Repetitive Elements In Genome Research Articles

ScNanoSeq-CUT&Tag: a single-cell long-read CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells.

Chromatin modifications are fundamental epigenetic marks that determine genome functions, but it remains challenging to profile those of repetitive elements and complex genomic regions. Here, we develop scNanoSeq-CUT&Tag, a streamlined method, by adapting modified cleavage under targets and tagmentation (CUT&Tag) to the nanopore sequencing platform for genome-wide chromatin modification profiling within individual cells. We show that scNanoSeq-CUT&Tag can accurately profile histone marks and transcription factor occupancy patterns at single-cell resolution as well as distinguish different cell types. scNanoSeq-CUT&Tag efficiently maps the allele-specific chromatin modifications and allows analysis of their neighboring region co-occupancy patterns within individual cells. Moreover, scNanoSeq-CUT&Tag can accurately detect chromatin modifications for individual copies of repetitive elements in both human and mouse genomes. Overall, we prove that scNanoSeq-CUT&Tag is a valuable single-cell tool for efficiently profiling histone marks and transcription factor occupancies, especially for previously poorly studied complex genomic regions and blacklist genomic regions.

GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida.

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.

Enhancer activation from transposable elements in extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of MYC-amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs. To characterize a potential regulatory role of EIEs, we focused on a fragment of the L1M4a1#LINE/L1 which we found to be co-amplified with MYC on ecDNA, gaining enhancer-associated chromatin marks in contrast to its normally silenced state. This EIE, in particular, existed as a naturally occurring structural variant upstream of MYC, gaining oncogenic potential in the transcriptionally permissive ecDNA environment. This EIE sequence is sufficient to enhance MYC expression and is required for cancer cell fitness. These findings suggest that silent repetitive genomic elements can be reactivated on ecDNA, leading to functional cooption and amplification. Repeat element activation on ecDNA represents a mechanism of accelerated evolution and tumor heterogeneity and may have diagnostic and therapeutic potential.

DANTE and DANTE_LTR: lineage-centric annotation pipelines for long terminal repeat retrotransposons in plant genomes.

Long terminal repeat (LTR) retrotransposons constitute a predominant class of repetitive DNA elements in most plant genomes. With the increasing number of sequenced plant genomes, there is an ongoing demand for computational tools facilitating efficient annotation and classification of LTR retrotransposons in plant genome assemblies. Herein, we introduce DANTE, a computational pipeline for Domain-based ANnotation of Transposable Elements, designed for sensitive detection of these elements via their conserved protein domain sequences. The identified protein domains are subsequently inputted into the DANTE_LTR pipeline to annotate complete element sequences by detecting their structural features, such as LTRs, in adjacent genomic regions. Leveraging domain sequences allows for precise classification of elements into phylogenetic lineages, offering a more granular annotation compared with coarser conventional superfamily-based classification methods. The efficiency and accuracy of this approach were evidenced via annotation of LTR retrotransposons in 93 plant genomes. Results were benchmarked against several established pipelines, showing that DANTE_LTR is capable of identifying significantly more intact LTR retrotransposons. DANTE and DANTE_LTR are provided as user-friendly Galaxy tools accessible via a public server (https://repeatexplorer-elixir.cerit-sc.cz), installable on local Galaxy instances from the Galaxy tool shed or executable from the command line.

Neotelomeres and telomere-spanning chromosomal arm fusions in cancer genomes revealed by long-read sequencing

Alterations in the structure and location of telomeres are pivotal in cancer genome evolution. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeats, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. These results provide a framework for the systematic study of telomeric repeats in cancer genomes, which could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

Revealing the Satellite DNA Content in Ancistrus sp. (Siluriformes: Loricariidae) by Genomic and Bioinformatic Analysis

Introduction: Eukaryotic genomes are composed of simple, repetitive sequences, including satellite DNAs (satDNA), which are noncoding sequences arranged in tandem arrays. These sequences play a crucial role in genomic functions and innovations, influencing processes such as the maintenance of nuclear material, the formation of heterochromatin and the differentiation of sex chromosomes. In this genomic era, advances in next-generation sequencing and bioinformatics tools have facilitated the exhaustive cataloging of repetitive elements in genomes, particularly in non-model species. This study focuses on the satDNA content of Ancistrus sp., a diverse species of fish from the Loricariidae family. The genus Ancistrus shows significant karyotypic evolution, with extensive variability from the ancestral diploid number. Methods: By means of bioinformatic approaches, 40 satDNA families in Ancistrus sp., constituting 5.19% of the genome were identified. Analysis of the abundance and divergence landscape revealed diverse profiles, indicating recent amplification and homogenization of these satDNA sequences. Results: The most abundant satellite, AnSat1-142, constitutes 2.1% of the genome, while the least abundant, AnSat40-52, represents 0.0034%. The length of the monomer repeat varies from 16 to 142 base pairs, with an average length of 61 bp. These results contribute to understanding the genomic dynamics and evolution of satDNAs in Ancistrus sp. Conclusion: The study underscores the variability of satDNAs between fish species and provides valuable information on chromosome organization and the evolution of repetitive elements in non-model organisms.

Transposable elements as tissue-specific enhancers in cancers of endodermal lineage

Transposable elements (TE) are repetitive genomic elements that harbor binding sites for human transcription factors (TF). A regulatory role for TEs has been suggested in embryonal development and diseases such as cancer but systematic investigation of their functions has been limited by their widespread silencing in the genome. Here, we utilize unbiased massively parallel reporter assay data using a whole human genome library to identify TEs with functional enhancer activity in two human cancer types of endodermal lineage, colorectal and liver cancers. We show that the identified TE enhancers are characterized by genomic features associated with active enhancers, such as epigenetic marks and TF binding. Importantly, we identify distinct TE subfamilies that function as tissue-specific enhancers, namely MER11- and LTR12-elements in colon and liver cancers, respectively. These elements are bound by distinct TFs in each cell type, and they have predicted associations to differentially expressed genes. In conclusion, these data demonstrate how different cancer types can utilize distinct TEs as tissue-specific enhancers, paving the way for comprehensive understanding of the role of TEs as bona fide enhancers in the cancer genomes.

Improving Cancer Epigenetic Therapy; A Glimpse of NRF2

One of the mechanisms used by epigenetic therapy is the elevation of host cell-derived double stranded RNA (dsRNA) baseline levels through overexpression of genomic repetitive elements especially Alu retroelements. The dsRNAs trigger immunogenic responses since immune system cannot distinguish between endogenous and exogenous dsRNAs derived from viral infections; hence called “Viral mimicry response”. These dsRNAs are recognized by pattern recognition receptors (PRRs) such as MDA-5 which further induce inflammatory responses through interferon secretion. However, the response is limited through the function of some editing enzymes such as ADAR1 which destabilizes the formation of dsRNAs and renders the therapy less efficient through attenuating interferon secretion by immune cells. Since, some cancer cells can survive even after ADAR1 inhibition, it is speculated that there might be other mechanism which contribute to dsRNA destabilization. Since dsRNA formation derived from retroelement transcripts mimics viral infections, we tried to review the mechanistic approaches applied during host-pathogen interaction to highlight a possible candidate which might be cogitable for further investigations in epigenetic therapy. dsRNAs produced by RNA viruses are sensed by PRRs and activate nuclear factor erythroid 2 p45-related factor 2 (NRF2) which further downregulates STING protein and attenuates IFN release. RNA viruses such as SARS-CoV-2 have the potential to impair NRF2 signaling and eliminate its inhibitory effect from STING, leading to excessive release of IFNs and destroy pulmonary cells through cytokine release storm (CRS). Here, we briefly explain that NRF2, in a very downstream side of anti-viral response, might be a potential candidate target in combination with epigenetic therapy to circumvent the limitations in cancer epigenetic therapy.

Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

Abstract 6020: The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment

Abstract The purpose of this study was to understand the effect of p53 mutation status in ovarian cancer cells on the expression of repetitive elements (REs) following epigenetic treatment. High-grade serous ovarian carcinoma is the most aggressive subtype of ovarian cancer and the 5 year survival rate has remained unchanged for over two decades. Over 90% of high-grade serous ovarian carcinoma (OC) patients have a mutation in the TP53 gene, which encodes the p53 protein. P53 is a transcription factor primarily involved in maintaining genome integrity, but it can also regulate the expression of REs in the genome. REs account for more than half of the genome and these repetitive genomic elements are epigenetically silenced in healthy cells to prevent genome instability. The expression of some REs, such as long interspersed nuclear elements and endogenous retroviruses, are upregulated in human OC patients and cell lines. The mechanism by which p53 regulates expression of REs is not well-understood; the literature documents some instances of p53 activating RE transcription and others of p53 repressing RE transcription, depending on the target elements. We have previously shown that RE expression can be induced by treating OC cells with low doses of DNA methyltransferase inhibitors (DNMTis). Recently, we showed that OC cell lines with mutant p53 have higher baseline expression of REs compared to wild type (WT) cell lines. Treatment of TP53 mutant OC cell lines with DNMTis had a muted effect on RE expression compared to treatment of WT OC cell lines. To explore the relationship between TP53 mutation status and RE expression after DNMTi treatment, OC cell lines with different p53 mutation statuses were treated with 500 nM of the DNMTi 5-azacytidine. We then analyzed the expression of several REs via RT-qPCR and western blot. We hypothesized that TP53 mutant cell lines would have lower DNMTi upregulation of REs than TP53 WT cell lines. While at the RNA level RE expression was variable, at the protein level, we found that DNMTi treatment increased protein levels of several REs in TP53 WT and mutant, but not null, OC cell lines. Future work will examine the feasibility of utilizing these RE proteins as immunotherapy targets in OC. Citation Format: Khadra K. Omar, Erin E. Grundy, Olivia Cox, Melissa Hadley, Katherine B. Chiappinelli. The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6020.

CAULIFINDER: a pipeline for the automated detection and annotation of caulimovirid endogenous viral elements in plant genomes

Plant, animal and protist genomes often contain endogenous viral elements (EVEs), which correspond to partial and sometimes entire viral genomes that have been captured in the genome of their host organism through a variety of integration mechanisms. While the number of sequenced eukaryotic genomes is rapidly increasing, the annotation and characterization of EVEs remains largely overlooked. EVEs that derive from members of the family Caulimoviridae are widespread across tracheophyte plants, and sometimes they occur in very high copy numbers. However, existing programs for annotating repetitive DNA elements in plant genomes are poor at identifying and then classifying these EVEs. Other than accurately annotating plant genomes, there is intrinsic value in a tool that could identify caulimovirid EVEs as they testify to recent or ancient host-virus interactions and provide valuable insights into virus evolution. In response to this research need, we have developed CAULIFINDER, an automated and sensitive annotation software package. CAULIFINDER consists of two complementary workflows, one to reconstruct, annotate and group caulimovirid EVEs in a given plant genome and the second to classify these genetic elements into officially recognized or tentative genera in the Caulimoviridae. We have benchmarked the CAULIFINDER package using the Vitis vinifera reference genome, which contains a rich assortment of caulimovirid EVEs that have previously been characterized using manual methods. The CAULIFINDER package is distributed in the form of a Docker image.

A de novo assembled high-quality chromosome-scale Trifolium pratense genome and fine-scale phylogenetic analysis

BackgroundRed clover (Trifolium pratense L.) is a diploid perennial temperate legume with 14 chromosomes (2n = 14) native to Europe and West Asia, with high nutritional and economic value. It is a very important forage grass and is widely grown in marine climates, such as the United States and Sweden. Genetic research and molecular breeding are limited by the lack of high-quality reference genomes. In this study, we used Illumina, PacBio HiFi, and Hi-C to obtain a high-quality chromosome-scale red clover genome and used genome annotation results to analyze evolutionary relationships among related species.ResultsThe red clover genome obtained by PacBio HiFi assembly sequencing was 423 M. The assembly quality was the highest among legume genome assemblies published to date. The contig N50 was 13 Mb, scaffold N50 was 55 Mb, and BUSCO completeness was 97.9%, accounting for 92.8% of the predicted genome. Genome annotation revealed 44,588 gene models with high confidence and 52.81% repetitive elements in red clover genome. Based on a comparison of genome annotation results, red clover was closely related to Trifolium medium and distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum among legumes. Analyses of gene family expansions and contractions and forward gene selection revealed gene families and genes related to environmental stress resistance and energy metabolism.ConclusionsWe report a high-quality de novo genome assembly for the red clover at the chromosome level, with a substantial improvement in assembly quality over those of previously published red clover genomes. These annotated gene models can provide an important resource for molecular genetic breeding and legume evolution studies. Furthermore, we analyzed the evolutionary relationships among red clover and closely related species, providing a basis for evolutionary studies of clover leaf and legumes, genomics analyses of forage grass, the improvement of agronomic traits.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

DeepGRP: engineering a software tool for predicting genomic repetitive elements using Recurrent Neural Networks with attention

BackgroundRepetitive elements contribute a large part of eukaryotic genomes. For example, about 40 to 50% of human, mouse and rat genomes are repetitive. So identifying and classifying repeats is an important step in genome annotation. This annotation step is traditionally performed using alignment based methods, either in a de novo approach or by aligning the genome sequence to a species specific set of repetitive sequences. Recently, Li (Bioinformatics 35:4408–4410, 2019) developed a novel software tool dna-brnn to annotate repetitive sequences using a recurrent neural network trained on sample annotations of repetitive elements.ResultsWe have developed the methods of dna-brnn further and engineered a new software tool DeepGRP. This combines the basic concepts of Li (Bioinformatics 35:4408–4410, 2019) with current techniques developed for neural machine translation, the attention mechanism, for the task of nucleotide-level annotation of repetitive elements. An evaluation on the human genome shows a 20% improvement of the Matthews correlation coefficient for the predictions delivered by DeepGRP, when compared to dna-brnn. DeepGRP predicts two additional classes of repeats (compared to dna-brnn) and is able to transfer repeat annotations, using RepeatMasker-based training data to a different species (mouse). Additionally, we could show that DeepGRP predicts repeats annotated in the Dfam database, but not annotated by RepeatMasker. DeepGRP is highly scalable due to its implementation in the TensorFlow framework. For example, the GPU-accelerated version of DeepGRP is approx. 1.8 times faster than dna-brnn, approx. 8.6 times faster than RepeatMasker and over 100 times faster than HMMER searching for models of the Dfam database.ConclusionsBy incorporating methods from neural machine translation, DeepGRP achieves a consistent improvement of the quality of the predictions compared to dna-brnn. Improved running times are obtained by employing TensorFlow as implementation framework and the use of GPUs. By incorporating two additional classes of repeats, DeepGRP provides more complete annotations, which were evaluated against three state-of-the-art tools for repeat annotation.

Impact of Repetitive DNA Elements on Snake Genome Biology and Evolution.

The distinctive biology and unique evolutionary features of snakes make them fascinating model systems to elucidate how genomes evolve and how variation at the genomic level is interlinked with phenotypic-level evolution. Similar to other eukaryotic genomes, large proportions of snake genomes contain repetitive DNA, including transposable elements (TEs) and satellite repeats. The importance of repetitive DNA and its structural and functional role in the snake genome, remain unclear. This review highlights the major types of repeats and their proportions in snake genomes, reflecting the high diversity and composition of snake repeats. We present snakes as an emerging and important model system for the study of repetitive DNA under the impact of sex and microchromosome evolution. We assemble evidence to show that certain repetitive elements in snakes are transcriptionally active and demonstrate highly dynamic lineage-specific patterns as repeat sequences. We hypothesize that particular TEs can trigger different genomic mechanisms that might contribute to driving adaptive evolution in snakes. Finally, we review emerging approaches that may be used to study the expression of repetitive elements in complex genomes, such as snakes. The specific aspects presented here will stimulate further discussion on the role of genomic repeats in shaping snake evolution.

A first look at the ‘repeatome’ of Benedenia humboldti, a major pathogen in yellowtail aquaculture: Repetitive element characterization, nuclear rRNA operon assembly, and microsatellite discovery

The monogenean Benedenia humboldti is a pathogen of the yellowtail Seriola lalandi in the South-Eastern Pacific ocean. Using low-coverage short Illumina 150bp pair-end reads sequencing, this study examines, for the first time, the 'repeatome' (= repetitive genomic elements), including the 45S ribosomal RNA DNA operon and microsatellites, in B. humboldti. Repetitive elements comprised a large fraction of the nuclear genome and a considerable proportion of them could not be assigned to known repeat element families. Taking into account only annotated repetitive elements, the most frequent belonged to the 45S ribosomal RNA operon or were classified as satellite DNA and Class I - Long Interspersed Nuclear Elements (LINEs) which were considerably more abundant than Class I - LTR elements. The ribosomal RNA gene operon in B. humboldti is comprised of, in the following order, a 5' ETS (length=233bp), ssrDNA (2082bp), ITS1 (346bp), 5.8S rDNA (150bp), ITS2 (572bp), lsrDNA (3887bp), and a 3' ETS (1097bp). A total of 15 SSRs were identified. These newly developed genomic resources will contribute to the better understanding of meta-population connectivity in this species, cryptic species in the genus, and will advance pest management strategies.

Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number.

The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as ‘back-up’ copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.

Endogenous retroviruses drive KRAB zinc-finger protein family expression for tumor suppression.

Gene expression aberration is a hallmark of cancers, but the mechanisms underlying such aberrations remain unclear. Human endogenous retroviruses (HERVs) are genomic repetitive elements that potentially function as enhancers. Since numerous HERVs are epigenetically activated in tumors, their activation could cause global gene expression aberrations in tumors. Here, we show that HERV activation in tumors leads to the up-regulation of hundreds of transcriptional suppressors, namely, Krüppel-associated box domain-containing zinc-finger family proteins (KZFPs). KZFP genes are preferentially encoded nearby the activated HERVs in tumors and transcriptionally regulated by these adjacent HERVs. Increased HERV and KZFP expression in tumors was associated with better disease conditions. Increased KZFP expression in cancer cells altered the expression of genes related to the cell cycle and cell-matrix adhesion and suppressed cellular growth, migration, and invasion abilities. Our data suggest that HERV activation in tumors drives the synchronized elevation of KZFP expression, presumably leading to tumor suppression.

The mechanisms underlying antigenic variation and maintenance of genomic integrity in Mycoplasma pneumoniae and Mycoplasma genitalium.

Mycoplasma pneumoniae and Mycoplasma genitalium are important causative agents of infections in humans. Like all other mycoplasmas, these species possess genomes that are significantly smaller than that of other prokaryotes. Moreover, both organisms possess an exceptionally compact set of DNA recombination and repair-associated genes. These genes, however, are sufficient to generate antigenic variation by means of homologous recombination between specific repetitive genomic elements. At the same time, these mycoplasmas have likely evolved strategies to maintain the stability and integrity of their 'minimal' genomes. Previous studies have indicated that there are considerable differences between mycoplasmas and other bacteria in the composition of their DNA recombination and repair machinery. However, the complete repertoire of activities executed by the putative recombination and repair enzymes encoded by Mycoplasma species is not yet fully understood. In this paper, we review the current knowledge on the proteins that likely form part of the DNA repair and recombination pathways of two of the most clinically relevant Mycoplasma species, M. pneumoniae and M. genitalium. The characterization of these proteins will help to define the minimal enzymatic requirements for creating bacterial genetic diversity (antigenic variation) on the one hand, while maintaining genomic integrity on the other.

A chromosome-scale assembly of the black gram (Vigna mungo) genome.

Black gram (Vigna mungo) is an important short duration grain legume crop. Black gram seeds provide an inexpensive source of dietary protein. Here, we applied the 10X Genomics linked-read technology to obtain a de novo whole genome assembly of V.mungo cultivated variety Chai Nat 80 (CN80). The preliminary assembly contained 12,228 contigs and had an N50 length of 5.2Mb. Subsequent scaffolding using the long-range Chicago and HiC techniques yielded the first high-quality, chromosome-level assembly of 499Mb comprising 11 pseudomolecules. Comparative genomics analyses based on sequence information from single-copy orthologous genes revealed that black gram and mungbean (Vignaradiata) diverged about 2.7 million years ago . The transversion rate (4DTv) analysis in V.mungo revealed no evidence supporting a recent genome-wide duplication event observed in the tetraploid créole bean (Vigna reflexo-pilosa). The proportion of repetitive elements in the black gram genome is slightly lower than the numbers reported for related Vigna species. The majority of long terminal repeat retrotransposons appeared to integrate into the genome within the last five million years. We also examined alternative splicing events in V.mungo using full-length transcript sequences. While intron retention was the most prevalent mode of alternative splicing in several plant species, alternative 3' acceptor site selection represented the majority of events in black gram. Our high-quality genome assembly along with the genomic variation information from the germplasm provides valuable resources for accelerating the development of elite varieties through marker-assisted breeding and for future comparative genomics and phylogenetic studies in legume species.