Small Transposons Research Articles

Genomic Diversity of Methicillin-Resistant Staphylococcus aureus CC398 Isolates Collected from Diseased Swine in the German National Resistance Monitoring Program GERM-Vet from 2007 to 2019.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) isolates (n = 178) collected in the national resistance monitoring program GERM-Vet from diseased swine in Germany from 2007 to 2019 were investigated for their genomic diversity with a focus on virulence and antimicrobial resistance (AMR) traits. Whole-genome sequencing was followed by molecular typing and sequence analysis. A minimum spanning tree based on core-genome multilocus sequence typing was constructed, and antimicrobial susceptibility testing was performed. Most isolates were assigned to nine clusters. They displayed close phylogenetic relationships but a wide molecular variety, including 13 spa types and 19 known and four novel dru types. Several toxin-encoding genes, including eta, seb, sek, sep, and seq, were detected. The isolates harbored a wide range of AMR properties mirroring the proportions of the classes of antimicrobial agents applied in veterinary medicine in Germany. Multiple novel or rare AMR genes were identified, including the phenicol-lincosamide-oxazolidinone-pleuromutilin-streptogramin A resistance gene cfr, the lincosamide-pleuromutilin-streptogramin A resistance gene vga(C), and the novel macrolide-lincosamide-streptogramin B resistance gene erm(54). Many AMR genes were part of small transposons or plasmids. Clonal and geographical correlations of molecular characteristics and resistance and virulence genes were more frequently observed than temporal relations. In conclusion, this study provides insight into population dynamics of the main epidemic porcine LA-MRSA lineage in Germany over a 13-year-period. The observed comprehensive AMR and virulence properties, most likely resulting from the exchange of genetic material between bacteria, highlighted the importance of LA-MRSA surveillance to prevent further dissemination among swine husbandry facilities and entry into the human community. IMPORTANCE The LA-MRSA-CC398 lineage is known for its low host specificity and frequent multiresistance to antimicrobial agents. Colonized swine and their related surroundings represent a considerable risk of LA-MRSA-CC398 colonization or infection for occupationally exposed people through which such isolates might be further disseminated within the human community. This study provides insight into the diversity of the porcine LA-MRSA-CC398 lineage in Germany. Clonal and geographical correlations of molecular characteristics and resistance and virulence traits were detected and may be associated with the spread of specific isolates through livestock trade, human occupational exposure, or dust emission. The demonstrated genetic variability underlines the lineage's ability to horizontally acquire foreign genetic material. Thus, LA-MRSA-CC398 isolates have the potential to become even more dangerous for various host species, including humans, due to increased virulence and/or limited therapeutic options for infection control. Full-scale LA-MRSA monitoring at the farm, community, and hospital level is therefore essential.

The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

Genome Size Evolution: Small Transposons with Large Consequences

Transposable elements (TEs) heavily influence genome size variation between organisms. A new study on larvacean tunicates now shows that even non-autonomous TEs - small TEs that parasitize the enzymatic machinery of large, autonomous TEs - can have a large impact on genome size.

A transposable element within the Non-canonical telomerase RNA of Arabidopsis thaliana modulates telomerase in response to DNA damage [corrected

Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3’ terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2Δ) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault.

Harnessing mobile genetic elements to explore gene regulation

Sequences that regulate expression of a gene in cis but are located at large distances along the DNA from the gene, as found with most developmentally regulated genes in higher vertebrates, are difficult to identify if those sequences are not conserved across species. Mutating suspected gene-regulatory sequences to alter expression then becomes a hit-or-miss affair. The relaxed specificity of transposon insertions offers an opportunity to develop alternate strategies, to scan in an unbiased manner, pieces of chromosomal DNA cloned in BACs for transcription enhancing elements. This article illustrates how insertions of Tn10 with enhancer-traps into BAC DNA containing the gene, and its germ-line expression in zebrafish, have identified distal regulatory elements functionally. Transposition of Tn10 first introduces the enhancer-trap with a loxP site randomly into BAC DNA. Cre-recombination between the inserted loxP and the loxP endogenous to a BAC-end positions the enhancer-trap to the newly created truncated end of BAC DNA. The procedure generates a library of integration-ready enhancer-trap BACs with progressive truncations from an end in a single experiment. Individual enhancer-trap BACs from the library can be evaluated functionally in zebrafish or mice. Furthermore, the ability to readily alter sequences in a small transposon plasmid containing a regulatory domain of the gene allows re-introduction of altered parts of a BAC back into itself. It serves as a useful strategy to functionally dissect multiple discontinuous regulatory domains of a gene quickly. These methodologies have been successfully used in identifying novel regulatory domains of the Amyloid Precursor Protein (appb) gene in zebrafish, and provided important clues for regulation of the gene in humans.

Factors acting on Mos1 transposition efficiency

BackgroundMariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo.ResultsThe transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency.ConclusionThe study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.

Transcripts of the transposon mariner are present in epileptic brain.

Mobile genetic elements termed transposons have been increasingly implicated in human disease. The small transposon mariner is widespread within non-vertebrate genomes and causes mutation by replication, excision, and insertion of itself without an RNA intermediate. We find that human DNA contains about 60 copies of this gene. Mariner transcripts are abundant in RNA prepared from sclerotic epileptic hippocampi. In contrast, typically no mariner-specific RNA is detected in non-sclerotic hippocampi from other epileptic patients or from autopsies. A complete but non-functional copy was obtained using rapid amplification of cDNA ends (RACE). This human mariner transcript is approximately 45% homologous to a functional counterpart active in Drosophila, with a coding region of 1035 bases flanked by 32 base inverted terminal repeats. The differential expression of mariner transcripts within sclerotic hippocampi suggests the probable activity of an autonomous element which by mutating critical genes could establish an epileptogenic substrate in the hippocampus.

Insertional polymorphism in introns 4 and 10 of the maize beta-glucosidase gene glu1.

The major beta-glucosidase isozyme Glu1 is encoded by a highly polymorphic gene (glu1) in maize. The glu1 gene comprises 12 exons and 11 introns. Two of these introns, introns 4 and 10, show insertional polymorphism: those in allele glu1-1 (represented by inbred line OH7B) are longer than those in other inbred genotypes and in two teosintes (Zea mexicana and Zea parviglumis) surveyed. Sequence data revealed that an increase in the length of intron 4 from 150 to 477 bp in OH7B is due to a short (11 bp) tandem duplication and a large insertion sequence of 313 bp plus a 4-bp (5' ATAG 3') direct repeat. The 313-bp insertion sequence (referred to as mzsTn-1) has all the features of a transposon, having a 25-bp well-conserved (3/25 mismatches) inverted repeat sequence at its termini flanked by a 4-bp direct repeat. The increase in length from 1041 to 1302 bp in intron 10 of OH7B is due to a 259-bp insertion sequence (referred to as mzsTn-2) plus a 2-bp (5' TA 3') direct repeat. The mzsTn-2 element also possesses all the hallmarks of a transposon: a 34-bp well-conserved (3/34 mismatches) inverted repeat sequence at its termini flanked by a 2-bp direct repeat. The mzsTn-1 element belongs to a new family of inverted repeat elements, while mzsTn-2 belongs to the Stowaway family of inverted repeat elements. Analysis of PCR products from amplifications off genomic-DNA templates, using primers derived from the inverted repeat termini, and Southern blotting data suggest that both small transposons are members of a multigene family. The occurrence of two different small transposons in introns of the same glu1 allele in inbred OH7B and their absence in other genotypes suggest that they have moved into this glu1 allele recently through mediation of their autonomous counterparts that are active in OH7B or in its ancestry.

Genetic evidence for absence of transposition functions from the internal part of Tn981 a relative of Tn9.

Inverse transposition of the DNA of pBR322 was found to be mediated by the small transposon Tn981 a relative of Tn9 flanked by direct repeats of IS1. Since the resulting structure IS1::pBR322::IS1 (Tn983) is transposed in a second step in the absence of Tn981, it is concluded that all the functions necessary for transposition of IS1 flanked transposons are coded for by IS1 itself or the E. coli chromosome, respectively.