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Nature Jun 2024Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes. We recently discovered that IS110 family elements encode...
Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC-Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5'-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.
Topics: Catalytic Domain; Cryoelectron Microscopy; DNA; DNA Transposable Elements; Models, Molecular; Nucleic Acid Conformation; Protein Multimerization; Recombinases; Recombination, Genetic; RNA, Untranslated; Substrate Specificity
PubMed: 38926616
DOI: 10.1038/s41586-024-07570-2 -
Nature Jun 2024Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These...
Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes that are involved in fundamental DNA repair processes, such as homologous recombination, or in the transposition of foreign genetic material by viruses and mobile genetic elements. Here we report that IS110 insertion sequences, a family of minimal and autonomous mobile genetic elements, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and the donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables the insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion. The IS110 bridge recombination system expands the diversity of nucleic-acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements-insertion, excision and inversion-that are required for genome design.
Topics: Base Pairing; Base Sequence; DNA; DNA Transposable Elements; Mutagenesis, Insertional; Recombinases; Recombination, Genetic; RNA, Untranslated
PubMed: 38926615
DOI: 10.1038/s41586-024-07552-4 -
Nature Jun 2024Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated...
Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function.
Topics: AAA Domain; Adenosine Triphosphatases; Catalytic Domain; Cryoelectron Microscopy; DNA; DNA Transposable Elements; Enzyme Activation; Models, Molecular; Protein Multimerization; Transposases
PubMed: 38926614
DOI: 10.1038/s41586-024-07550-6 -
Methods in Molecular Biology (Clifton,... 2024CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the...
CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the generation of CHO cell pools using the piggyBac transposon system for mediating gene integration. The method describes the co-transfection of cells with the donor plasmid (coding for the gene of interest) and the helper plasmid (coding for the transposase) using polyethyleneimine (PEI). This is followed by a genetic selection for the generation of a cell pool. The resulting cell pool can be used to start a batch or fed-batch culture. Alternatively, it can be used for generation of clonal cell lines or generation of cell banks for future use.
Topics: Animals; CHO Cells; Cricetulus; DNA Transposable Elements; Transfection; Plasmids; Recombinant Proteins; Polyethyleneimine; Transposases; Genetic Vectors
PubMed: 38926277
DOI: 10.1007/978-1-0716-3878-1_9 -
Methods in Molecular Biology (Clifton,... 2024The production of recombinant proteins has helped in understanding of their function and developing new therapies. However, one of the major bottlenecks for protein...
The production of recombinant proteins has helped in understanding of their function and developing new therapies. However, one of the major bottlenecks for protein production is the establishment of reliable mammalian cell lines with high expression levels. In this chapter, we describe a simple and robust system that allows for the quick establishment of stable transgenic 293 cell lines with reproducible and high protein expression levels. This methodology is based on the piggyBac transposon system and enables the inducible production of the protein of interest. Finally, this methodology can easily be used in conventional laboratory cell culture settings without requiring specialized devices.
Topics: DNA Transposable Elements; Humans; Recombinant Proteins; HEK293 Cells; Transfection; Genetic Vectors
PubMed: 38926276
DOI: 10.1007/978-1-0716-3878-1_8 -
Systems Biology in Reproductive Medicine Dec 2024PIWI-interacting RNAs (piRNAs) are 24-32 nucleotide RNA sequences primarily expressed in germ cells and developing embryos that suppress transposable element expression...
PIWI-interacting RNAs (piRNAs) are 24-32 nucleotide RNA sequences primarily expressed in germ cells and developing embryos that suppress transposable element expression to protect genomic integrity during epigenetic reprogramming events. We characterized the expression of piRNA sequences and their encoding clusters in sperm samples from an idiopathic fertility model of Holstein bulls with high and low Sire Conception Rates. The piRNA populations were determined to be mostly similar between fertility conditions when investigated by principal component and differential expression analysis, suggesting that a high degree of conservation in the piRNA system is likely necessary for the production of viable sperm. Both fertility conditions demonstrated evidence of 'ping-pong' activity - a secondary biogenesis pathway associated with active transposable element targeting and suppression. Most sperm-borne piRNAs were between 29-30 nucleotides in length and originated from 226 clusters across the genome, with the exception of chromosome 20. Mapping analysis revealed abundant targeting of several transposable element families, suggesting a suppressive function of sperm piRNAs consistent with their established roles. Expression of genes targeted by sperm-borne piRNAs is significantly reduced throughout early embryogenesis compared to the mRNA population. Limited transposable element expression is known to be essential for spermatogenesis, thus epigenetic regulation of this pathway is likely to influence sperm quality and fertilizing capacity.
Topics: Male; Animals; Cattle; RNA, Small Interfering; Spermatozoa; Fertility; DNA Transposable Elements; Piwi-Interacting RNA
PubMed: 38924761
DOI: 10.1080/19396368.2024.2364742 -
BMC Veterinary Research Jun 2024Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate...
Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.
Topics: Animals; DNA Transposable Elements; Green Fluorescent Proteins; Rats, Transgenic; Rats; Gene Transfer Techniques; Transgenes; Male; Gene Silencing; Female; Promoter Regions, Genetic
PubMed: 38918814
DOI: 10.1186/s12917-024-04123-7 -
Foodborne Pathogens and Disease Jun 2024The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in isolates from a Brazilian...
