It Is Just a Matter of Time: Balancing Homologous Recombination and Non-homologous End Joining at the rDNA Locus During Meiosis

Ribosomal RNA genes (rDNAs) are located in large domains of hundreds of rDNA units organized in a head-to-tail manner. The proper and stable inheritance of rDNA clusters is of paramount importance for survival. Yet, these highly repetitive elements pose a potential risk to the genome since they can undergo non-allelic exchanges. Here, we review the current knowledge of the organization of the rDNA clusters in Arabidopsis thaliana and their stability during meiosis. Recent findings suggest that during meiosis, all rDNA loci are embedded within the nucleolus favoring non-homologous end joining (NHEJ) as a repair mechanism, while DNA repair via homologous recombination (HR) appears to be a rare event. We propose a model where (1) frequent meiotic NHEJ events generate abundant single nucleotide polymorphisms and insertions/deletions within the rDNA, resulting in a heterogeneous population of rDNA units and (2) rare HR events dynamically change rDNA unit numbers, only to be observed in large populations over many generations. Based on the latest efforts to delineate the entire rDNA sequence in A. thaliana, we discuss evidence supporting this model. The results compiled so far draw a surprising picture of rDNA sequence heterogeneity between individual units. Furthermore, rDNA cluster sizes have been recognized as relatively stable when observing less than 10 generations, yet emerged as major determinant of genome size variation between different A. thaliana ecotypes. The sequencing efforts also revealed that transcripts from the diverse rDNA units yield heterogenous ribosome populations with potential functional implications. These findings strongly motivate further research to understand the mechanisms that maintain the metastable state of rDNA loci.

Establishment of a near-contiguous genome sequence of the citric acid producing yeast Yarrowia lipolytica DSM 3286 with resolution of rDNA clusters and telomeres

Yarrowia lipolytica is an oleaginous yeast that is particularly suitable for the sustainable production of secondary metabolites. The genome of this yeast is characterized by its relatively large size and its high number of different rDNA clusters located in its telomeric regions. However, due to the presence of long repetitive elements in the sub-telomeric regions, rDNA clusters and telomeres are missing in current genome assemblies of Y. lipolytica. Here, we present the near-contiguous genome sequence of the biotechnologically relevant strain DSM 3286. We employed a hybrid assembly strategy combining Illumina and nanopore sequencing reads to integrate all six rDNA clusters as well as telomeric repeats into the genome sequence. By fine-tuning of DNA isolation and library preparation protocols, we were able to create ultra-long reads that not only contained multiples of mitochondrial genomes but also shed light on the inter- and intra-chromosomal diversity of rDNA cluster types. We show that there are ten different rDNA units present in this strain that additionally appear in a predefined order in a cluster. Based on single reads, we also demonstrate that the number of rDNA repeats in a specific cluster varies from cell to cell within a population.

The human ribosomal DNA array is composed of highly homogenized tandem clusters

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

Genomic architecture of 5S rDNA cluster and its variations within and between species

Ribosomal genes (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosome. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. Here, we generate ultra-long Nanopore and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform manual assembly of the cluster using the long reads that carry these units, which led to an assembly of rDNA cluster consisting of up to 167 5S rDNA units. The ordering and copy number of various rDNA units are indicative of separation time between strains. Surprisingly, we observed a drastically lower level of variation in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than C. elegans N2 strain, suggesting a unique mechanism in maintaining the rDNA cluster stability in the N2. Single-copy transgenes landed into the rDNA cluster shows the expected expression in the soma, supporting that rDNA genomic environment is transcriptionally compatible with RNA polymerase II. Delineating the structure and variation of rDNA cluster paves the way for its functional and evolutionary studies.

Prevalence of Malassezia species on the skin of HIV-seropositive patients

Malassezia is a genus of lipophilic yeasts residing on the skin of warm-blooded animals. The correlation between specific species and their involvement in skin diseases has been well researched. However, only very few studies have investigated the distribution of Malassezia spp. on the healthy skin of patients infected with human immunodeficiency virus (HIV). The purpose of this work was to analyze whether the composition of Malassezia spp. isolated from the skin of the HIV-infected patients differs from that of healthy individuals. The study included a total of 96 subjects, who were divided into two equally sized groups: HIV-seropositive and HIV-seronegative. The specimens were collected from the subjects by swabbing four anatomical sites (face, chest, back, and scalp). Species were identified using phenotype-based methods, and the identification of strains isolated from the HIV-seropositive patients was confirmed by PCR sequencing of the rDNA cluster. Malassezia spp. were isolated from 33 (69%) HIV-seropositive patients and 38 (79%) healthy volunteers. It was found that men were much more likely to have their heads colonized with Malassezia spp. than women. The most prevalent species on the skin of both HIV-seropositive and HIV-seronegative individuals were Malassezia sympodialis, M. globosa, and M. furfur, albeit at different proportions in the two populations. The diversity of Malassezia spp. was the highest on the face of the HIV-seropositive patients (Shannon–Weiner Index H = 1.35) and lowest on the back of the healthy volunteers (H = 0.16). The phenotype- and molecular-based identification methods were congruent at 94.9%. It was observed a tendency that the HIV-seropositive patients had higher CD4+ cell counts, indicating higher colonization with Malassezia spp.

