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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.

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.

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.

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.

Summary The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion remain unclear. We show that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. We find that temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. We propose that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution.

The simultaneous overexpression of multiple copies of Mn superoxide dismutase (SOD) and ectopic catalase (mtCat) transgenes in the mitochondria of the fruit fly, Drosophila melanogaster, was shown previously to diminish the life span. The hypothesis tested in the present study was that this effect was due primarily to the presence of one or the other transgene. An alternative hypothesis was that both transgenes have additive, negative effects. Crosses were performed between five pairs of transgenic lines containing single-copy insertions of either mtCat, Mn SOD, or P element vector control transgenes at unique loci, and the life spans of progeny containing two mtCat, Mn SOD or vector insertions were determined. Increasing amounts of mitochondrial catalase activity tended to be associated with decreases in mean life span. Overexpression of two copies of the genomic Mn SOD transgene had no effect on life span. The results do not support the hypothesis that enhanced mitochondrial SOD or catalase activity promotes longevity in flies.

Although mariner transposons are widespread in animal genomes, the vast majority harbor multiple inactivating mutations and only two naturally occurring elements are known to be active. Previously, we discovered a mariner-family transposon, Mboumar, in the satellite DNA of the ant Messor bouvieri. Several copies of the transposon contain a full-length open reading frame, including Mboumar-9, which has 64% nucleotide identity to Mos1 of Drosophila mauritiana. To determine whether Mboumar is currently active, we expressed and purified the Mboumar-9 transposase and demonstrate that it is able to catalyze the movement of a transposon from one plasmid to another in a genetic in vitro hop assay. The efficiency is comparable to that of the well-characterized mariner transposon Mos1. Transposon insertions were precise and were flanked by TA duplications, a hallmark of mariner transposition. Mboumar has been proposed to have a role in the evolution and maintenance of satellite DNA in M. bouvieri and its activity provides a means to examine the involvement of the transposon in the genome dynamics of this organism.

Little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Using a structure-based approach, nine ts mutants of Gal4 were generated in yeast by mutating four DNA binding residues. Two of these nine yeast ts mutants were cloned into P element vectors under control of the Elav and GMR promoters and transgenic Drosophila lines were generated. These were crossed to UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. Both of these yeast ts mutants show a ts phenotype in Drosophila and result in rapid induction of reporter gene expression upon shifting to the permissive temperature. Exposed, functional residues involved in protein-ligand or protein-protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms.

We have carried out detailed analyses of Drosophila P-transposon-homologous Pdre elements in zebrafish (Danio rerio) and present a model to explain the evolutionary origin of the complete and terminally truncated copies. Analysis of the most complex terminally truncated element led to the identification of novel DNA transposons, referred to as demio, hexi, and lady elements. We also show that the existence of a P-homologous stable gene can be explained in two ways: it is a P-transposon source gene, and insertion into or recombination with another transposon can lead to mobilization by recruitment of structures necessary for transposition (known as the source gene hypothesis), or it is the result of a domestication event involving a P transposon (domestication hypothesis). Our results indicate that both scenarios are possible and that they reflect different stages of the P-element life cycle.

Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.

Transposable elements have enormous potential to overcome one of the major hurdles in nonviral gene delivery, namely the lack of long-term gene expression. The Sleeping Beauty (SB) transposon is a promising vector system for nonviral gene therapy as it has the highest transposition activity of all known DNA transposons within mammalian cells. In an effort to generate a more efficient delivery vehicle, we conducted a systematic evaluation of several novel and previously identified SB transposase mutants. The results indicate that certain combinations of mutants do not enhance transposition, whereas others give a synergistic response. The most active mutant, designated HSB17, shows nearly 17-fold higher transposition activity compared to the original transposase SB10 when tested within the same expression cassette. In addition, synergistic activity is observed when this hyperactive mutant is combined with an improved transposon. Animal studies utilizing the hyperactive transposase show enhanced long-term reporter gene expression. These modifications further expand the utility of this transposon-based gene transfer system.

