Intraspecies Variation in Tetrahymena rostrata

Two distinct isolates of the facultative parasite, Tetrahymena rostrata were compared, identifying and utilising markers that are useful for studying clonal variation within the species were identified and utilised. The sequences of mitochondrial genomes and several nuclear genes were determined using Illumina short read sequencing. The two T. rostrata isolates had similar morphology. The linear mitogenomes had the gene content and organisation typical of the Tetrahymena genus, comprising 8 tRNA genes, 6 ribosomal RNA genes and 45 protein coding sequences (CDS), twenty-two of which had known function. The two isolates had nucleotide identity within common nuclear markers encoded within the histone H3 and H4 and small subunit ribosomal RNA genes and differed by only 2–4 nucleotides in a region of the characterised actin genes. Variation was observed in several mitochondrial genes and was used to determine intraspecies variation and may reflect the natural history of T. rostrata from different hosts or the geographic origins of the isolates.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

Background: Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics.Methods: To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results: The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions: This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11–RAD50–NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease.