Target Site Duplications Research Articles

The Birth and Demise of the ISApl1-mcr-1-ISApl1 Composite Transposon: the Vehicle for Transferable Colistin Resistance.

ABSTRACTThe origin and mobilization of the ~2,609-bp DNA segment containing the mobile colistin resistance gene mcr-1 continue to be sources of uncertainty, but recent evidence suggests that the gene originated in Moraxella species. Moreover mcr-1 can be mobilized as an ISApl1-flanked composite transposon (Tn6330), but many sequences have been identified without ISApl1 or with just a single copy (single ended). To further clarify the origins and mobilization of mcr-1, we employed the Geneious R8 software suite to comprehensively analyze the genetic environment of every complete mcr-1 structure deposited in GenBank as of this writing (September 2017) both with and without associated ISApl1 (n = 273). This revealed that the 2,609-bp mcr-1 structure was likely mobilized from a close relative of a novel species of Moraxella containing a chromosomal region sharing >96% nucleotide identity with the canonical sequence. This chromosomal region is bounded by AT and CG dinucleotides, which have been described on the inside ends (IE) of all intact Tn6330 described to date and represent the ancestral 2-bp target site duplications (TSDs) generated by ISApl1 transposition. We further demonstrate that all mcr-1 structures with just one ISApl1 copy or with no ISApl1 copies were formed by deletion of ISApl1 from the ancestral Tn6330, likely by a process related to the “copy-out–paste-in” transposition mechanism. Finally, we show that only the rare examples of single-ended structures that have retained a portion of the excised downstream ISApl1 including the entire inverted right repeat might be capable of mobilization.

Characterization of pEncl-30969cz, a novel ColE1-like plasmid encoding VIM-1 carbapenemase, from an Enterobacter cloacae sequence type 92 isolate

A VIM-1-producing ST92 Enterobacter cloacae was isolated in a Czech hospital. blaVIM-1 was part of the class 1 integron In110 carried by a Tn1721-like transposon. Tn1721-like was located on a ColE1-like plasmid, pEncl-30969cz (33,003 bp). Target site duplications at the boundaries of Tn1721-like suggested its transposition into the pEncl-30969cz backbone.

Helitron’s Periodicities Identification in C.Elegans based on the Smoothed Spectral Analysis and the Frequency Chaos Game Signal Coding

Helitrons are typical rolling circle transposons which make up the bulk of eukaryotic genomes. Unlike of other DNA transposons, these transposable elements (TEs), don’t create target site duplications or end in inverted repeats, which make them particular challenge to identify and more difficult to annotate. To date, these elements are not well studied; they only attracted the interest of researchers in biology. The focus of this paper is oriented towards identifying the helitrons in C.elegans genome in the perspective of signal processing. Aiming at the helitron's identification, a novel methodology including two steps is proposed: the coding and the spectral analysis. The first step consists in converting DNA into a 1-D signal based on the statistical features of the sequence (FCGS coding). As for the second step, it aims to identify the global periodicities in helitrons using the Smoothed Fourier Transform. The resulting spectrum and spectrogram are shown to present a specific signature of each helitron’s type.

McClintock: An Integrated Pipeline for Detecting Transposable Element Insertions in Whole-Genome Shotgun Sequencing Data.

Transposable element (TE) insertions are among the most challenging types of variants to detect in genomic data because of their repetitive nature and complex mechanisms of replication . Nevertheless, the recent availability of large resequencing data sets has spurred the development of many new methods to detect TE insertions in whole-genome shotgun sequences. Here we report an integrated bioinformatics pipeline for the detection of TE insertions in whole-genome shotgun data, called McClintock (https://github.com/bergmanlab/mcclintock), which automatically runs and standardizes output for multiple TE detection methods. We demonstrate the utility of McClintock by evaluating six TE detection methods using simulated and real genome data from the model microbial eukaryote, Saccharomyces cerevisiae. We find substantial variation among McClintock component methods in their ability to detect nonreference TEs in the yeast genome, but show that nonreference TEs at nearly all biologically realistic locations can be detected in simulated data by combining multiple methods that use split-read and read-pair evidence. In general, our results reveal that split-read methods detect fewer nonreference TE insertions than read-pair methods, but generally have much higher positional accuracy. Analysis of a large sample of real yeast genomes reveals that most McClintock component methods can recover known aspects of TE biology in yeast such as the transpositional activity status of families, target preferences, and target site duplication structure, albeit with varying levels of accuracy. Our work provides a general framework for integrating and analyzing results from multiple TE detection methods, as well as useful guidance for researchers studying TEs in yeast resequencing data.

