Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites

Intergeneric crosses between Brassica species and Raphanus sativus have produced crops with prominent shoot and root systems of Brassica and R. sativus, respectively. It is necessary to discriminate donor genomes when studying cytogenetic stability in distant crosses to identify homologous chromosome pairing, and microsatellite repeats have been used to discriminate subgenomes in allopolyploids. To identify genome-specific microsatellites, we explored the microsatellite content in three Brassica species (B. rapa, AA, B. oleracea, CC, and B. nigra, BB) and R. sativus (RR) genomes, and validated their genome specificity by fluorescence in situ hybridization. We identified three microsatellites showing A, C, and B/R genome specificity. ACBR_msat14 and ACBR_msat20 were detected in the A and C chromosomes, respectively, and ACBR_msat01 was detected in B and R genomes. However, we did not find a microsatellite that discriminated the B and R genomes. The localization of ACBR_msat20 in the 45S rDNA array in ×Brassicoraphanus 977 corroborated the association of the 45S rDNA array with genome rearrangement. Along with the rDNA and telomeric repeat probes, these microsatellites enabled the easy identification of homologous chromosomes. These data demonstrate the utility of microsatellites as probes in identifying subgenomes within closely related Brassica and Raphanus species for the analysis of genetic stability of new synthetic polyploids of these genomes.

A minimal rDNA array replicates early, delays genome replication, and uncouples anaphase entry from S phase completion

ABSTRACTRibosomal DNA (rDNA) copy number varies widely among individuals in many species, but the phenotypic consequences of such copy number fluctuations remain largely unexplored. In the yeast Saccharomyces cerevisiae, each rDNA repeat contains an origin of replication. Previous studies have demonstrated that the yeast rDNA locus can be a significant competitor for replication resources, suggesting that rDNA copy number variation may affect timely completion of genome-wide replication. We hypothesized that reduction in rDNA copy number and thus rDNA replication origins would reduce competition from the rDNA locus and thereby improve non-rDNA genome replication. To test this hypothesis, we engineered yeast strains with short rDNA arrays of 35 copies, a minimal copy number that still maintains wild type level ribosome function. Contrary to our hypothesis, the minimal rDNA strain displayed classic replication defects: decreased plasmid maintenance, delayed completion of chromosomal replication, and increased sensitivity to replication stress agonists. Although a normal rDNA array replicates late in S phase, the minimal rDNA array initiated replication in early S phase, resulting in delayed replication across the non-rDNA portions of the genome. Moreover, we discovered that absence of the rDNA fork barrier protein Fob1p increased DNA damage sensitivity in strains with early replicating rDNA. We present evidence that this increased sensitivity may be due to compromised regulation of cyclin phosphatase Cdc14p and premature entry into anaphase. Our results indicate that precocious rDNA replication, rather than total number of rDNA origins, compromises replication of the genome. Taken together, we suggest that the rDNA’s large, late-replicating state is evolutionarily conserved to promote genome stability through timely genome replication and coordination of S phase completion with anaphase entry.

A Fob1 independent role for the Smc5/6 complex in the rDNA that modulates lifespan

SUMMARYThe ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two spacer regions within each repetitive element, called non-transcribed spacer 1 (NTS1) and NTS2, are important in nucleolar organization. Smc5/6 localizes to both NTS1 and NTS2 and is involved in the regulation of Sir2 and Cohibin binding at NTS1, whereas Fob1 and Sir2 are required for optimal binding of the complex to NTS1 and NTS2, respectively. We demonstrate that Smc5/6 functions in chromatin silencing at NTS1 independently of its role in homologous recombination (HR) when forks pause at the replication fork barrier (RFB). In contrast, when the complex does not localize to the rDNA in nse3-1 mutants, the shortened lifespan correlates with NTS2 homeostasis independently of FOB1 status. Our data identify the importance of Smc5/6 integrity in NTS2 transcriptional silencing and repeat tethering, which in turn underscores rDNA stability and replicative lifespan.HighlightsSmc5/6 is important for transcriptional silencing in the rDNA.Smc5/6 tethers the rDNA array to the periphery.Transcriptional silencing of ncRNA at NTS1 and NTS2 is differentially regulated.Smc5/6 has a role in rDNA maintenance independent of HR processing at the RFB.Fob1-independent disruption of Smc5/6 at NTS2 leads to lifespan reduction.

