976

rDNA and acrocentric chromosomes in man. I. rDNA levels in a subject carrier of a 8p/13p balanced translocation and in his unbalanced son.

A malformed female infant was found to have a 46,XX complement with a chromosome 8 shorter than normal with a secondary constriction and satellites on the short arm. Chromosome studies on the clinically normal father showed a balanced translocation between chromosome 8 and 13, i.e., 46,XY,t(8;13) (p21 p12). The proposita, carrier of the unbalanced form of the translocation, resulted partially monosomic for short arm of chromosome 8 (8p-) and partially trisomic for short arm of chromosome 13. The levels of DNA complementary to rRNA (normal in the father who had 10 NOR and increased in the proposita who had 11 NOR) confirmed our interpretation of the rearrangement.

Adult↗

PubMed2025

Hypermethylation at 45S rDNA promoter in cancers.

The ribosomal genes (rDNA genes) encode 47S rRNA which accounts for up to 80% of all cellular RNA. At any given time, no more than 50% of rDNA genes are actively transcribed, and the other half is silent by forming heterochromatin structures through DNA methylation. In cancer cells, upregulation of ribosome biogenesis has been recognized as a hallmark feature, thus, the reduced methylation of rDNA promoter has been thought to support conformational changes of chromatin accessibility and the subsequent increase in rDNA transcription. However, an increase in the heterochromatin state through rDNA hypermethylation can be a protective mechanism teetering on the brink of a threshold where cancer cells rarely successfully proliferate. Hence, clarifying hypo- or hypermethylation of rDNA will unravel its additional cellular functions, including organization of genome architecture and regulation of gene expression, in response to growth signaling, cellular stressors, and carcinogenesis. Using the bisulfite-based quantitative real-time methylation-specific PCR (qMSP) method after ensuring unbiased amplification and complete bisulfite conversion of the minuscule DNA amount of 1 ng, we established that the rDNA promoter was significantly hypermethylated in 107 breast, 65 lung, and 135 colon tumour tissue samples (46.81%, 51.02% and 96.60%, respectively) as compared with their corresponding adjacent normal samples (26.84%, 38.26% and 77.52%, respectively; p < 0.0001). An excessive DNA input of 1 μg resulted in double-stranded rDNA remaining unconverted even after bisulfite conversion, hence the dramatic drop in the single-stranded DNA that strictly required for bisulfite conversion, and leading to an underestimation of rDNA promoter methylation, in other words, a faulty hypomethylation status of the rDNA promoter. Our results are in line with the hypothesis that an increase in rDNA methylation is a natural pathway protecting rDNA repeats that are extremely sensitive to DNA damage in cancer cells.

DNA Methylation↗

PubMed1979

Length heterogeneity of amplified circular rDNA molecules in oocytes of the house cricket Acheta domesticus (Orthoptera: Gryllidae).

Amplification of the genes coding for rRNA occurs in the oocytes of a wide variety of organisms. The amplification process appears to be mediated through a rolling-circle mechanism. The approximate molecular weight of the smallest rDNA circles is equivalent to the estimated combined molecular weight of DNA which codes for a single ribosomal RNA precursor molecule and an associated non-transcribed spacer DNA sequence. RNA-DNA hybridization studies carried out on oocytes of the house cricket, Acheta domesticus, suggest that DNA coding for rRNA accounts for only a small fraction of the rDNA satellite, all of which is amplified in the oocyte. In order to test the possibility that the remainder of the amplified rDNA represents spacer and to determine whether a rolling-circle mechanism might also be involved in amplification in A. domesticus oocytes, rDNA was isolated from ovaries of A. domesticus and spread for electron microscopy. A large proportion of the rDNA isolated from ovaries is circular, while main-band DNA and rDNA prepared from other tissues demonstrates few if any circles. The mean size of the smallest rDNA circles is approximately 8 times longer than the length estimated for DNA which codes for 18S and 28 S rRNA. Denaturation mapping shows the rDNA circles to contain two major readily denaturing regions located about equidistant from one another on the circle. Each readily denaturing region accounts for 4--6% of the total DNA in the circle. The fact that only 12% of the average molecule is required to code for A. domesticus 18S and 28S rRNA is consistent with the hybridization data. Considerable size heterogeneity exists in the length of the smallest class of rDNA molecules. In the rDNA of other species such heterogeneity has been shown to reside in the non-transcribed spacer.

Animals↗

PubMed1992

The flax ribosomal RNA-encoding genes are arranged in tandem at a single locus interspersed by 'non-rDNA' sequences.

The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.

Blotting, Southern↗

PubMed2026

Visualization and quantification of rDNA instabilities in mammalian cells and mouse models.