The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in isolates from a Brazilian pork production chain and associate them with their antibiotic resistance pattern. A total of 41 whole-genome sequencing data of nontyphoidal were analyzed using PlasmidSPAdes and IntegronFinder software. Nine isolates (21.9%) had some integrons identified (complete and/or incomplete). Six complete class 1 integrons were found, with streptomycin resistance genes (, ) alone or downstream of a trimethoprim resistance gene (, , and some also containing resistance genes for sulfonamides (, ) and chloramphenicol (). Class 2 integron was detected in only one isolate, containing -- gene cassettes. Five isolates harbored CALINs-clusters but lacking integrases-with antimicrobial resistance genes typically found in integron structures. In all, integrons were observed among four serotypes: Derby, Bredeney, Panama, and monophasic var. Typhimurium I 4,[5],12:i:-. The association of integrons with antibiotic resistance phenotype showed that these elements were predominantly identified in multidrug resistance isolates, and six of the seven gentamicin-resistant isolates had integrons. So, surveillance of integrons in should be performed to identify the potential for the spread of antimicrobial resistance genes among bacteria.
Topics: Integrons; Brazil; Animals; Swine; Salmonella; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Microbial Sensitivity Tests; Phenotype; Food Microbiology; Whole Genome Sequencing; Computer Simulation; Pork Meat
PubMed: 38917456
DOI: 10.1089/fpd.2023.0118 -
F1000Research 2023Mobile elements (MEs) constitute a major portion of the genome in primates and other higher eukaryotes, and they play important role in genome evolution and gene...
BACKGROUND
Mobile elements (MEs) constitute a major portion of the genome in primates and other higher eukaryotes, and they play important role in genome evolution and gene function. MEs can be divided into two fundamentally different classes: DNA transposons which transpose in the genome in a "cut-and-paste" style, and retrotransposons which propagate in a "copy-and-paste" fashion via a process involving transcription and reverse-transcription. In primate genomes, DNA transposons are mostly dead, while many retrotransposons are still highly active. We report here the identification of a unique group of MEs, which we call "retro-DNAs", for their combined characteristics of these two fundamentally different ME classes.
METHODS
A comparative computational genomic approach was used to analyze the reference genome sequences of 10 primate species consisting of five apes, four monkeys, and marmoset.
RESULTS
From our analysis, we identified a total of 1,750 retro-DNAs, representing 748 unique insertion events in the genomes of ten primate species including human. These retro-DNAs contain sequences of DNA transposons but lack the terminal inverted repeats (TIRs), the hallmark of DNA transposons. Instead, they show characteristics of retrotransposons, such as polyA tails, longer target-site duplications (TSDs), and the "TT/AAAA" insertion site motif, suggesting the use of the L1-based target- primed reverse transcription (TPRT) mechanism. At least 40% of these retro-DNAs locate into genic regions, presenting potentials for impacting gene function. More interestingly, some retro-DNAs, as well as their parent sites, show certain levels of expression, suggesting that they have the potential to create more retro-DNA copies in the present primate genomes.
CONCLUSIONS
Although small in number, the identification of these retro-DNAs reveals a new mean for propagating DNA transposons in primate genomes without active canonical DNA transposon activity. Our data also suggest that the TPRT machinery may transpose a wider variety of DNA sequences in the genomes.
Topics: Animals; Retroelements; Primates; Humans; Genome; DNA Transposable Elements; Genomics; Evolution, Molecular
PubMed: 38915770
DOI: 10.12688/f1000research.130043.3 -
Current Microbiology Jun 2024The study focuses on the in silico genomic characterization of Sphingobium indicum B90A, revealing a wealth of genes involved in stress response, carbon monoxide...
The study focuses on the in silico genomic characterization of Sphingobium indicum B90A, revealing a wealth of genes involved in stress response, carbon monoxide oxidation, β-carotene biosynthesis, heavy metal resistance, and aromatic compound degradation, suggesting its potential as a bioremediation agent. Furthermore, genomic adaptations among nine Sphingomonad strains were explored, highlighting shared core genes via pangenome analysis, including those related to the shikimate pathway and heavy metal resistance. The majority of genes associated with aromatic compound degradation, heavy metal resistance, and stress response were found within genomic islands across all strains. Sphingobium indicum UT26S exhibited the highest number of genomic islands, while Sphingopyxis alaskensis RB2256 had the maximum fraction of its genome covered by genomic islands. The distribution of lin genes varied among the strains, indicating diverse genetic responses to environmental pressures. Additionally, in silico evidence of horizontal gene transfer (HGT) between plasmids pSRL3 and pISP3 of the Sphingobium and Sphingomonas genera, respectively, has been provided. The manuscript offers novel insights into strain B90A, highlighting its role in horizontal gene transfer and refining evolutionary relationships among Sphingomonad strains. The discovery of stress response genes and the czcABCD operon emphasizes the potential of Sphingomonads in consortia development, supported by genomic island analysis.
Topics: Phylogeny; Sphingomonadaceae; Genome, Bacterial; Hexachlorocyclohexane; Computer Simulation; Biodegradation, Environmental; Genomic Islands; Gene Transfer, Horizontal
PubMed: 38904756
DOI: 10.1007/s00284-024-03762-1