Lost in Translation: population genomics and long-read sequencing reveals relaxation of concerted evolution of the ribosomal DNA cistron

Concerted evolution of the ribosomal DNA array has been studied in numerous eukaryotic taxa, yet is still poorly understood. rDNA genes are repeated dozens to hundreds of times in the eukaryotic genome [1] and it is believed that these arrays are homogenized through concerted evolution [2, 3] preventing the accumulation of intragenomic, and intraspecific, variation. However, numerous studies have reported rampant intragenomic and intraspecific variation in the rDNA array [4–10], contradicting our current understanding of concerted evolution. The internal transcribed spacers (ITS) of the rDNA cistron are the most commonly used DNA barcoding region in Fungi [11], and rely on concerted evolution to homogenize the rDNA array leading to a “barcode gap” [12]. Here we show that in Boletus edulis Bull., ITS intragenomic variation persists at low allele frequencies throughout the rDNA array, this variation does not correlate with genomic relatedness between populations, and rDNA genes may not evolve in a strictly concerted fashion despite the presence of unequal recombination and gene conversion. Under normal assumptions, heterozygous positions found in ITS sequences represent hybridization between populations, yet through allelic mapping of the rDNA array we found numerous heterozygous alleles to be stochastically introgressed throughout, presenting a dishonest signal of gene flow. Moreover, despite the signal of gene flow in ITS, our organisms were highly inbred, indicating a disconnect between true gene flow and barcoding signals. In addition, we show that while the mechanisms of concerted evolution are ongoing in pseudo-heterozygous individuals, they are not fully homogenizing the ITS array. Concerted evolution of the rDNA array may insufficiently homogenize the ITS gene, allowing for misleading signals of gene flow to persist, vastly complicating the use of the ITS locus for DNA barcoding in Fungi.

Genomic copy-number loss is rescued by self-limiting production of DNA circles

SummaryCopy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC production is proportional to repeat loss and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Lastly, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

Genetic context and proliferation determine contrasting patterns of copy number amplification and loss for 5S and 45S ribosomal DNA arrays in cancer

The multicopy 45S ribosomal DNA (45S rDNA) array gives origin to the nucleolus, the first discovered nuclear organelle, site of Poll I 45S rRNA transcription and key regulator of cellular metabolism, DNA repair response, genome stability, and global epigenetic states. The multicopy 5S ribosomal DNA array (5S rDNA) is located on a separate chromosome, encodes the 5S rRNA transcribed by Pol III, and exhibits concerted copy number variation (cCNV) with the 45S rDNA array in human blood. Here we combined genomic data from >700 tumors and normal tissues to provide a portrait of ribosomal DNA variation in human tissues and cancers of diverse mutational signatures. We show that most cancers undergo coupled 5S rDNA array amplification and 45S rDNA loss, with abundant inter-individual variation in rDNA copy number of both arrays, and concerted modulation of 5S-45S copy number in some but not all tissues. Analysis of genetic context revealed associations between the presence of specific somatic alterations, such as P53 mutations in stomach and lung adenocarcinomas, and coupled 5S gain / 45S loss. Finally, we show that increased proliferation rates along cancer lineages can partially explain contrasting copy number changes in the 5S and 45S rDNA arrays. We suggest that 5S rDNA amplification facilitates increased ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in highly proliferating tumor cells. Our results highlight the tissue- specificity of concerted copy number variation and uncover contrasting changes in 5S and 45S rDNA copy number along rapidly proliferating cell lineages.Lay SummaryThe 45S and 5S ribosomal DNA (rDNA) arrays contain hundreds of rDNA copies, with substantial variability across individuals and species. Although physically unlinked, both arrays exhibit concerted copy number variation. However, whether concerted copy number is universally observed across all tissues is unknown. It also remains unknown if rDNA copy number may vary in tissues and cancer lineages. Here we showed that most cancers undergo coupled 5S rDNA array amplification and 45S rDNA loss, and concerted 5S-45S copy number variation in some but not all tissues. The coupled 5S amplification and 45S loss is associated with the presence of certain somatic genetic variations, as well as increased cancerous cell proliferation rate. Our research highlights the tissue- specificity of concerted copy number variation and uncover contrasting changes in rDNA copy number along rapidly proliferating cell lineages. Our observations raise the prospects of using 5S and 45S ribosomal DNA states as indicators of cancer status and targets in new strategies for cancer therapy.

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.

Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA ofSaccharomyces cerevisiae. Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT (regulation ofnucleolarsilencing andtelophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions intransare downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).

Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome.