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BACKGROUND Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. RESULTS Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. CONCLUSION Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.

Transposable elements (TEs) are repetitive regions of DNA that are able to self-replicate and reinsert themselves throughout host genomes. Since the discovery of TEs, a prevalent question has been whether increasing TE copy number has an effect on the fitness of their hosts. P-elements (PEs) in Drosophila are a well-studied TE that has strong phenotypic effects. When a female without PEs (M) is crossed to a male with them (P), the resulting females are often sterile, a phenomenon called hybrid dysgenesis (HD). Here, we used short- and long-read sequencing to infer the number of PEs in the genomes of dozens of isofemale lines from two Drosophila species and measured whether the magnitude of HD was correlated with the number of PEs in the paternal genome. Consistent with previous reports, we find evidence for a positive correlation between the paternal PE copy number and the magnitude of HD in progeny from ♀M × ♂ P crosses for both species. Other crosses are not affected by the number of PE copies. We also find that the correlation between the strength of HD and PE copy number differs between species, which suggests that there are genetic differences that might make some genomes more resilient to the potentially deleterious effects of TEs. Our results suggest that PE copy number interacts with other factors in the genome and the environment to cause HD and that the importance of these interactions is species specific.

The invasion of transposable elements (TEs) in mammals and invertebrates, is likely stopped by piRNAs that emerge after insertion of the TE into a piRNA cluster (the trap model). It remains, however, still unclear which factors influence the dynamics of TE invasions. The activity of the TE (i.e. transposition rate) is one of the frequently discussed key factors. Here we take advantage of the temperature-dependent activity of the P-element, a widely studied eukaryotic TE, to test how TE activity affects the dynamics of a TE invasion. Taking advantage of the temperature-specific activity of the P-element, we monitored its invasion dynamics in experimentalDrosophila simulanspopulations at hot and cold culture conditions. Despite marked differences in transposition rates, the P-element reached very similar copy numbers at both temperatures. The reduction of the insertion rate upon approaching the copy number plateau was accompanied by the emergence of similar amounts of piRNAs against the P-element at both temperatures. Interestingly, the ping-pong cycle, which degrades TE transcripts, becomes only active after the copy number has reached the plateau. We show that the P-element abundance is an order of magnitude lower and fewer P-element insertions in piRNA clusters were observed than expected under the trap model. We conclude that the transposition rate has at the most only a minor influence on TE abundance, but other factors, such as paramutations or selection against TE insertions are shaping the TE composition of organism.

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

Peer reviewed: True, P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

BackgroundActive DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerisation as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterised homologue of DmTNP. ResultsHere we report (using co-IP, pull down, co-localization, PLA) that both the full length as well as truncated hTHAP9 and DmTNP (corresponding to amino-terminal DNA binding and Leucine-rich coiled coil domains) undergo homo-oligomerisation, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerisation is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerisation. We also report that Hcf-1, THAP1, THAP10 and THAP11 are possible protein interaction partners of hTHAP9. ConclusionsThus, we highlight the importance of DNA as well as the amino-terminal regions of both hTHAP9 and DmTNP, for their ability to form higher order oligomeric states. Elucidating the functional relevance of the different putative oligomeric state/s of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

Transposable elements not only can change genomic positions and disperse across the gene pool, but also can jump to another species through horizontal transmission. Of late, the P element, a DNA transposon in insects, was shown to cross the genetic boundary from Drosophila melanogaster to D. simulans in Europe around 2006. To understand the dynamics of transposable elements, especially in the early stages of invasion, we examined 63 lines of D. simulans from 11 natural populations in Japan established in 1976-2015. Based on PCR analyses, P elements were demonstrated to exist in Japan in 2008 and later. One copy of the full-length P element was identified and mapped to a site on chromosome 3 L in a genome. All of 18 copies of P elements examined shared "A" at the nucleotide position 2040, which is representative of the direct descendants of the original P element that invaded in D. simulans. We also found that some lines having P elements can induce intensive gonadal dysgenesis in D. simulans at 29°C. Our present results imply that P elements in D. simulans arrived at the east end of Asia just a few years later than or almost simultaneously to the initial invasion in Europe, Africa, and North America, suggesting a more astonishingly rapid spread than previously assumed.

Let V be a vector space over a field of characteristic p. In this paper we complete the classification of all irreducible subgroups G of GL ⁢ ( V ) {\mathrm{GL}(V)} that contain a p-element whose Jordan normal form has exactly one non-trivial block, and possibly multiple trivial blocks. Broadly speaking, such a group acting primitively is a classical group acting on a symmetric power of a natural module, a 7-dimensional orthogonal group acting on the 8-dimensional spin module, a complex reflection group acting on a reflection representation, or one of a small number of other examples, predominantly with a self-centralizing cyclic Sylow p-subgroup.

Objective P-element Induced WImpy protein-like RNA-mediated gene silencing 2 (PIWIL2) is a reported oncogene strongly associated with tumorigenesis and cancer progression. However, the potential function of PIWIL2 in oral cancer is still largely unclear. Methods In this study, we investigated the clinical significance of PIWIL2 expression in human oral squamous cell carcinoma (OSCC) cell lines and tissues. We also examined its function in OSCC pathogenesis by knocking down PIWIL2 expression with short hairpin RNAs, followed by phenotypic experiments focused on cell migration, invasion, proliferation, and apoptosis rates. Results We found that PIWIL2 was overexpressed in OSCC cell lines and tissues and significantly correlated with the malignancy stage. Furthermore, knockdown of PIWIL2 in a human OSCC cell line Tca8113 induced cell cycle arrest and apoptosis. Silencing PIWIL2 expression also significantly suppressed the migration and invasion abilities of Tca8113 cells. Conclusions Collectively, our results suggest a functional role of PIWIL2 in regulating OSCC pathogenesis. Our data imply that PIWIL2 could serve as a potential therapeutic target for OSCC treatment.

Using the FISH method and PCR analysis, the presence of canonical P element was studied in the genomes of a number of laboratory lines isolated from nature in different years from the Zaprionus genus (Z. indianus) and Drosophila genus, Sophophora subgenus (D. ananassae, D. eugracilis, D. simulans, D. immigrans), Drosophila subgenus (D. virilis, D. mercatorum, D. hydei, D. funebris, D. pseudoobscura), Lordiphosa subgenus (L. magnipectinata), and Dorsilopha subgenus (D. busckii) in a search for new cases of horizontal transfer. According to our data, the L. magnipectinata genome contains sequences homologous to terminal regions of canonical P element, as well as the sequence with a weak homology from the central part of canonical P element. The P-element hybridization sites adjacent to the chromocenter were found in the D. pseudoobscura genome; this can indicate an ancient origin of the sequences homologous to the P element. The P element is absent in old D. simulans lines, except for the line isolated from nature in 2014 (in which the P element was found); this confirms data of other researchers about recent cases of horizontal P-element transfer in this species. No new cases of horizontal transfer were detected in the analyzed lines.

Transposable elements (TEs) are virtually ubiquitous components of genomes, yet they often impose significant fitness consequences on their hosts. In addition to producing specific deleterious mutations by insertional inactivation, TEs also impose general fitness costs by inducing DNA damage and participating in ectopic recombination. These latter fitness costs are often assumed to be dosage-dependent, with stronger effects occurring in the presence of higher TE copy numbers. We test this assumption in Drosophila melanogaster by considering the relationship between the copy number of two active DNA transposons, P-element and hobo element, and the incidence of hybrid dysgenesis, a sterility syndrome associated with transposon activity in the germline. By harnessing a subset of the Drosophila Genetic Reference Panel (DGRP), a group of fully-sequenced D. melanogaster strains, we describe quantitative and structural variation in P-elements and hobo elements among wild-derived genomes and associate these factors with hybrid dysgenesis. We find that the incidence of hybrid dysgenesis is associated with both P-element and hobo element copy number in a dosage-dependent manner. However, the relationship is weak for both TEs, suggesting that dosage alone explains only a small part of TE-associated fitness costs.

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), a novel class of noncoding RNAs, are involved in the carcinogenesis. However, the functional significance of piRNAs in oral squamous cell carcinoma (OSCC) remains unknown. In the present study, we used chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) induced OSCC mouse model. piRNAs and mRNAs were profiled using next-generation sequencing in the tongue tumor tissues from 4NQO induction and healthy tongue tissues from control mice. Furthermore, we analyzed the differential gene expression of human OSCC in Gene Expression Omnibus (GEO) database. According to the common differentially expressed genes in the 4NQO model and human OSCC tissues, piRNAs and mRNAs network were established based on informatics method. A total of 14 known piRNAs and 435 novel predicted piRNAs were differently expressed in tumor tissue compared to healthy tissue. Among differently expressed piRNAs 260 were downregulated, and 189 were upregulated. The mRNA targets for the differentially expressed piRNAs were identified using RNAhybrid software. Primary immunodeficiency and herpes simplex infection were the most enriched pathways. A total of 22 mRNAs overlapped in human and mice OSCC. Moreover, we established the regulatory network of 11 mRNAs, including Tmc5, Galnt6, Spedf, Mybl2, Muc5b, Six31, Pigr, Lamc2, Mmp13, Mal, and Mamdc2, and 11 novel piRNAs. Our data showed the interaction between piRNAs and mRNAs in OSCC, which might provide new insights in the development of diagnostic biomarkers and therapeutic targets of OSCC.

Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation. Time-lapsed imaging of GFP-laced rodlets in human cells revealed that they formed quickly in a dynamic process involving fusion and fission. We delayed the availability of the transposon DNA and found that transposition declined after transposase concentrations became high enough for visible transposase rodlets to appear. In combination with earlier findings for maize Ac elements, these results give insight into transposase overproduction inhibition by demonstrating that the appearance of transposase protein structures and the end of active transposition are simultaneous, an effect with implications for genetic engineering and horizontal gene transfer.

In Drosophila , P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein–DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed “Inverted Repeat Binding Protein (IRBP) 18” and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

In Drosophila melanogaster, the mini-w+transgene in Pci is normally expressed throughout the adult eye; however, when other P or KP elements are present, a variegated-eye phenotype results, indicating random w+silencing during development called P-element-dependent silencing (PDS). Mutant Su(var)205 and Su(var)3-7 alleles act as haplo-suppressors/triplo-enhancers of this variegated phenotype, indicating that these heterochromatic modifiers act dose dependently in PDS. Previously, we recovered a spontaneous mutation of P{lacW}ciDplaccalled P{lacW}ciDplacE1(E1) that variegated in the absence of P elements, presumably due to the insertion of an adjacent gypsy element. From a screen for genetic modifiers of E1 variegation, we describe here the isolation of five mutations in ash1 and three in trx that enhance the E1 variegated phenotype in a dose-dependent and cumulative manner. These mutant alleles enhance PDS at E1, and in E1/P{lacW}ciDplac, but suppress position effect variegation (PEV) at In(1)wm4. This opposite action is consistent with a model where ASH1 and TRX mark transcriptionally active chromatin domains. If ASH1 or TRX function is lost or reduced, heterochromatin can spread into these domains creating a sink that diverts heterochromatic proteins from other variegating locations, which then may express a suppressed phenotype.

P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP. These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs.

When Drosophila mothers lacking P-element-piRNAs (i.e. ISO1) mate with certain fathers harboring active P-elements (i.e. Harwich, Har), hybrid daughters suffer from gonadal dysgenesis (GD), where ovaries collapse from uncontrolled P-element transposition. To discover the P-element driving GD, we selected hybrid D. melanogaster lines with portions of Har DNA within the ISO1 genome. We generated HISR-D and HISR-N lines retaining or losing GD induction, respectively. Despite greatly-reduced numbers of P-elements, HISR-D lines produced as many P-element piRNAs as parental Har. In these lines, we discovered a highly-truncated P-element variant: “Har-P”, which mobilized de novo in all HISR lines. Crossing P-transposase with Har-Ps in HISR-N lines restored GD and revealed a paternal P-element-piRNA-directed imprint on Har-Ps to suppress lethal somatic transposition. Har-P may weaponize P-transposase during catastrophic transposition.

Background P-element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. Maternally deposited piRNAs suppress P-element transposition in the progeny, linking them to P-M phenotypes; however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. Results To elucidate the molecular basis of P-element suppression by zygotic factors, we investigated the genomic constitution and P-element piRNA production derived from fathers. As a result, we characterized males of naturally derived Q, M’ and P strains, which show different capacities for the P-element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M’-strain fathers restrict the P-element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M’ strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P-element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. Conclusions The piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny. Expression levels of the P-element piRNA and KP-element mRNA vary among F1 progeny due to the constitution of the paternal genome, and are involved in phenotypic variation in the subsequent generation. Electronic supplementary material The online version of this article (10.1186/s13100-018-0110-y) contains supplementary material, which is available to authorized users.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.

Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.