Ribosomal DNA (rDNA) encodes the 18S, 5.8S, and 28S rRNA, accounting for ∼70% of cellular transcription. Despite its essential role and links to cancer and aging, quantifying rDNA instability in mammals remains challenging due to its repetitive organization and inherent heterogeneity. Here, we developed a murine rDNA FISH probe and genomic tools tailored for laboratory mouse strains. The results confirmed rDNA cluster locations, revealed substantial inter- and intra-strain as well as intercellular heterogeneity in rDNA organization within inbred mice and unstressed cells, and identified sources of spontaneous and replication-associated DNA double-strand breaks in the rDNA transcription termination region. Using mouse embryonic stem cells, we showed that BRCA1-mediated homologous recombination promotes rDNA instability, the non-homologous end joining factor XRCC1, but not Ku, suppresses intra-cluster deletions, and ATM kinase preserves rDNA cluster stability. Together, these findings establish a platform and tools for studying rDNA instability in animal models relevant to aging and cancer research.

Animals↗

PubMed1977

A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA.

The sequences corresponding to the 18S and 28S rRNAs have been mapped within a cloned 17 kilobase (kb) fragment formed by Eco R1 cleavage of Drosophila melanogaster rDNA. This fragment, Dm103, represents the longer of two major types of repeating units that are present in the rDNA of this fly, and was cloned as a hybrid plasmid, pDm103, consisting of Dm103 inserted at the Eco R1 site of the pSC101 vector (Glover et al., 1975). Mapping of the 18S and 28S rDNA in Dm103 was accomplished by quantitative determination of the amount of these rDNAs in each member of an ordered set of restriction fragments obtained by Hind III and Eco R1 ccleavage of pDm103. The amounts of 18S and 28S rDNAs were determined by hybridization of the rRNAs to fragments that were purified by cloning, and an unambiguous order of the fragments within pDm103 was established by heteroduplex mapping and from the stoichiometry of the fragment lengths. The resulting map revealed that the 4 kb of 28S rDNA within the long repeating unit represented by Dm103 is divided into two blocks that are separated by 5.4 kb of DNA of unknown function. It is this unusual arrangement of the 28S rDNA that distinguishes the long repeating units (17 kb) from the short units (11.5) kb), whose 4 kb of 28S rDna is confined to a single block, as is shown in the accompanying paper (White and Hogness, 1977). The remainder of the DNA in this long unit appears to be typically arranged, with the 2 kb of 18S rDNA confined to a single block that is separated by about 1 kb from the closest block of 28S rDNA.

Animals↗

PubMed1976

Subunit structure of rDNA-containing chromatin.

Recent studies indicate that chromatin has a repeating subunit structure. In an attempt to relate this organization to chromatin's role in selective gene transcription we have begun to examine the subunit structure of a specific gene. Tetrahymena pyriformis preferentially replicates the genes coding for rRNA (rDNA) during refeeding after prolonged starvation. By prelabeling cultures during exponential growth with [14C]thymidine and pulse-labeling during refeeding with [3H]thymidine, we have been able to differentially label bulk chromatin and rDNA-containing chromatin. Nuclei which contained at least 78% of their 3H label in rDNA were digested with staphylococcal nuclease, and the DNA digestion products analyzed on agarose gels. Both the kinetics of digestion and the digestion products were similar for 14C- and 3H-labeled chromatin. In order to monitor protein exchange, digestions were also performed on partially purified rDNA-containing chromatin or free rDNA in the presence of nuclei. While the chromatin had a digestion pattern like nuclei, the rDNA was afforded no protection from digestion. Our conclusion is that the chromatin containing rDNA (a repeated, extrachromosomal gene in Tetrahymena) exhibits a particulate structure very similar to that of bulk chromatin. This organization does not exist in free rDNA and is not the result of protein exchange during the nuclease digestion.

Animals↗

PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗

PubMed1976

Increase of rDNA redundancy in bb females of Drosophila melanogaster.

The purpose of this work was to analyze the difference between males and females with respect to rDNA magnification. To study eventual rDNA variations in females aYbb chromosome was chosen since it can magnify in males but not show phenomena of rDNA dosage compensation. The authors have observed an increase of rDNA pertaining to the Ybb chromosome in females of XXNO-/Ybb genotype with respect to the same Ybb chromosome studied in XXbb+/Ybb females. This non-inheritable rDNA increase cannot be explained in terms of compensatory increase reported in X/O males nor can it fit in the magnification scheme. The possibility might be entertained that some mechanism is missing in females which cannot complete a magnification cycle. The rDNA increase that we called rDNA magnification in males occurs in the germ line and in the soma, whereas the evidence, here reported, suggest that magnification in females occurs only in the soma.