The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K and R, respectively. The effects of these mutations on the transpositional activities of Mos1 -derived transposon constructs were evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti . The transpositional activities of two Mos1 transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3′ ITRs, were increased when the mutant transposases were supplied in trans in E. coli . The use of the perfect repeat transposon with wild type transposase did not result in an increase in transposition frequency in Ae. aegypti . However, an improvement in the integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular evolution of Mos1 , and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1 mediated germ line transformation of Ae. aegypti .

P-element transposition in Drosophila is regulated by tissue-specific alternative splicing of the P-element transposase pre-mRNA. In somatic cells, the P-element somatic inhibitor (PSI) protein binds to exon 3 of the pre-mRNA and recruits U1 small nuclear ribonucleoprotein (snRNP) to the F1 pseudo-splice site. This abrogates binding of U1 snRNP to the genuine 5' splice site, thereby preventing excision of the third intron. Two homologous short sequences, referred to as the A and B boxes, near the C terminus of PSI bind to U1-70k protein within U1 snRNP. We have now mapped the AB box-binding site of U1-70k to a short proline-rich sequence at the C terminus. Our NMR study shows that the B box forms an anti-parallel helical hairpin in which four highly conserved aromatic residues form a cluster on one face of the first helix. This hydrophobic cluster interacts extensively with the proline-rich region of the U1-70k protein.

The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis in vertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes. A mutagenesis approach was undertaken to improve various aspects of the transposon, including safety and overall efficiency of gene transfer in human cells. Deletional analysis of transposon sequences within first-generation SB vectors showed that the inverted repeats of the element are necessary and sufficient to mediate high-efficiency transposition. We constructed a "sandwich" transposon, in which the DNA to be mobilized is flanked by two complete SB elements arranged in an inverted orientation. The sandwich element has superior ability to transpose >10-kb transgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactive versions of the SB transposase by single-amino-acid substitutions. These mutations act synergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressed HMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition by almost an order of magnitude compared to the first-generation transposon system. The improved vector system should prove useful for efficient gene transfer in vertebrates.

The α-glycerophosphate (GP) cycle in dipteran insect flight muscle is composed of two enzyme, the cytoplasmic α-glycerophosphate dehydregenase (α GPDH) and the mitochondrial α-glycerophosphate oxidase (α GPO). In order to analyze the enzyme activity in transformant, nine strains of Drosophila melanogaster have been constructed to have the flight muscle-specific α -glycerophosphate dehydrogenase (α GPDH) cDNA under the control of the Drosophila heat shock promoter (hsp70) on the third chromosome. These strains were compared with α GPDH null mutant line in the enzyme activity. All the transgenic strains exhibited higher enzymatic activities than null mutant line, but the amount of activity was still very low even at heat shock condition showing 30% in α GPDH enzyme amount, compared with wild type.

Insertion of a P element in one copy of the Drosophila Indy gene results in a significant increase in Drosophila lifespan. In order to examine whether mutations or polymorphisms in the human Indy gene promoted human longevity, the Indy gene was sequenced in 13 subjects of age 90-99, 12 subjects between age 38-49 and 18 subjectsage 23. Three coding region polymorphisms resulting in an amino acid change, F62L, V80L and I550V, two silent coding region polymorphisms, A99A and I551I, and three intron polymorphisms, IVS2+10 C deletion, IVS9+40T/A and IVS10+38C/T were identified. The I550V polymorphism occurred in whites, Hispanics, Asians and African-Americans at allele frequencies of 0.3514, 0.5104, 0.4274 and 0.5375, respectively. The I550V polymorphism was genotyped in 2366 subjects and examined for its association with age and weight.

Background: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5′ of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation.Results: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci.Conclusions: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.

The need for genetically engineered animals for drug target discovery, development and validation is growing. Transgenic animals, including mice and rats, and knockout and knock-in animals, provide useful animal models on which to test hypotheses and therapies. Tosk, Inc. has developed two gene delivery vectors based on the Drosophila P element transposon: StealthGene™ and a targeted gene delivery (TGD) vector that works via homologous recombination. The promise of these new vectors will be assessed for applications ranging from replacing current transgenic and knockout technologies, to creating large transgenic animals such as goats, pigs, sheep and chickens, to human gene therapy.