Comparative cytogenetic analyses in Ancistrus species (Siluriformes: Loricariidae)

ABSTRACT Ancistrus is a specious genus of armored catfishes that has been extensively used for cytogenetic studies in the last 17 years. A comparison of the extensive karyotypic plasticity within this genus is presented with new cytogenetic analysis for Ancistrus cf. multispinis and Ancistrus aguaboensis. This study aims to improve our understanding of chromosomal evolution associated with changes in the diploid number (2n) and the dispersion of ribosomal DNAs (rDNAs) within Ancistrus. Ancistrus cf. multispinis and A. aguaboensis exhibit 2n of 52 and 50 chromosomes, respectively. Given that A. cf. multispinis shares a 2n = 52 also found in Pterygoplichthyini, the sister group for Ancistrini, a Robertsonian (Rb) fusion event is proposed for the 2n reduction in A. aguaboensis. 5S rDNAs pseudogenes sites have already been associated with Rb fusion in Ancistrus and our analysis suggests that the 2n reduction in A. aguaboensis was triggered by double strand breaks (DSBs) and chromosomal rearrangements at 5S rDNA sites. The presence of evolutionary breakpoint regions (EBRs) into rDNA cluster is proposed to explain part of the Rb fusion in Ancistrus. Cytogenetic data presented extends the diversity already documented in Ancistrus to further understand the role of chromosomal rearrangements in the diversification of Ancistrini.

New insights into the karyotype evolution of the genus Gampsocleis (Orthoptera, Tettigoniinae, Gampsocleidini)

Five species belonging to the genusGampsocleisFieber, 1852 were analyzed using fluorescencein situhybridization (FISH) with 18S rDNA and telomeric probes, as well as C-banding, DAPI/CMA3staining and silver impregnation. The studied species showed two distinct karyotypes, with 2n = 31 (male) and 2n = 23 (male) chromosomes. The drastic reduction in chromosome number observed in the latter case suggests multiple translocations and fusions as the main responsible that occurred during chromosome evolution. Two groups of rDNA distribution were found inGampsocleisrepresentatives analyzed. Group 1, with a single large rDNA cluster on the medium-sized autosome found in four species, carried in the haploid karyotype. Group 2, represented only byG.abbreviata, was characterized by the presence of two rDNA signals. TTAGG telomeric repeats were found at the ends of chromosome arms as expected. The rDNA clusters coincided with active NORs and GC-rich segments.

cytbas a New Genetic Marker for Differentiation ofProtothecaSpecies

ABSTRACTAchlorophyllous unicellular microalgae of the genusPrototheca(Trebouxiophyceae,Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused byProtothecawickerhamii, followed byProtothecazopfiiand, sporadically, byProtothecacutisandProtothecamiyajii. In veterinary medicine, however,P. zopfiiis a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification ofProtothecaspp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among allProtothecaspp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of allProtothecaspp. We propose the mitochondrialcytbgene as a new and robust marker for diagnostics and phylogenetic studies of theProtothecaalgae. Thecytbgene displayed important advantages over the rDNA markers. Not only did thecytbgene have the highest discriminatory capacity for resolving allProtothecaspecies, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partialcytbgene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of allProtothecaspecies described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Redescription, molecular features, and neotype deposition of Rhipicephalus pusillus Gil Collado and Ixodes ventalloi Gil Collado (Acari, Ixodidae)

Two species of ticks, Rhipicephalus pusillus Gil-Collado and Ixodes ventalloi Gil-Collado are redescribed, their molecular features (16S rDNA) compared with near species, and neotypes named and deposited. The male of R. pusillus is characterized by a smooth dorsal surface, with relatively short marginal grooves, not reaching the eyes and reaching the first festoon, longer than wide adanal plates, internally concave and widely rounded posteriorly, without internal spurs, accessory adanal plates not projecting over the cuticle, and a narrow, long and straight process on the spiracular plate. The female has very small porose areas, separated by 2.5-3 diameters, and a large triangular spur on the ventro-internal side of the palpal article 1. The nymph has lateral processes curved anteriorly in the ventral aspect of the basis capituli, long auriculae, and internal spurs on the four coxae. The larva of R. pusillus is separated by the presence of spurs on the ventral surface of palpal segment I, together with the rounded apices of the palpi. Features of 16S rDNA cluster this species near the R. sanguineus s.l. complex of species. The distinctive feature of the female of I. ventalloi is the long and curved auriculae, which are absent in every other species of Ixodes reported in the Western Palaearctic. The male is characterized by relatively long conscutal setae and deep and well defined cervical fields. The nymph is unique by having slightly apparent lateral carinae and small cornua, very abundant and long alloscutal setae, about six times longer than those on the scutum and internal spur of coxa I reaching coxa II. The larva of I. ventalloi has Md1-Md3 setae of the same size as the rest of the conscutal setae, which are only slightly longer than the scutal setae, cornua present, and anal groove clearly divergent. In addition to the unique combination of morphological features, the molecular 16rDNA sequence clearly separates I. ventalloi from other species of the I. ricinus group. We named and deposited in an internationally accessible collection the neotypes of both species, since the holotypes were lost.

Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae)

The eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). In recent years, an increasing number of rDNA sequence polymorphisms have been identified in numerous species. In the present study, we provide 33 complete ITS (ITS1-5.8S-ITS2) sequences from two Symphurus plagiusa individuals. To the best of our knowledge, these sequences are the first detailed information on ITS sequences in Pleuronectiformes. Here, two divergent types (Type A and B) of the ITS1-5.8S-ITS2 rDNA sequence were found, which mainly differ in sequence length, GC content, nucleotide diversity (π), secondary structure and minimum free energy. The ITS1-5.8S-ITS2 rDNA sequence of Type B was speculated to be a putative pseudogene according to pseudogene identification criteria. Cluster analysis showed that sequences from the same type clustered into one group and two major groups were formed. The high degree of ITS1-5.8S-ITS2 sequence polymorphism at the intra-specific level indicated that the S. plagiusa genome has evolved in a non-concerted evolutionary manner. These results not only provide useful data for ribosomal pseudogene identification, but also further contribute to the study of rDNA evolution in teleostean genomes.