Insertionally polymorphic sites of human endogenous retrovirus-K (HML-2) with long target site duplications

BackgroundHuman endogenous retroviruses (HERVs) belong to the LTR-retrotransposon family, where the complete HERV sequence contains two long terminal repeats (LTRs) located at each end. Intact LTRs possess highly conserved transcriptional promoter and enhancer sequences, so analyses of HERV insertional polymorphisms are expected to provide greater insights into human genomic variation compared with the conventional analysis of single nucleotide variations. High-throughput sequencing technology is developing but genome-wide investigations of HERVs are methodically challenging, and thus a comprehensive understanding of HERV insertional polymorphisms and target site duplications (TSDs) remains elusive.ResultsWe identified five human-specific insertionally polymorphic sites in HERVK (HML-2), one of the HERV subgroups, by extracting HML-2-deleted sequences from the genomic structural variation database, which we successfully characterized and then updated the existing catalogue of HML-2 insertional polymorphisms. The insertionally polymorphic states were confirmed in a small Japanese population by genomic PCR analysis for four of the five sites identified. Sequencing of the preintegration sites clearly showed that the HML-2 site located at 7p21.2 had 250-base pair (bp) TSDs, which is one of the longest TSDs in HML-2. In addition to these five sites, another insertionally polymorphic site for a non-human-specific HML-2 site was also identified at 6p25.2, which was flanked by 111-bp TSDs and the corresponding ERV locus was also annotated in the genome of non-human primates.ConclusionsOur analysis demonstrated the existence of HERV insertions flanked by unconventionally long TSDs, including those with lengths as high as 250 bp. This suggests that the length range of retroviral TSDs is larger than considered previously, which might help to understand how retroviral integration occurs in the host genome.

Insertion and deletion polymorphisms of the ancient AluS family in the human genome

BackgroundPolymorphic Alu elements account for 17% of structural variants in the human genome. The majority of these belong to the youngest AluY subfamilies, and most structural variant discovery efforts have focused on identifying Alu polymorphisms from these currently retrotranspositionally active subfamilies. In this report we analyze polymorphisms from the evolutionarily older AluS subfamily, whose peak activity was tens of millions of years ago. We annotate the AluS polymorphisms, assess their likely mechanism of origin, and evaluate their contribution to structural variation in the human genome.ResultsOf 52 previously reported polymorphic AluS elements ascertained for this study, 48 were confirmed to belong to the AluS subfamily using high stringency subfamily classification criteria. Of these, the majority (77%, 37/48) appear to be deletion polymorphisms. Two polymorphic AluS elements (4%) have features of non-classical Alu insertions and one polymorphic AluS element (2%) likely inserted by a mechanism involving internal priming. Seven AluS polymorphisms (15%) appear to have arisen by the classical target-primed reverse transcription (TPRT) retrotransposition mechanism. These seven TPRT products are 3′ intact with 3′ poly-A tails, and are flanked by target site duplications; L1 ORF2p endonuclease cleavage sites were also observed, providing additional evidence that these are L1 ORF2p endonuclease-mediated TPRT insertions. Further sequence analysis showed strong conservation of both the RNA polymerase III promoter and SRP9/14 binding sites, important for mediating transcription and interaction with retrotransposition machinery, respectively. This conservation of functional features implies that some of these are fairly recent insertions since they have not diverged significantly from their respective retrotranspositionally competent source elements.ConclusionsOf the polymorphic AluS elements evaluated in this report, 15% (7/48) have features consistent with TPRT-mediated insertion, thus suggesting that some AluS elements have been more active recently than previously thought, or that fixation of AluS insertion alleles remains incomplete. These data expand the potential significance of polymorphic AluS elements in contributing to structural variation in the human genome. Future discovery efforts focusing on polymorphic AluS elements are likely to identify more such polymorphisms, and approaches tailored to identify deletion alleles may be warranted.

