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BioRxiv : the Preprint Server For... May 2024Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse...
Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF () whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
PubMed: 38766058
DOI: 10.1101/2024.05.08.593200 -
Communications Biology May 2024In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also...
In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.
Topics: Klebsiella pneumoniae; Drug Resistance, Multiple, Bacterial; DNA Transposable Elements; Rec A Recombinases; Plasmids; Anti-Bacterial Agents
PubMed: 38762617
DOI: 10.1038/s42003-024-06312-4 -
Water Research Jul 2024The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12...
The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs' concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs' content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, bla and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes bla and bla, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.
Topics: Integrons; Wastewater; Seasons; Drug Resistance, Microbial; Waste Disposal, Fluid; RNA, Ribosomal, 16S; Genes, Bacterial; Anti-Bacterial Agents; Bacteria
PubMed: 38761599
DOI: 10.1016/j.watres.2024.121784 -
American Journal of Human Genetics Jun 2024Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes...
Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.
Topics: Humans; Spermatogenesis; Male; DNA Methylation; Genome, Human; Spermatids; Spermatocytes; DNA Transposable Elements; Spermatozoa; Meiosis; Transcription Factors
PubMed: 38759652
DOI: 10.1016/j.ajhg.2024.04.017 -
Zoological Research May 2024Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly...
Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( , ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( ). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
Topics: Animals; Zebrafish; DNA Transposable Elements; Transgenes; Humans; Animals, Genetically Modified; Gene Transfer Techniques
PubMed: 38757224
DOI: 10.24272/j.issn.2095-8137.2024.026 -
Communications Biology May 2024The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline....
The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals.
Topics: Animals; Retroelements; DNA Methylation; Mice; DNA (Cytosine-5-)-Methyltransferases; CpG Islands; Male
PubMed: 38755427
DOI: 10.1038/s42003-024-06252-z -
PloS One 2024Human Endogenous Retroviruses (HERVs) are fossil viruses that composes 8% of the human genome and plays several important roles in human physiology, including muscle...
BACKGROUND
Human Endogenous Retroviruses (HERVs) are fossil viruses that composes 8% of the human genome and plays several important roles in human physiology, including muscle repair/myogenesis. It is believed that inflammation may also regulate HERV expression, and therefore may contribute in the muscle repair, especially after training exercise. Hence, this study aimed to assess the level of HERVs expression and inflammation profile in practitioners' resistance exercises after an acute strength training session.
METHODS
Healthy volunteers were separated in regular practitioners of resistance exercise training group (REG, n = 27) and non-trained individuals (Control Group, n = 20). All individuals performed a strength exercise section. Blood samples were collected before the exercise (T0) and 45 minutes after the training session (T1). HERV-K (HML1-10) and W were relatively quantified, cytokine concentration and circulating microparticles were assessed.
RESULTS
REG presented higher level of HERV-W expression (~2.5 fold change) than CG at T1 (p<0.01). No difference was observed in the levels of HERV-K expression between the groups as well as the time points. Higher serum TNF-α and IL-10 levels were verified post-training session in REG and CG (p<0.01), and in REG was found a positive correlation between the levels of TNF-α at T1 and IL-10 at T0 (p = 0.01). Finally, a lower endothelial microparticle percentage was observed in REG at T1 than in T0 (p = 0.04).
CONCLUSION
REG individuals exhibited a significant upregulation of HERV-W and modulation of inflammatory markers when compared to CG. This combined effect could potentially support the process of skeletal muscle repair in the exercised individuals.
Topics: Humans; Endogenous Retroviruses; Resistance Training; Biomarkers; Male; Adult; Inflammation; Female; Young Adult; Tumor Necrosis Factor-alpha; Exercise; Interleukin-10
PubMed: 38753716
DOI: 10.1371/journal.pone.0303798 -
Cell Reports May 2024R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise...
R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.
Topics: Retroelements; Humans; DNA; Zinc Fingers; Protein Domains; Protein Binding
PubMed: 38753487
DOI: 10.1016/j.celrep.2024.114239 -
Genome Biology and Evolution Jun 2024Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the...
Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius, while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes.
Topics: Animals; DNA Transposable Elements; Genome Size; Evolution, Molecular; Anura; Genome; Poison Frogs
PubMed: 38753031
DOI: 10.1093/gbe/evae109 -
Fungal Genetics and Biology : FG & B Jun 2024Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have...
Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 10 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.
Topics: Retroelements; Terminal Repeat Sequences; Genome, Fungal; Anaerobiosis; Neocallimastigomycota; Gene Expression Regulation, Fungal; Phylogeny; Transcription, Genetic; Transcriptome
PubMed: 38750926
DOI: 10.1016/j.fgb.2024.103897