Ribosomal DNA Copy Research Articles

A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR.

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation.

Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy.

Ribosomal DNA copy number loss and sequence variation in cancer.

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Single Cell Analysis Linking Ribosomal (r)DNA and rRNA Copy Numbers to Cell Size and Growth Rate Provides Insights into Molecular Protistan Ecology.

Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)‐based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single‐cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per‐cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV 0.14. The maximum growth rate could be well predicted by a combination of per‐cell ribotype CN and temperature. Our empirical data and modeling on single‐cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance‐based interpretation of quantitative ribotype data in population and community ecology of protists.

Nutrition and epigenetics link found in mice

Pregnant mice fed low-protein diets have progeny that are underweight all the way through adulthood, a new study shows. These results hold true, however, only if the mice have relatively more adenine than cytidine bases in a certain region of their genomes. The work directly shows how early environmental impacts could interact with an individual’s DNA to alter long-term health. “People debate nature versus nurture, but our work shows how separating genetics and environment is artificial—and impossible,” says Vardhman K. Rakyan at Queen Mary University of London, who led the research. Rakyan’s team carefully scrutinized a section of the rodents’ DNA that promotes RNA production. This RNA, in turn, forms the ribosome, a protein synthesis machine (Science 2016, DOI: 10.1126/science.aaf7040). Mice—and humans—have many copies of ribosomal DNA in their genomes. If the section of the rodents’ genome preceding a copy of ribosomal DNA—called the promoter region—had an adenosine rather than

Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae.

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

Regulation of ribosomal DNA amplification by the TOR pathway

Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions.

Administration of probiotics Lactobacillus rhamnosus GG and Lactobacillus gasseri K7 during pregnancy and lactation changes mouse mesenteric lymph nodes and mammary gland microbiota

The milk and mammary gland (MG) microbiome can be influenced by several factors, such as mode of delivery, breastfeeding, maternal lifestyle, health status, and diet. An increasing number of studies show a variety of positive effects of consumption of probiotics during pregnancy and breastfeeding on the mother and the newborn. The aim of this study was to investigate the effect of oral administration of probiotics Lactobacillus gasseri K7 (LK7) and Lactobacillus rhamnosus GG (LGG) during pregnancy and lactation on microbiota of the mouse mesenteric lymph nodes (MLN), MG, and milk. Pregnant FVB/N mice were fed skim milk or probiotics LGG or LK7 resuspended in skim milk during gestation and lactation. On d 3 and 8 postpartum, blood, feces, MLN, MG, and milk were analyzed for the presence of LGG or LK7. The effects of probiotics on MLN, MG, and milk microbiota was evaluated by real-time PCR and by 16S ribosomal DNA 454-pyrosequencing. In 5 of 8 fecal samples from the LGG group and in 5 of 8 fecal samples from the LK7 group, more than 1×103 of live LGG or LK7 bacterial cells were detected, respectively, whereas no viable LGG or LK7 cells were detected in the control group. Live lactic acid bacteria but no LGG or LK7 were detected in blood, MLN, and MG. Both probiotics significantly increased the total bacterial load as assessed by copies of 16S ribosomal DNA in MLN, and a similar trend was observed in MG. Metagenomic sequencing revealed that both probiotics increased the abundance of Firmicutes in MG, especially the abundance of lactic acid bacteria. The Lactobacillus genus appeared exclusively in MG from probiotic groups. Both probiotics influenced MLN microbiota by decreasing diversity (Chao1) and increasing the distribution of species (Shannon index). The LGG probiotic also affected the MG microbiota as it increased diversity and distribution of species and proportions of the genera Lactobacillus and Bifidobacterium. These results provide evidence that probiotics can modulate the bacterial composition of MLN and MG microbiota in ways that could improve the health of the MG and, ultimately, the health of the newborn.

Detection rate of diarrhoea-causing Kudoa hexapunctata in Pacific bluefin tuna Thunnus orientalis from Japanese waters

Diffuse outbreaks of food poisoning with unknown aetiologies leading to diarrhoea and vomiting within a short time after ingesting flatfish (Paralichthys olivaceus), tuna (Thunnus spp.), or amberjack (Seriola dumerili) have occurred nationwide in Japan, including the Tokyo metropolitan area. In this study, we surveyed the detection rates of kudoid parasites in 12 tuna samples that caused clinical diarrhoea from 2009 to 2012; we assessed 104 samples of whole juvenile Pacific bluefin tuna (PBT, Thunnus orientalis) and 153 block samples of other tuna distributed in the Tokyo Metropolitan Central Wholesale Market. The survey revealed that more than 70% of clinical diarrhoea cases due to tuna ingestion occurred between June and September, and Kudoa hexapunctata were detected in 9 of 12 tuna samples associated with clinical diarrhoea cases. The numbers of spores and 18S ribosomal DNA (rDNA) copies per gram of fish in 8 of 9 samples were more than 1×106 spores and 1×109 copies, respectively. Market research revealed that the K. hexapunctata-positive rate in juvenile PBT from Japanese waters was 64.4% (67/104) but that in adult PBT was 10.4% (7/67). The numbers of K. hexapunctata 18S rDNA copies in 64.5% (20/31) samples and 72.7% (16/22) of <5kg fish samples collected between May and July were more than 1×109copies/g. On the other hand, kudoid parasites were not detected from 73 tuna samples except for a single sample of Thunnus albacares. Cell monolayer permeability assays performed to examine the toxicity of K. hexapunctata against Caco-2 cells revealed that the transepithelial electrical resistance (TER) in 5×107K. hexapunctata spores decreased by 80% within 2–4h. In conclusion, K. hexapunctata was commonly detected in juvenile PBT from Japanese waters and are a likely cause of the diarrhoea outbreaks.