Aneuploidy↗

PubMed1992

Clonal variation of chromosome size derived from the rDNA cluster region in Candida albicans.

Of the eight Candida albicans chromosomes, chromosome 2, assigned by the MGL1 probe, is more variable in size than the other chromosomes among strains. We found that the clonal variation of chromosome 2, which carries a rDNA gene, occurred at a frequency of up to 10% of the progeny clones. After total chromosomal digestion with XhoI, which has no recognition sites within the rDNA repeat unit, the fragments containing the rDNA cluster were detected by Southern hybridization. The difference in fragment sizes corresponded to the clonal size variation of chromosome 2. The intensity of hybridization with rDNA also correlated with the difference in size. In addition, there was no size change in the non-rDNA region as detected by NotI digestion of chromosome 2, and there was no observed change in the individual rDNA basic repeat unit size. From these lines of evidence, we confirmed that the clonal size variation of chromosome 2 which occurs at high frequency is derived from the size change of the rDNA cluster.

Candida albicans↗

PubMed1979

Quantitative determination of amplified rDNA and its distribution during oogenesis in Xenopus laevis.

The number of extra-chromosomal nucleoli and their rDNA content were determined during oogenesis in Xenopus laevis. The highly variable number of nucleoli (500 to 2,500) in oocytes of the same stage and from the same female or of different stages or from different females is not a measure of the extent of amplification. In all oocytes examined, a inversely proportional relation was found between the number of nucleoli in an oocyte and their mean rDNA content. These results indicate that there is no variation of the rDNA content of oocytes during oogenesis nor between oocytes of different females. The varying nucleolar numbers found in oocytes result thus from fusion and fission of pre-existing nucleoli. The determination of the rDNA content, in absolute units (35 pg), after amplification which occurs at the beginning of oogenesis, makes it possible to calculate the rDNA content of one nucleolus. This ranged from 0.7.10(-2) pg to 15.10(-2) pg, corresponding to about 500--11,000 cistrons of rDNA. No distinct size classes between these two extremes were observed.

Animals↗

PubMed1979

Genetic modulation of RNA metabolism in Drosophila. III. Requirement for an rDNA-deficient X chromosome in YbbSuVar-3-mediated increases in RNA synthesis.

Males of the genotype car bb/YbbSuVar-5 have an approximately two-fold increase in the rate of accumulation of 4S, 5S, 18S plus 28S and poly-A+ RNA molecules when compared to males of the genotype car bb/Ybb- (CLARK, STRAUSBAUGH AND KIEFER 1977; CLARK and KIEFER 1977). Experiments were designed to determine if the modulation of RNA metabolism by YbbSuVar-5 requires an rDNA deficiency on the X chromosome. Synthetic rates for 4S, 5S, and 18S plus 28S RNA's were measured in the genotypes Sam + iso/Ybb- and Sam + iso/YbbSuVar-5 by injecting adult males with 3H-uridine, allowing them to incorporate label for two hours, fractionating extracted RNA by polyacrylamide gel electrophoresis and determining the specific activities of RNA's (dpm/microgram RNA). The results of these determination showed that the synthetic rates for 4S, 5S, and 18S plus 28S RNA's are the same in these two genotypes, demonstrating that the YbbSuVar-5 chromosome does not increase transcription when paired with an X chromosome that is wild type for rDNA. Saturation hybridization was used to measure the rDNA content in several genotypes. These results showed that Sam + iso/YbbSuVar-5 males and Sam + iso/Ybb- males have equivalent amounts of rDNA.--These results are consistent with the suggestion that the Ybb- and YbbSuVar-5 chromosomes have similar amounts of rDNA. The nature of compensatory strategies in rDNA-deficient genotypes is discussed.

Animals↗

PubMed1977

The chromosomal location of rDNA in selected lower primates.

Hybridization in stiu was used to identify the chromosomes that carry rDNA in representative lower primates, including the baboons, Papio cynocephalus and Papio hamadryas; the colobus monkey, Colobus polykomos; the tree shrew, Tupaia glis; the lemur, Lemur fulvis; the saki, Pithecia pithecia; the marmoset, Saguinus nigricollis, and the spider monkey, Ateles geoffroyi. The marker chromosome, common to the Cercopithecines studied to date, carries the rDNA in the baboons. Another marker chromosome carries rDNA in a South American species, the spider monkey. A multichromosomal distribution of rDNA was demonstrated in the tree shrew, lemur, saki, and marmoset. None of the rDNA-containing chromosomes in the prosimians and New World monkeys show homology to the chromosomes that carry rDNA in the Hominids, Pongids, or Old World monkeys.

Animals↗

PubMed1992

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Blotting, Northern↗