RelocaTE2: a high resolution transposable element insertion site mapping tool for population resequencing.

BackgroundTransposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE polymorphisms, particularly transposition events or non-reference insertion sites can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools.MethodsWe have developed the tool RelocaTE2 for identification of TE insertion sites at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision.Results and DiscussionThe performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrate high level of sensitivity and specificity, particularly when the sequence coverage is not shallow. In comparison to other tools tested, RelocaTE2 achieves the best balance between sensitivity and specificity. In particular, RelocaTE2 performs best in prediction of TSDs for TE insertions. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE insertion sites and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing.

Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

BackgroundMutator-like transposable elements (MULEs) are widespread with members in fungi, plants, and animals. Most of the research on the MULE superfamily has focused on plant MULEs where they were discovered and where some are extremely active and have significant impact on genome structure. The maize MuDR element has been widely used as a tool for both forward and reverse genetic studies because of its high transposition rate and preference for targeting genic regions. However, despite being widespread, only a few active MULEs have been identified, and only one, the rice Os3378, has demonstrated activity in a non-host organism.ResultsHere we report the identification of potentially active MULEs in the mosquito Aedes aegypti. We demonstrate that one of these, Muta1, is capable of excision and reinsertion in a yeast transposition assay. Element reinsertion generated either 8 bp or 9 bp target site duplications (TSDs) with no apparent sequence preference. Mutagenesis analysis of donor site TSDs in the yeast assay indicates that their presence is important for precise excision and enhanced transposition. Site directed mutagenesis of the putative DDE catalytic motif and other conserved residues in the transposase protein abolished transposition activity.ConclusionsCollectively, our data indicates that the Muta1 transposase of Ae. aegypti can efficiently catalyze both excision and reinsertion reactions in yeast. Mutagenesis analysis reveals that several conserved amino acids, including the DDE triad, play important roles in transposase function. In addition, donor site TSD also impacts the transposition of Muta1.

Templated Sequence Insertion Polymorphisms in the Human Genome

Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including (1) target-site duplication (TSD), (2) polyadenylation 10–30 nucleotides downstream of a “cryptic” polyadenylation signal, and (3) preference for insertion at a 5′-TTTT/A-3′ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break (DSB) via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25–30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

Analysis of pCERC7, a small antibiotic resistance plasmid from a commensal ST131 Escherichia coli, defines a diverse group of plasmids that include various segments adjacent to a multimer resolution site and encode the same NikA relaxase accessory protein enabling mobilisation

The ampicillin resistance plasmid pCERC7, carrying transposon Tn2 with an IS4 insertion, was detected in the draft genome of a commensal Escherichia coli isolate. The genome data also revealed that this isolate belongs to ST131, clade B. pCERC7 is 9712bp comprised of a 3319bp backbone, Tn2::IS4 (6388bp) and 5bp of target site duplication, and was present at a copy number of 40. pCERC7 is related to several plasmids composed of only the backbone, or the backbone with the Tn2 insertion in the same position. These plasmids have been found previously in Escherichia coli or Salmonella enterica recovered in several different countries from as early as the 1970s. This group was named the NTP16 group after the best studied example. pCERC7 was annotated using available information about plasmids in this group and additional analyses. The backbone includes genes for RNA I and RNA II to initiate replication and the Tn2 interrupts a gene found here to encode a protein 66% identical to the Rom regulatory protein of ColE1. NTP16 family plasmids include a gene, previously designated mobA, that was found to encode a homologue (53% identical) of the NikA relaxase accessory protein of the conjugative IncI1 plasmid R64, which is known to bind to the R64 oriT. However, a nikB relaxase gene is not present, indicating that a relaxase must be supplied in trans for mobilisation by R64 to occur, as demonstrated previously for NTP16. Hence, MobA of NTP16 and relatives was renamed NikA. Upstream of nikA, we found a region closely related to the oriT of R64. pCERC7 and all members of the NTP16 family also include a multimer resolution site, nmr, similar to the cer site of ColE1. The backbone of the NTP16 family also includes genes for a demonstrated toxin-antitoxin system, LsoAB. Several more distantly related groups of plasmids that include a very closely related nmr-nikA-oriT segment (99.4–93.7% DNA identity) were identified in the GenBank non-redundant DNA database. All use an RNA I/RNA II-Rom system for replication initiation, but each contains a unique fragment adjacent to the nmr site. The segment of the NTP16/pCERC7 group that encodes the LsoAB toxin-antitoxin system is replaced by a different segment in other family groups. The point at which the sequences diverge is between the XerC and XerD sites of the dif site at one end of nmr, suggesting that the evolution of this broad group of plasmids involves XerC/XerD recombination.

Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293.

Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4–14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140–493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3’-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50–65% and 60–75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259–343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.

Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems.

Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei. Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3′ end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1–Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.

The characteristics and functions of a miniature inverted-repeat transposable element TaMITE81 in the 5' UTR of TaCHS7BL from Triticum aestivum.

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in eukaryotic and prokaryotic genomes. In this article, a MITE, TaMITE81, was isolated from the 5' untranslated region (UTR) of TaCHS7BL, chalcone synthase (CHS) catalyzing the first committed step of anthocyanin biosynthesis, in the wheat cultivar 'Opata' with white grain. TaMITE81 was only 81 nucleotides, including a terminal inverted repeat with 39 nucleotides and was flanked by two nucleotides, "TA", target site duplications that were typical features of stowaway-like MITEs. Compared with the wheat cultivar 'Gy115' with purple grain, which is without the insertion, the expression of TaCHS7BL was lower in several organs of 'Opata'. The insertion of TaMITE81 into the 5' UTR of the GUS gene also reduced the transient expression of GUS on the coleoptiles of 'Opata', which means the insertion of TaMITE81 was the reason for the low expression of TaCHS7BL in 'Opata'. But the genotype of TaCHS7BL was not linked to phenotype of grain color in the RILs derived from a cross 'Gy115' and 'Opata'. The TaMITE81 density of the hexaploid variety of T. aestivum was more than 10 times that of diploid relatives, which implies that polyploidization caused the amplification of TaMITE81 homologous sequences. Further research should be conducted on decoding the relationship between TaCHS7BL and other traits relative to anthocyanin biosynthesis in wheat, and discovering the mechanism of TaMITE81 transposon action.

Dynamics of a Novel Highly Repetitive CACTA Family in Common Bean (Phaseolus vulgaris).

Transposons are ubiquitous genomic components that play pivotal roles in plant gene and genome evolution. We analyzed two genome sequences of common bean (Phaseolus vulgaris) and identified a new CACTA transposon family named pvCACTA1. The family is extremely abundant, as more than 12,000 pvCACTA1 elements were found. To our knowledge, this is the most abundant CACTA family reported thus far. The computational and fluorescence in situ hybridization (FISH) analyses indicated that the pvCACTA1 elements were concentrated in terminal regions of chromosomes and frequently generated AT-rich 3 bp target site duplications (TSD, WWW, W is A or T). Comparative analysis of the common bean genomes from two domesticated genetic pools revealed that new insertions or excisions of pvCACTA1 elements occurred after the divergence of the two common beans, and some of the polymorphic elements likely resulted in variation in gene sequences. pvCACTA1 elements were detected in related species but not outside the Phaseolus genus. We calculated the molecular evolutionary rate of pvCACTA1 transposons using orthologous elements that indicated that most transposition events likely occurred before the divergence of the two gene pools. These results reveal unique features and evolution of this new transposon family in the common bean genome.