Four kinds of robust elements have been recognised inAmorphognathus quinquiradiatusMoskalenko, 1977 (inKanyginet al. 1977) from the early Late Ordovician of Siberia, indicating that at least 17 elements were present in the apparatus, one of them similar to the P1element of the Early SilurianDistomodus. The new generic nameMoskalenkodusis proposed for these conodonts with a pterospathodontid-like S series element morphology. This implies that the relatedDistomodus,PterospathodusandGamachignathuslineages had a long cryptic evolutionary history, probably ranging back to the early Ordovician, when they split from the lineage ofIcriodella, having a duplicated M location in common. The balognathidPromissum, with a 19-element apparatus, may have shared ancestry withIcriodellain Ordovician high latitudes, withSagittodontina,Lenodus,TrapezognathusandPhragmodusas possible connecting links. The pattern of the unbalanced contribution ofBaltonioduselement types to samples suggests that duplication of M and P2series elements may have been an early event in the evolution of balognathids. The proposed scenario implies a profound transformation of the mouth region in the evolution of conodonts. The probable original state was a chaetognath-like arrangement of coniform elements; all paired and of relatively uniform morphology. This was modified at the origin of protopanderodontids by the introduction of a medial S0element, which resulted in the separation of the exposed unit of M and S series elements from the P series elements hidden in the throat. A rotation of the S series elements to an almost horizontal position in early prioniodontids may have promoted duplication of the M element pair. InGamachignathus,IcriodellaandPterospathoduslineages, these elements are differentiated morphologically. Subsequent anteriorward bending of the P element series caused duplication of the balognathid P2element pair, but they remained undifferentiated, even in the otherwise elaboratePromissum. The whole clade of conodonts with supernumerary element pairs in the apparatus has its roots in high latitudes of the Ordovician.

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.

Transposable elements not only can change genomic positions and disperse across the gene pool, but also can jump to another species through horizontal transmission. Of late, the P element, a DNA transposon in insects, was shown to cross the genetic boundary from Drosophila melanogaster to D. simulans in Europe around 2006. To understand the dynamics of transposable elements, especially in the early stages of invasion, we examined 63 lines of D. simulans from 11 natural populations in Japan established in 1976–2015. Based on PCR analyses, P elements were demonstrated to exist in Japan in 2008 and later. One copy of the full‐length P element was identified and mapped to a site on chromosome 3 L in a genome. All of 18 copies of P elements examined shared “A” at the nucleotide position 2040, which is representative of the direct descendants of the original P element that invaded in D. simulans. We also found that some lines having P elements can induce intensive gonadal dysgenesis in D. simulans at 29°C. Our present results imply that P elements in D. simulans arrived at the east end of Asia just a few years later than or almost simultaneously to the initial invasion in Europe, Africa, and North America, suggesting a more astonishingly rapid spread than previously assumed.

Background Transposition of P elements in the genome causes P–M hybrid dysgenesis in Drosophila melanogaster. For the P strain, the P–M phenotypes are associated with the ability to express a class of small RNAs, called piwi-interacting small RNAs (piRNAs), that suppress the P elements in female gonads. However, little is known about the extent to which piRNAs are involved in the P–M hybrid dysgenesis in M′ and Q strains, which show different abilities to regulate the P elements from P strains. Results To elucidate the molecular basis of the suppression of paternally inherited P elements, we analyzed the mRNA and piRNA levels of P elements in the F1 progeny between males of a P strain and nine-line females of M′ or Q strains (M′ or Q progenies). M′ progenies showed the hybrid dysgenesis phenotype, while Q progenies did not. Consistently, the levels of P-element mRNA in both the ovaries and F1 embryos were higher in M′ progenies than in Q progenies, indicating that the M′ progenies have a weaker ability to suppress P-element expression. The level of P-element mRNA was inversely correlated to the level of piRNAs in F1 embryos. Importantly, the M′ progenies were characterized by a lower abundance of P-element piRNAs in both young ovaries and F1 embryonic bodies. The Q progenies showed various levels of piRNAs in both young ovaries and F1 embryonic bodies despite all of the Q progenies suppressing P-element transposition in their gonad. Conclusions Our results are consistent with an idea that the level of P-element piRNAs is a determinant for dividing strain types between M′ and Q and that the suppression mechanisms of transposable elements, including piRNAs, are varied between natural populations. Electronic supplementary material The online version of this article (10.1186/s13100-017-0096-x) contains supplementary material, which is available to authorized users.