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

Many vectors are available for expression of cloned genes in the Drosophila germ line and eye. Nevertheless, for experiments that require female germ line expression, it is often problematic to find a vector that directs transcription suitably early and at high enough levels. In addition, a vector specific for undifferentiated cells in the Drosophila eye has yet to be described. Here, we have used the enhancer and promoter sequences of the fat facets gene to construct a P element transformation vector, pFAF, that provides high levels of gene expression in the female germ line. We present evidence that pFAF is a more potent activator of transcription in the female germ line than other commonly used vectors. In addition, in the eye, pFAF is unique in that it activates transcription specifically in undifferentiated cells.

Microsomal glutathione S-transferase-I (MGST-I) has been thought to be important for protecting the cell from oxidative damages and/or xenobiotics. We have previously identified the Microsomal glutathione S-transferase-like (Mgstl) gene, a Drosophila homologue of human MGST-I. To investigate the function of the enzyme using Drosophila as a model system, we examined the expression pattern of Mgstl during development, and generated loss-of-function mutants to assess its in-vivo function. Mgstl was expressed in all developmental stages. It is expressed ubiquitously with the highest expression in the larval fat body, an insect organ thought to be functionally corresponding to mammalian liver, while relatively low in the central nervous system. This tissue distribution is consistent with that of MGST-I in humans or Rats. Mgstl null mutants generated from a P element insertion line showed no obvious defects in morphology, indicating that it is not essential for the development. However, their life span was significantly reduced compared to control flies, suggesting that the MGSTL protein is involved in processes somehow contributing to aging. We found an Mgstl pseudogene, which is apparently derived through the reverse transcription of Mgstl mRNA and subsequent integration into the genome.

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.

Interleukin 4 (IL-4) gene expression is controlled at the level of transcription by the complex interactions of multiple factors that bind to a proximal promoter region. Nuclear factor of activated T cells (NFAT) can bind up to five purine-rich sequences in the IL-4 promoter termed the P elements (P0-P4). In this paper, we characterize a novel P element in the upstream region of the human IL-4 promoter that we term P5. P5 shares a core NFAT motif ((-353)GGAAA(-357)) and additional sequence similarity with the other P elements and supported strong interactions between the NFATp DNA-binding domain (DBD) and the AP-1 proteins cFos and cJun in DNA-binding assays. Inducibility of the IL-4 promoter was significantly impaired in a reporter construct in which the P5 element was mutated in the context of the full-length promoter. We conclude that P5 represents a novel IL-4 promoter P element that contributes to IL-4 promoter inducibility.

Work on how transposable elements are maintained and spread by virtue of their transposition processes have produced many theoretical studies of their evolutionary dynamics. But recent studies, which have experimentally identified some of these mechanisms, have not been taken into account. We present an integrated model of P transposable element regulation. It includes, at an individual level, the various mechanisms of regulation and the transposition events, that have been experimentally identified, recording specifically the chromosomal localisations of the inserted copies. It attempts to define the minimum conditions for explaining the regulation and spread of the P transposable element in Drosophila melanogaster natural populations. One test of this model is that it must explain the different population states found in the wild. A program that simulates the changes in Drosophila populations during the invasion of P elements was developed; the simulated populations were then compared to natural population data at the molecular and genetic levels. The model was validated by testing the dynamics of P element invasion in populations. It could explain the different natural population states with a recurrent invasion process. The simulations show that migration reduces the total number of copies, increases the number of defective copies, decreases P-activity and increases P-susceptibility, shifting equilibrium states from P to M′. They also show that the copies determining P-cytotype regulation spread faster by selection when located on the X chromosome. This result could account for the unexplained accumulation of P copies on the X chromosomes of some natural populations. Moreover the simulations predict a novel equilibrium state, called P′, not yet characterized in natural populations but that can be found in natural population data.

It was about 25 years ago that Hiraizumi noticed that the males of some strains of flies transmitted recombinant progeny[1]. This was in apparent contrast to Morgan's 1914 observation that recombination was absent in the Drosophila melanogaster male[2]. Were the rules of recombination changing in Drosophila , or was there an external agent at work? Kidwell et al. linked the male recombination phenomenon to a syndrome of other traits including sterility, chromosome rearrangements and mutations[3]. Investigation of mutations caused by this syndrome led to the identification of the P family of transposable elements, but it was not clear how P elements related to male recombination[4]. Two groups led by Sved and Engels recently solved this enigma[5–7]and, as so often happens in science, the answer to one problem gives insights into seemingly unrelated areas. The work initiated by Hiraizumi is providing a new understanding of how somatic genomes might maintain their integrity.

New P element plasmids containing the organophosphate-degrading gene opd as a dominant selectable marker were tested as transformation vectors in Drosophila melanogaster. One of these vectors was modified by the addition of the D. melanogaster mini-white gene as a comarker. When transformed individuals were identified using paraoxon selection for opd alone, results were similar to those obtained with mini-white. No false positives were recovered, however one strain contained the mini-white gene but inadequate resistance to survive our screening regimen due to a defective Hsp70-opd gene. Results suggest that Hsp70-opd is similar to mini-white for distinguishing transformed individuals but does not require time-consuming individual examination. Due to the mode of action of organophosphorus nerve agents, Hsp70-opd has potential as a selectable marker in numerous animals beside fruit flies.

The numbers of sensory hairs of Drosophila melanogaster present an ideal model system to elucidate the genetic basis of morphological quantitative variation. Loci affecting bristle number can be identified and their properties studied by accumulating spontaneous mutations, by P element mutagenesis, by mapping factors causing divergence between selection lines and by the association of phenotypic variation with molecular variation at candidate neurogenic loci. The consensus emerging from the application of all approaches is that much of the mutational and segregating variation affecting bristle number is attributable to alleles with large phenotypic effects at a small number of candidate loci.

Transposable elements are a major source of genetic change, including the creation of novel genes, the alteration of gene expression in development, and the genesis of major genomic rearrangements. They are ubiquitous among contemporary organisms and probably as old as life itself. The long coexistence of transposable elements in the genome would be expected to be accompanied by host-element coevolution. Indeed, the important role of host factors in the regulation of transposable elements has been illuminated by recent studies of several systems in Drosophila. These include host factors that regulate the P element, a host mutation that renders the genome permissive for gypsy mobilization and infection, and newly induced mutations that affect the expression of transposon insertion mutations. The finding of a type of hybrid dysgenesis in D. virilis, in which multiple unrelated transposable elements are mobilized simultaneously, may also be relevant to host-factor regulation of transposition.

Distribution and variation of two P-element subfamilies designated M-type and O-type elements were investigated in Drosophila bifasciata (Db) and its relatives. PCR screening revealed that full-sized and internally deleted elements of both types occur in three geographic Db strains and in the related species, D. imaii (D1). Molecular analyses indicate differences in the evolutionary behaviour of the two P-element types. Internally deleted M-type elements fall into two size classes present in all three Db strains. In contrast, internally deleted O-type elements vary between the strains in number and length. With respect to genomic location, M-type elements seem to be restricted to conserved euchromatic sites, whereas the positions of O-type elements appear to be geographically variable. In one strain of Db (Italy), O-type elements seem to accumulate in the heterochromatin. Sequencing of a 397-bp segment shows intra- and interspecific divergence of M-type elements. In a 452-bp segment of the O-type elements, no substitutions were found, neither within nor between species. This finding suggests recent introgression of O-type elements via hybridization between Db and Dl. Sequence identity and variation in chromosomal locations among different copies imply that O-type elements are transpositionally active. For M-type elements, genomic mobility cannot be proved. In a survey of several other taxa, no O-type-related sequences were detected so far. Therefore, the origin of the O-type subfamily remains unknown, whereas the source of M-type elements can be traced back to the genus Scaptomyza.

Sequence relationships and functional aspects were analysed in the P element homologues of Drosophila subobscura (Ds) and D. guanche (Dg) . In both species, the P homologues are clustered at a single genomic position. They lack the characteristic terminal structures of actively transposing P elements, but they have the coding capacity for a 66-kDa ‘repressor-like’ protein. Two different types of cluster units (G-type and A-type) can be distinguished. The A-type unit, which is present in multiple copies, is transcribed in adult flies. In contrast, the G-type unit has a much lower copy number and is apparently not expressed. In Dg , the isolated G-type sequence carries a 420-bp insertion in the promoter region, which is probably responsible for inactivation. Sequence comparisons of different cluster units show that differentiation of the two types preceeds the lineage split of these species. Substitution rates of the deduced proteins reveal two distinct subregions: high variability at the N terminus and strong sequence conservation in the rest of the protein. The variable region contains motifs characteristic of DNA-binding proteins. Adaptive diversification of the cluster units towards specific binding properties might be a plausible explanation for variability in the N-termini. Both unit types have lost the weak promoter region characteristic of P transposons. In the A-type unit, a new promoter has been formed which is apparently composed of parts of insertion sequences derived from two different mobile elements. Immobilization, change of the control unit, transcriptional activity and diversification at the amino acid level suggest that, besides the possible conserved repressor function, the P homologues of Ds and Dg may have acquired a novel assignment in the genome