The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.

Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination

Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen froma transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair ofterminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germlineand that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar tothose used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon.

The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

BackgroundWith the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach.MethodsTargeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle.ResultsHH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%).ConclusionWith the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations.

LINE-1-like retrotransposons contribute to RNA-based gene duplication in dicots.

RNA-based duplicated genes or functional retrocopies (retrogenes) are known to drive phenotypic evolution. Retrogenes emerge via retroposition, which is mainly mediated by long interspersed nuclear element 1 (LINE-1 or L1) retrotransposons in mammals. By contrast, long terminal repeat (LTR) retrotransposons appear to be the major player in plants, although an L1-like mechanism has also been hypothesized to be involved in retroposition. We tested this hypothesis by searching for young retrocopies, as these still retain the sequence features associated with the underlying retroposition mechanism. Specifically, we identified polymorphic retrocopies (retroCNVs) by analyzing public Arabidopsis (Arabidopsis thaliana) resequencing data. Furthermore, we searched for recently originated retrocopies encoded by the reference genome of Arabidopsis and Manihot esculenta. Across these two datasets, we found cases with L1-like hallmarks, namely, the expected target site sequence, a polyA tail and target site duplications. Such data suggest that an L1-like mechanism could operate in plants, especially dicots.

ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs.

ABSTRACTBacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3′ termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication. We also identify autonomous ISC transposons that encode TnpA-like Y1 transposases. Thus, the nonautonomous ISC transposons could be mobilized in trans either by Y1 transposases of other, autonomous ISC transposons or by Y1 transposases of the more abundant IS605 transposons. These findings imply an evolutionary scenario in which the ISC transposons evolved from IS605 family transposons, possibly via insertion of a mobile group II intron encoding the HNH domain, and Cas9 subsequently evolved via immobilization of an ISC transposon.IMPORTANCE Cas9 endonucleases, the effectors of type II CRISPR-Cas systems, represent the new generation of genome-engineering tools. Here, we describe in detail a novel family of transposable elements that encode the likely ancestors of Cas9 and outline the evolutionary scenario connecting different varieties of these transposons and Cas9.

Evolution of AbGRI2-0, the Progenitor of the AbGRI2 Resistance Island in Global Clone 2 of Acinetobacter baumannii.

A320, isolated in the Netherlands in 1982 and also known as RUH134, is the earliest available multiply antibiotic-resistant (MAR) Acinetobacter baumannii isolate belonging to global clone 2 (GC2) and is the reference strain for this clone. The draft genome sequence of A320 was used to investigate the original location and configuration of the IS26-bounded AbGRI2 resistance island found in current GC2 isolates. PCR mapping and sequencing were used to order contigs composing the resistance islands. A320 contains two IS26-bounded resistance islands, AbGRI2-0a and AbGRI2-0b, of 7.8 kb and 25.4 kb, respectively. Together they contain blaTEM, aacC1, aadA1, sul1, catA1, and aphA1b genes, which confer resistance to antibiotics used clinically in the 1970s, as well as an incomplete mercury resistance module. Tracking the continuity of the chromosome and the target site duplications revealed that the two resistance islands were originally together as AbGRI2-0, an island of 32.4 kb, and were subsequently separated via an IS26-mediated intramolecular inversion that reversed the orientation of 1.54 Mb of the chromosome and duplicated an IS26. A320 contains an ancestral form of AbGRI2, and the original insertion site of the AbGRI2 island was identified. Many of the AbGRI2 versions present in the completed GC2 genomes can be derived from it via the variant AbGRI2-1. IS26-mediated inversions have also played a part in forming AbGRI2-0, and, upon reversal, large regions of AbGRI2-0 are identical to parts of AbaR0, the ancestral version of the AbaR islands present in GC1 isolates. This indicates a common source.