Hybrid dysgenesis is a sterility syndrome resulting from the mobilization of certain transposable elements in theDrosophilagermline. Particularly extreme is the hybrid dysgenesis syndrome caused byP-element DNA transposons, in which dysgenic female ovaries often contain few or no germline cells. Those offspring that are produced from dysgenic germlines exhibit high rates ofde novomutation and recombination, implicating transposition-associated DNA damage as the cause of germline loss. However, how this loss occurs, in terms of the particular cellular response that is triggered (cell cycle arrest, senescence, or cell death) remains poorly understood. We demonstrate that two components of the DNA damage response, Checkpoint kinase 2 and its downstream target p53, are determine the frequency of ovarian atrophy that is associated withP-element hybrid dysgenesis. We further show that p53 is strongly induced in the germline stem cells (GSCs) of dysgenic females, and is required for their maintenance. Our observations support the critical role for p53 in conferring tolerance of transposable element activity in stem cells.

The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library. TIS-Seq also facilitates identification of TIS in up to 96 individual/hand-picked mutants in a single MiniSeq/MiSeq run. TIS-Seq is a versatile method that can be easily modified to identify TIS from any kind of transposon mutant, as long as one end of the DNA sequence is known. Therefore, TIS-Seq is a promising method for transposon mutant screening. © 2017 by John Wiley & Sons, Inc. Keywords: PiggyBac; transposon insertion; transposon mutant; high throughput; mutant screening

The Drosophila melanogaster P transposable element provides one of the best cases of horizontal transfer of a mobile DNA sequence in eukaryotes. Invasion of natural populations by the P element has led to a syndrome of phenotypes known as P-M hybrid dysgenesis that emerges when strains differing in their P element composition mate and produce offspring. Despite extensive research on many aspects of P element biology, many questions remain about the genomic basis of variation in P-M dysgenesis phenotypes across populations. Here we compare estimates of genomic P element content with gonadal dysgenesis phenotypes for isofemale strains obtained from three worldwide populations of D. melanogaster to illuminate the molecular basis of natural variation in cytotype status. We show that P element abundance estimated from genome sequences of isofemale strains is highly correlated across different bioinformatics approaches, but that abundance estimates are sensitive to method and filtering strategies as well as incomplete inbreeding of isofemale strains. We find that P element content varies significantly across populations, with strains from a North American population having fewer P elements but a higher proportion of full-length elements than strains from populations sampled in Europe or Africa. Despite these geographic differences in P element abundance and structure, neither the number of P elements nor the ratio of full-length to internally-truncated copies is strongly correlated with the degree of gonadal dysgenesis exhibited by an isofemale strain. Thus, variation in P element abundance and structure across different populations does not necessarily lead to corresponding geographic differences in gonadal dysgenesis phenotypes. Finally, we confirm that population differences in the abundance and structure of P elements that are observed from isofemale lines can also be observed in pool-seq samples from the same populations. Our work supports the view that genomic P element content alone is not sufficient to explain variation in gonadal dysgenesis across strains of D. melanogaster, and informs future efforts to decode the genomic basis of geographic and temporal differences in P element induced phenotypes.

TheDrosophila melanogaster Ptransposable element provides one of the best cases of horizontal transfer of a mobile DNA sequence in eukaryotes. Invasion of natural populations by thePelement has led to a syndrome of phenotypes known as P-M hybrid dysgenesis that emerges when strains differing in theirPelement composition mate and produce offspring. Despite extensive research on many aspects ofPelement biology, many questions remain about the genomic basis of variation in P-M dysgenesis phenotypes in natural populations. Here we compare gonadal dysgenesis phenotypes and genomicPelement predictions for isofemale strains obtained from three worldwide populations ofD. melanogasterto illuminate the molecular basis of natural variation in cytotype status. We show that the number of predictedPelement insertions in genome sequences from isofemale strains is highly correlated across different bioinformatics methods, but the absolute number of insertions per strain is sensitive to method and filtering strategies. Regardless of method used, we find that the number of euchromaticPelement insertions predicted per strain varies significantly across populations, with strains from a North American population having fewerPelement insertions than strains from populations sampled in Europe or Africa. Despite these geographic differences, numbers of euchromaticPelement insertions are not strongly correlated with the degree of gonadal dysgenesis exhibited by an isofemale strain. Thus, variation inPelement insertion numbers across different populations does not necessarily lead to corresponding geographic differences in gonadal dysgenesis phenotypes. Additionally, we show that pool-seq samples can uncover population differences in the number ofPelement insertions observed from isofemale lines, but that efforts to rigorously detect differences in the number ofPelements across populations using pool-seq data must properly control for read depth per strain. Our work supports the view that euchromaticPelement copy number is not sufficient to explain variation in gonadal dysgenesis across strains ofD. melanogaster, and informs future efforts to decode the genomic basis of geographic and temporal differences inPelement induced phenotypes.