Several CaCu 5 -type compounds in which Ce is combined with a 3d element (or Cu) and an s, p element have been studied by time-of-flight neutron diffraction at room temperature. A strong site preference of the s, p elements was observed. It is shown that Al and Ga occupy almost exclusively the 3g site. This behaviour is in contrast to that of B, which is known to substitute almost exclusively into the 3g site in CeCo 5 .

The PiggyBac (PB) transposon system is a potent nonviral gene delivery tool with relevance in both gene therapy and cell reprogramming for production of induced pluripotent stem cells (iPSCs). Moreover, by taking advantage of the ability of the PB transposase to excise transposon-embedded gene cassettes from the genome without leaving footprints, the PB system is unique in facilitating seamless genome editing. The need for intracellular production of the transposase, however, raises concerns related to delivery and to cytotoxicity caused by sustained transposase expression and insertional mutagenesis. Furthermore, transposon re-integration may decrease the overall efficiency of the PB-mediated excision as well as increasing the risk of adverse secondary insertions. Based on our previous work, we present a new approach for lentivirus-based delivery of PB transposase. By fusing the hyperactive PB transposase, hyPBase, to the C-terminus of the GagPol polyprotein, we show robust incorporation and subsequent release of the transposase in matured lentiviral particles. Furthermore, in an effort to limit transposon re-integration, we engineered a hyPBase variant carrying three missense mutations. This novel hyPBase variant, hyPBaseExc+/Int−, demonstrates integration levels very close to background levels, thus limiting the risk of reintegration. Notably, the ability of hyPBaseExc+/Int− to excise transposons from plasmid-borne as well as from genomically integrated PB transposon cassettes is increased up to 6.3-fold relative to the original hyPBase transposase. By fusing the hyPBaseExc+/Int− to the C-terminus of GagPol, transposase protein can be efficiently delivered to cells by lentiviral protein transduction and, in our model system, performs seamless genomic excision of PB transposon cassettes in a copy number and dose-dependent manner, resulting in excision in up to 23.6% of virus-treated cells. Using a transposon containing the puro-deltaTK transgene cassette, we furthermore show that cells with successful excision can be enriched 17-fold by negative selection with FIAU. We believe that protein transduction of hyPBaseExc+/Int− may increase the applicability and safety of transposase-directed genomic excision in iPSCs and in hard-to-transfect cell types including hematopoietic cells.

Variation in the number of gene copies can play a major role in changing the coding capacities of eukaryotic genomes. Different mechanisms, such as unequal recombination or transposon-induced chromosome rearrangements, are believed to be responsible for these events. We have used the direct tandem duplication at the complex locus polyhomeotic (ph) of Drosophila melanogaster as a model system to study functional redundancy associated with chromosomal rearrangements, such as duplications or deletions. The locus covers 28.6 kb and comprises two independent units, ph proximal and ph distal, which are not only similar on the molecular level, but appear to be functionally redundant [Dura et al., Cell 51 (1987) 829-839; Deatrick et al., Gene 105 (1991) 185-195]. We present a molecular and phenotypic analysis of two hypomorphic ph mutants, ph2 and ph4, induced during hybrid dysgenesis. Each corresponds to an internal deletion in the ph locus that overlaps both transcription units. We show that the deletions are likely due to a P/M hybrid dysgenesis-induced rearrangement between proximal and distal ph, that created a single new chimerical ph gene. At least one of the breakpoints must be located in a 1247-bp region that is rich in single sequence, and 100% identical between proximal and distal ph. Junction points between units are in the protein-coding regions, but could not be exactly localized on the genomic sequence of either mutant, because of the precise molecular mechanism that caused the deletions. Protein products of the hybrid genes contain the same functional domains as either wild-type (wt) product.(ABSTRACT TRUNCATED AT 250 WORDS)

The vestigial gene (vg+) is required for normal wing development and is expressed in a spatially distinct pattern in imaginal discs. We have exploited a general property of P element alleles to target an enhancer trap to the 5′ region of the gene. By replacing the P element resident at this site in vg21 with a P element carrying a lacZ reporter gene, the vglacZ1 allele was selected on the basis of its increased mutant phenotype. In contrast to vg+ expression, which occurs primarily in the presumptive wing margin and hinge, β-galactosidase expression in vglacZ1 wing discs is localized to the dorsal wing surface and displays homologous haltere expression. The targeting of P element enhancer traps could be readily extended to other genes with low rates of primary P element insertion.

A bang-sensitive enhancer trap line was isolated in a behavioral screen. The flies show a weak bang-sensitive paralysis, recovering after about 7 s. The P element insert is localized at 93131-2 on the salivary chromosomes, the site of the (Na + ,K + )ATPase a subunit gene. Molecular characterization demonstrates that the transposon is inserted into the first intron of this gene. This insertion leads to normal-sized transcripts, but reduced levels of expression. This change is also reflected in lower amounts of a normal-sized a subunit protein. Mutant flies show a much greater sensitivity to ouabain, likewise indicating, on a functional level, a reduction in Na + pump activity. Furthermore, the bang-sensitive behavior can also be mimicked by injecting sublethal doses of ouabain into wild-type flies. The molecular and functional evidence indicates that the insertion has produced a hypomorphic mutation of the (Na + ,K + )ATPase α subunit gene, opening the way to future studies of the regulation of the Na + pump.

Mutations within or affecting ion channel genes can cause insecticide-resistant or temperature-sensitive phenotypes. Some laboratory-isolated temperature-sensitive sodium channel mutants of Drosophila melanogaster (Meigen) have been shown to possess low levels of resistance to DDT and pyrethroids but the extent to which field-isolated insecticide-resistant mutants are temperature sensitive has not been investigated. We report here a temperature-sensitive phenotype associated with the cyclodiene insecticide resistant mutant Rdl . Following exposure to 38°C resistant flies show temporary paralysis and are unable to locomote. Rescue of this mutant phenotype by a P-element-mediated germline transformant carrying a wild type copy of Rdl proves that the phenotype is associated with this locus. The possible reduced fitness conferred upon resistant insects in the field by this sensitivity to high temperature is discussed.

A P element (PE)-induced Drosophila melanogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene (CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points (tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.

We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg +2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3′-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp) , a TGF-β family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp . Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-β superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.

The possibility of somatic mobilisation of P elements in Drosphila melanogaster was investigated. Flies, trans-heterozygous for the genetic markers mwh and flr 3 were obtained by crossing males containing transposition-competent P elements with females having M cytotype. The hybrid dysgenic flies were reared at 29°C and their wings examined for mutant clones. The frequency of mutant spots found on the wings of the female flies was significantly higher than that of female control flies. We postulate that this increase in frequency may be due to P element mobilisation at high temperature in the wing cells of dysgenic hybrids. This is in direct contrast to the large body of reasearch which indicates that P-transposition-mediated mutation is restricted to the germline.

P transposable elements in Drosophila melanogaster can undergo precise loss at a rate exceeding 13% per generation. The process is similar to gene conversion in its requirement for a homolog that is wild type at the insertion site and in its reduced frequency when pairing between the homologs is inhibited. However, it differs from classical gene conversion by its high frequency, its requirement for P transposase, its unidirectionality, and its occurrence in somatic and premeiotic cells. Our results suggest a model of P element transposition in which jumps occur by a "cut-and-paste" mechanism but are followed by double-strand gap repair to restore the P element at the donor site. The results also suggest a technique for site-directed mutagenesis in Drosophila.

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.