Determination of ribosomal DNA copy number and comparison among strains of Coccidioides.

The use of PCR-based assays to detect fungi and diagnose fungal infections as well as to monitor fungal organ burden with diseases such as coccidioidomycosis is becoming more common. The target of these assays is frequently one or more of the ribosomal DNA (rDNA) gene subunits. The multicopy nature of this gene affords greater sensitivity over single-copy genes. However, there are few studies reporting the precise number of copies of the rDNA gene per genome in pathogenic fungi. Quantitative PCR was used to determine the number of copies of rDNA as well as CTS1, a single-copy gene, in samples of Coccidioides genomic DNA by the absolute quantification method. Variability of rDNA genome copy number was determined using 13 different Coccidioides isolates and was found to vary between 20 and 146 copies per genome. This suggests that detection of rDNA will likely afford an increased sensitivity of at least 20-fold over single-copy genes. However, estimation of the number of organisms present by quantification of the rDNA cannot be made prior to knowledge of each isolate's rDNA copy number because of the strain variation.

Ribosomal DNA copy number is coupled with gene expression variation and mitochondrial abundance in humans.

Ribosomes are essential intracellular machines composed of proteins and RNA molecules. The DNA sequences (rDNA) encoding ribosomal RNAs (rRNAs) are tandemly repeated and give origin to the nucleolus. Here we develop a computational method for estimating rDNA dosage (copy number) and mitochondrial DNA abundance using whole-genome short-read DNA sequencing. We estimate these attributes across hundreds of human genomes and their association with global gene expression. The analyses uncover abundant variation in rDNA dosage that is coupled with the expression of hundreds of functionally coherent gene sets. These include associations with genes coding for chromatin components that target the nucleolus, including CTCF and HP1β. Finally, the data show an inverse association between rDNA dosage and mitochondrial DNA abundance that is manifested across genotypes. Our findings uncover a novel and cryptic source of hypervariable genomic diversity with global regulatory consequences (ribosomal eQTL) in humans. The variation provides a mechanism for cellular homeostasis and for rapid and reversible adaptation.

Accession-Specific Haplotypes of the Internal Transcribed Spacer Region in Arabidopsis thaliana--A Means for Barcoding Populations

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.

Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus

Enumerating Aspergillus fumigatus CFU can be challenging since CFU determination by plate count can be difficult. CFU determination by quantitative real-time PCR (qPCR), however, is becoming increasingly common and usually relies on detecting one of the subunits of the multicopy rRNA genes. This study was undertaken to determine if ribosomal DNA (rDNA) copy number was constant or variable among different A. fumigatus isolates. FKS1 was used as a single-copy control gene and was validated against single-copy (pyrG and ARG4) and multicopy (arsC) controls. The copy numbers of the 18S rDNA subunit were then determined for a variety of isolates and were found to vary with the strain, from 38 to 91 copies per genome. Investigation of the stability of the 18S rDNA copy number after exposure to a number of different environmental and growth conditions revealed that the copy number was stable, varying less than one copy across all conditions, including in isolates recovered from an animal model. These results suggest that while the ribosomal genes are excellent targets for enumeration by qPCR, the copy number should be determined prior to using them as targets for quantitative analysis.

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25μM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5μM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture×treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

Analysis of microbial community structure and diversity in the thermophilic anaerobic digestion of waste activated sludge

The microbial community structures in the thermophilic anaerobic digestion (TAD) of waste activated sludge (WAS) and WAS were analyzed with molecular biological techniques including real-time polymerase chain reaction (PCR) and cloning. The microbial community of TAD had less diversity than that of WAS, and the sequences obtained in WAS were not present in TAD by the cloning analysis. In the TAD bacterial clone library, 97.5% of total clones were affiliated with the phylum Firmicutes and 73.1% with the genus Coprothermobacter. Real-time PCR and cloning analysis revealed that the number of Methanosarcina thermophila, which is an acetoclastic methanogen, is larger than that of Methanoculleus thermophilus, which is a hydrogenotrophic methanogen, in terms of the numbers of copies of 16S ribosomal DNA (rDNA).

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae

BackgroundAll previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.ResultsOur present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.ConclusionBy virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

Hungry for life

Hungry for life

Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae.

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.