P element is a DNA transposon, known to spread in genome using transposase activity. Its activity is tissue-specific and normally observed at high temperatures within 24°C to 29°C. Here, we present a predicted RNA secondary structure domain of P element pre-mRNA which could potentially regulate the temperature sensitivity of the P element activity. In canonical P elements, the structure is a small hairpin with double-helical part interrupted by a symmetric loop and a mismatch. In M type P elements, the A.A mismatch is substituted by an A-U base pair, stabilizing the structure. The hairpin structure covers the region involving the IVS-3 5′ splice site and both pseudo-splice sites F1 and F2. While the IVS-3 and F1 binding sites of U1 snRNA are located in the double-stranded part of the structure, the F2 site is exposed in the hairpin loop. The formation of this structure may interfere with landing of U1 snRNA on IVS-3 site, while F2 is positioned for the interaction. Alignment of P element sequences supports the proposed existence of the hairpin, showing high similarity for this region. The hairpin structure, stable at low temperatures, may prevent correct IVS-3 splicing. Conversely, temperature-induced destabilization of the hairpin structure may result in the splicing at the proper IVS-3 splice site. Taking into account the increasing amount of data demonstrating the important influence of RNA folding on phenotypes determined by alternative splicing a model for possible regulation of the activity of mobile elements by pre-mRNA secondary structure seems intriguing. Предсказана вторичная структура пре-мРНК P-элемента, которая, возможно, регулирует его активность и температурную чувствительность. Структура представляет собой шпильку, более стабильную в Р-элементах М-типа, в сравнении с каноническими. Регион, образующий шпильку, находится в области 5’ сайта сплайсинга третьего интрона (IVS-3), включая оба описанных псевдо-сайта сплайсинга - F1 и F2. В то время как истинный сайт и F1 расположены, главным образом, в двухцепочечной области шпильки, F2 - экспонирован в петле. Выравнивание последовательностей Р-элементов продемонстрировало высокую степень сходства для указанного региона, что свидетельствует в пользу существования предсказанной структуры. Формирование шпильки, по нашему мнению, может препятствовать связыванию U1 snRNA с истинным сайтом сплайсинга, и, напротив, индуцированная температурой дестабилизация шпильки может приводить к корректному сплайсингу IVS-3. Таким образом, предложена гипотеза о влиянии вторичной структуры пре-мРНК мобильного элемента на его активность. Передбачена вторинна структура пре-мРНК Р-елемента, що, можливо, регулює його активність та температурну чутливість. Структура являє собою шпильку, більш стабільну у Р-елементах М-типу, порівняно з канонічними. Регіон, що утворює шпильку розташований в області 5’ сайта сплайсинга третього інтрону (IVS-3), включає обидва відомих псевдо-сайта сплайсингу - F1 та F2. При цьому 5’ сайт сплайсингу IVS-3 та F1псевдо-сайт знаходяться у дволанцюговій частині шпильки, а F2 є єкспонованим у петлі. Вирівнювання послідовностей Р-елементів продемонструвало високий рівень подібності для вказаного регіону, що свідчить на користь існування передбаченої структури. Формування шпильки, на нашу думку, може перешкоджати зв’язуванню U1 snRNA з 5’ сайтом сплайсингу IVS-3, і, навпаки, індукована температурою дестабілізація шпильки може сприяти корректному сплайсингу третього інтрону. Таким чином, запропонована гіпотеза про вплив вторинної структури пре-мРНК мобільного елементу на його активність.

The transposons of Drosophila melanogaster are regulated by small RNAs that interact with the Piwi family of proteins. These piRNAs are generated from transposons inserted in special loci such as the telomere-associated sequences at the left end of the X chromosome. Drosophila's P transposons can also be regulated by a polypeptide encoded by the KP element, a 1.15-kb-long member of the P family. Using piRNA-generating telomeric P elements (TPs) and repressor-producing transgenic KP elements, we demonstrate a functional connection between these two modes of regulation. By themselves, the TPs partially repress gonadal dysgenesis, a trait caused by rampant P-element activity in the germ line. This repression is manifested as a strictly maternal effect arising from the cytoplasmic transmission of P-specific piRNAs from mother to offspring. The repression is enhanced by genetic interactions between the TPs and other, non-telomeric P elements-a phenomenon attributable to ping-pong amplification of maternal piRNAs. KP elements, like other kinds of non-telomeric P elements, enhance regulation anchored in the TPs. However, with some TPs, the enhanced regulation is manifested as a strictly zygotic effect of the KP element. This effect is seen when the TP has few sequences in common with the KP element, a condition not conducive to ping-pong amplification of piRNAs; it can be attributed to the action of the KP repressor polypeptide. Because the effect is seen only when a TP was present in the mother's genotype, maternally generated P-element piRNAs could facilitate regulation by the KP repressor polypeptide.

Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.

Summary Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this. In this work we test the hypothesis that FB-NOF is an active transposon nowadays. We search for its expression product, obtaining its cDNA, and propose the ORF and the sequence of its potential protein. We found that the NOF protein is not a transposase as it lacks any of the motifs of known transposases and also shows structural homology with hydrolases, therefore FB-NOF cannot belong to the superfamily MuDR/foldback, as up to now it has been classified, and can be considered as a non-autonomous transposable element. The alignment with the published genomes of 12 Drosophila species shows that NOF presence is restricted only to the 6 Drosophila species belonging to the melanogaster group.

Transposable P elements are regulated in the germ line by piRNAs, which are small RNAs that associate with the Piwi class of proteins. This regulation, called the P cytotype, is enhanced by genetic interactions between P elements that are primary sources of these RNAs and other P elements. The enhanced regulation is thought to reflect amplification of the primary piRNAs by cleavage of mRNAs derived from the other P elements through a mechanism called the ping-pong cycle. We tested the transposase-encoding P element known as ∆2-3 for its ability to enhance cytotype regulation anchored in P elements inserted at the telomere of the left arm of the X chromosome (TP elements). The ∆2-3 P element lacks the intron between exons 2 and 3 in the structurally complete P element (CP). Unlike the CP element, it does not markedly enhance cytotype regulation anchored in TP elements, nor does it transmit transposase activity through the egg cytoplasm. However, mRNAs from both the CP and ∆2-3 elements are maternally deposited in embryos. These observations suggest that maternally transmitted CP mRNA enhances cytotype regulation by participating in the ping-pong cycle and that it encodes the P transposase in the embryonic germ line, whereas maternally transmitted ∆2-3 mRNA does not, possibly because it is not efficiently directed into the primordial embryonic germ line. Strong transposon regulation may, therefore, require ping-pong cycling with maternally inherited mRNAs in the embryo.

The hypothesis tested in this study was that single-gene mutations found previously to extend the life span of Drosophila melanogaster could do so consistently in both long-lived y w and standard w (1118) genetic backgrounds. GAL4 drivers were used to express upstream activation sequence (UAS)-responder transgenes globally or in the nervous system. Transgenes associated with oxidative damage prevention (UAS-hSOD1 and UAS-GCLc) or removal (EP-UAS-Atg8a and UAS-dTOR (FRB) ) failed to increase mean life spans in any expression pattern in either genetic background. Flies containing a UAS-EGFP-bMSRA (C) transgene associated with protein repair were found not to exhibit life extension or detectable enhanced green fluorescent protein (EGFP) activity. The presence of UAS-responder transgenes was confirmed by PCR amplification and sequencing at the 5' and 3' end of each insertion. These results cast doubt on the robustness of life extension in flies carrying single-gene mutations and suggest that the effects of all such mutations should be tested independently in multiple genetic backgrounds and laboratory environments.

SummaryThe X-linked telomericPelements (TPs)TP5andTP6regulate the activity of the entirePelement family because they are inserted in a major locus for the production of Piwi-interacting RNAs (piRNAs). The potential for this cytotype regulation is significantly strengthened when eitherTP5orTP6is combined with a non-telomeric X-linked or autosomal transgene that contains aPelement. By themselves, none of the transgenicPelements have any regulatory ability. Synergism between the telomeric and transgenicPelements is much greater when the TP is derived from a female. Once an enhanced regulatory state is established in a female, it is transmitted to her offspring independently of either the telomeric or transgenicPelements – that is, it works through a strictly maternal effect. Synergistic regulation collapses when either the telomeric or the transgenicPelement is removed from the maternal genotype, and it is significantly impaired when the TPs come from stocks heterozygous for mutations in the genesaubergine,piwiorSu(var)205. The synergism between telomeric and transgenicPelements is consistent with a model in whichPpiRNAs are amplified by alternating, or ping-pong, targeting of primary piRNAs to sense and antisensePtranscripts, with the sense transcripts being derived from the transgenicPelement and the antisense transcripts being derived from the TP.