A mariner-like element termed mle-1 was discovered in the parasitic nematode Trichostrongylus colubriformis. The mle-1 has features which support its assignment as a mariner-like transposable element. Cloned mle-1 was derived from an intron of the tar-1 gene. It comprises 893 bp, includes two 27 bp flanking perfect inverted repeats and is present at approximately 50 copies in the genome. The element contains a coding region which displays homology to transposases, with the greatest amino acid similarity to a Caenorhabditis elegans mariner-like transposase. The coding region contains two 12 bp repeats; these repeats flank an 11 bp segment which accounts for a frameshift in this region. As a candidate transposon, mle-1 provides potential for genetic manipulation of this and related organisms.

Transposon systems permit non-viral delivery of genetic cargo into the genome of human cells and hold promise for eventual use in gene therapy. Transposon systems with activity in vertebrates include Sleeping Beauty (SB) and Mos1 of the Tc1/Mariner family and piggyBac which represents a different class of transposable element. We undertook evaluation of these three different transposable elements in human cells in order to compare the properties between each transposon system. This permitted direct comparison of the activity of native and hyperactive SB, native and hyperactive Mos1, and native piggyBacin plasmid vectors differing only by the transposase cDNA and inverted repeat (IR) elements. Mos1 required full internal sequences to be present for measurable transposition activity in human cells. The piggyBac transposable element could be reduced to a 311bp left IR and a 236bp right IR with retention of full activity. Evaluation of overall transposable activity revealed Mos1 << SB < piggyBac in cultured human cells. Hyperactive mutants of the Mos1 transposase were evaluated and showed little increase in transposition over native Mos1 in human cells. Interestingly, piggyBac showed more activity than native or even hyperactive SB (two-to-ten fold more active than hyperactive SB12 transposase combined with hyperactive pT3 transposon). Additionally, overproduction inhibition, a known major limitation of SB, was lacking with piggyBac. We evaluated piggyBac transposition following transient transfection of 50ng, 100ng, and 2|[mu]|g transposon DNA while varying the transposase DNA amount. In all cases, piggyBac lacked overproduction inhibition while SB transposition could be driven to very low levels at higher amounts of transposase DNA. Such results revealed that piggyBac transposition was limited only by the amount of transposon DNA present. Finally, we evaluated the addition of a zinc finger DNA binding domain element to the N-terminus of the different transposase enzymes. Zinc finger DNA binding domain addition ablated Mos1 activity and decreased SB activity which was only reliably measured using a combination of hyperactive transposase and transposon mutants. However, chimeric-piggyBac transposase activity appeared unaffected by N-terminal zinc finger DNA binding domain addition when compared to the native system. These results demonstrate that the piggyBac transposon system has advantageous properties when compared to other transposon systems with activity in human cells thereby making it a viable alternative system for gene therapy.

The Sleeping Beauty (SB) transposon system is extremely effective at integrating precise DNA sequences into vertebrate genomes, including those of humans. One potential use of this system is for gene therapy. The SB vector system has 2 components: 1) a transposon containing genetic cargo and 2) the catalytic transposase enzyme. In most cases the source of SB transposase has been a plasmid. This delivery strategy may allow extended periods of transposase activity that could cause deleterious effects were the transposons to be multiply remobilized. We have made several constructs to determine the duration of expression of SB transposase when delivered via a plasmid to cells in livers of mice. Three constructs contained the transposon and transposase on the same plasmid (cis constructs): pT2/mCAGGS-IDUA//Ub-SB11, pT2/mCAGGS-IDUA//MT-SB11, pT2/mCAGGS-GUSB//CMV-SB11. Another two constructs contained only a transposase gene with the transposon placed on separate plasmids (trans constructs): pT2/CAGGS-GUSB with PGK-SB11 and pT2/CAGGS-GUSB with SB11 RNA. MCAGGS is a shortened version, for driving the iduronidase (IDUA) gene, of the CAGGS promoter, which we use to drive glucuronidase B (GUSB) gene expression. We used high-pressure (hydrodynamic), tail-vein injection to deliver these constructs to the livers of C57/BL6 mice. The injections were evaluated 24hr post injection by expression assays of either the GUSB or IDUA genes. Mice will be euthanized at 6,5,4,3,2 months 6,4,2,1 weeks and 6,5,4,3,2,1 days and livers will be extracted. SB expression and functionality will be measured respectively by Western blot and excision PCR analyses. Linker-mediated PCR will be done to clone transposon insertions and integration as evidence of SB-mediated transposition events.