We analyzed the P-M gonadal dysgenesis characteristics and the molecular patterns of P and KP elements of 35 populations from distinct locations of Turkey and Cyprus to find out P element dynamics in natural populations of Drosophila melanogaster. The results of Gonadal dysgenesis (GD) tests showed a predominat M' phenotype for this region. Besides Q populations were also found in less numbers. It was also noticed that there is a strong tendency for the presence of a Q type "buffer zone" in the Western part of Turkey. The variation in GD percentage (as a index for P element activity and susceptibility) observed among the populations did not show any clear relationship with some geoclimatic variables, but a significant correlation was found between longitude, and P element susceptibility. The P and KP element were also characterized by polymerase chain reaction amplification. The molecular analyses showed that all the populations examined have the entire of 1.15 kb KP element. The molecular patterns of KP elements have been found similar between the populations studied.

We have determined that two P elements, P[21-3] and P[21r36], residing in the 5′-UTR of the vestigial wing gene, encode functional repressors in eye tissue. However, neither element fits a previous categorization of repressor-making elements as Type I or II. Both elements encode polypeptides that are shorter than the canonical elements they most closely resemble. DNA sequencing reveals that P[21r36] encodes an intact THAP domain that is missing in the P[21] element, which does not encode a functional repressor. Recovery of P[21-3] at sites other than vestigial (where it causes the wing mutant, vg21-3) reveals that the element can make repressor in wing tissue of sufficient activity to repress the mutant phenotype of vg21-3. Why the P[21-3] element fails to produce repressor when located at vestigial may be explained by our observation that three different mutants in the RNA interference pathway cause a partial reversion of vg21-3. We speculate that the vg and P-initiated transcripts that arise at the vg locus in the vg21-3mutant trigger an RNA interference response that results in the mutual degradation of both transcripts.

The P elements of Drosophila melanogaster are well-studied transposons with both mobilizing and repressor functions. P elements can also variably silence the expression of certain other transgenes through a phenomenon known as P element-dependent silencing (PDS). To examine the role of the P repressor in PDS, we have induced, isolated, and characterized 22 point mutations in an archetype P element called P[SalI]89D. All mutations showed a loss in the ability to silence one or more assays for the PDS phenotype. These mutants also lost the ability to induce the suppression of variegation in P[hsp26-pt-T]39C-12, another P element-dependent phenotype. A subgroup of 11 mutations was further assayed for their ability to act as a P repressor and silence the P element promoter transcribing a lacZ ( + ) gene, and this function was lost as well. Taken together, this study supports a model of PDS acting through protein interactions, not RNA, with heterochromatic proteins to modify the extent of variegation seen in PDS. Furthermore, the common loss of functions for PDS and P repressor silencing (from another P promoter) argues for a common role of the repressor. This makes the PDS model a good system for examining P repressor functions and how they relate to transposon-mediated gene silencing in general.

The P transposable element recently invaded wild Drosophila melanogaster strains worldwide. A single introduced copy can multiply and spread throughout the fly genome in just a few generations, even though its cut-and-paste transposition mechanism does not inherently increase copy number. P element insertions preferentially target the promoters of a subset of genes, but why these sites are hotspots remains unknown. We show that P elements selectively target sites that in tissue-culture cells bind origin recognition complex proteins and function as replication origins. The association of origin recognition complex-binding sites with selected promoters and their absence near clustered differentiation genes may dictate P element site specificity. Inserting at unfired replication origins during S phase may allow P elements to be both repaired and reduplicated, thereby increasing element copy number. The advantage transposons gain by moving from replicated to unreplicated genomic regions may contribute to the association of heterochromatin with late-replicating genomic regions.

The use of P element collections led to the discovery of unanticipated effects from common genetic background mutants white, brown, and rosy in our previously reported model of tauopathy that expresses full-length human tau in the fly eye, in which mutant rosy suppresses mutant white and brown worsening of tau-induced toxicity (Ambegaokar & Jackson, 2010, Genetics, v. 186, p. 435-42). Here we discuss further possible effects of mini-white and evidence for autophagy as a mediator of white enhancement of tau toxicity.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.