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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.

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.

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.

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.

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.

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.

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 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.

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.

Sexual recognition among individuals is crucial for the reproduction of animals. In Drosophila, like in many other animals, pheromones are suggested to play an important role in conveying information about an individual, such as sex, maturity and mating status. Sex-specific cuticular hydrocarbon components are thought to be major sex pheromones in Drosophila, and are postulated to act through the gustatory system, since they are mostly non-volatile chemicals. However, very little is known about the molecular and neural bases of gustatory pheromone reception. So far, a few putative gustatory receptors, including Gr32a and Gr68a, have been implicated in courtship behavior. Here, we examine another putative gustatory receptor, Gr39a, which shares a cluster with both Gr32a and Gr68a in a molecular phylogeny of the gustatory receptor family, for its potential role in courtship behavior. The Gr39a gene produces four isoforms through alternative splicing of different 5'-most exons. A quantitative real-time PCR analysis showed that the expression levels of all four splice variants of Gr39a were reduced in a fly line in which a P element was inserted into the Gr39a locus. Homozygous and hemizygous males for the P-element insertion, as well as males in which Gr39a was knocked down by RNAi, all showed reduced courtship levels toward females. The courtship levels returned to normal when the P element was excised out. A close analysis of courtship behavior of the mutant males revealed that the average duration of a continuous courtship bout was significantly shorter in the mutants than in the wild type. The results suggest that Gr39a has a role in sustaining courtship behavior in males, possibly through the reception of a stimulating arrestant pheromone.

Gene duplication is a major force driving genome evolution, and examples of this mode of evolution and of the functions of duplicated genes are needed to reveal general patterns. Here, our study focuses on a particular retrogene (i.e., CG9573) that originated about 5–13 million years ago that we have named Drcd-1 related. It originated in Drosophila through retroposition of the parental gene Required for cell differentiation 1 of Drosophila (Drcd-1; CG14213), which is a known transcription cofactor. Drcd-1r is only present in D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Drcd-1r is an X to autosome retroposition event. Many retrogenes are X to autosome copies and it has been shown that positive selection underlies this bias. We sought to understand Drcd-1r mode of evolution and function to contribute to the understanding of the selective pressures acting on X to autosome retrogenes. Drcd-1r overlaps with another gene, it is within the 3′ UTR of the gene CG13102 and is encoded in the opposite orientation. We have studied the characteristics of the transcripts and quantified expression of CG13102 and Drcd-1r in wild-type flies. We found that Drcd-1r is transcribed specifically in testes. We also studied the molecular evolution of Drcd-1r and Drcd-1 and found that the parental gene has evolved under very strong purifying selection but the retrogene has evolved very rapidly (Ka/Ks ~ 1) under both positive and purifying selection, as revealed using divergence and polymorphism data. These results indicate that Drcd-1r has a novel function in the Drosophila testes. To further explore Drcd-1r function we used a strain containing a P element inserted in the region where CG13102 and Drcd-1r are located that shows recessive male sterility. Analysis of this strain reveals the difficulties that can be encountered in studying the functions of genes with overlapping transcripts. Avenues for studying of the function of this gene are proposed.

Crossing the cyprinids diploid blunt snout bream Megalobrama amblycephala (BSB) and Carassius auratus red var. (RCC) generated sterile triploid (3nRB) and fertile tetraploid (4nRB) hybrid offspring. Utilizing inverted terminal repeats (ITRs) of transposon Tdr1 from Danio rerio as PCR primer, the results showed that evident change in the number of Tc1-like transposons in 4nRB relative to BSB occurred, whereas such change did not arise in 3nRB compared to BSB. No Tc1-like transposon was found in RCC. A novel transposon was isolated from both BSB and 3nRB and designated as Tma1, which consisted of multiple copies after dot-blot hybridization. Based on the analysis of PCR amplified flanking sequence, characterization of Tma1 indicated that this element flanked by a duplicated TA dinucleotide and harbored an ITR of about 224 bp. Tma1 also harbored an incomplete transposase gene. Another novel transposon designated as Tte1 was detected in 4nRB, which harbored an ITR of roughly 130 bp and consisted of multiple copies, but had no transposase gene. The analysis of PCR amplification and Southern blot hybridization showed that DNAs of 4nRB, which were hybridized to DIG-labeled pTma1, did not give band by PCR with Tma1 primer, on the other hand, 7 of 15 DNA samples from BSB, which were hybridized to DIG-labeled pTte1, did not produce band by PCR with Tte1 primer. These results suggest that Tte1 may be a recent invasion in BSB population and burst in 4nRB offspring. Our data provide clues as to the possible role of transposons as a driving mechanism for genomic evolution.

We compared a series of Drosophila strains with P element insertions from -28 to -144 nucleotides 5' to the transcription start site of the Hsp70A genes-corresponding to the range of naturally occurring P element insertion sites-to elucidate the consequences of insertion site for Hsp70A gene expression. Although all insertions reduced Hsp70A expression below that of a control strain, the magnitude of the reduction was inversely related to the number of nucleotides between the transcription start site and the insertion site. A pre-existing hypothesis is that naturally occurring transposable element insertions in Hsp promoters may be beneficial in some circumstances, which may account for their retention in natural populations. In the present study, in a control line heat shock reduced fecundity, whereas in lines with P element insertions heat shock typically increased fecundity. Finally, according to cluster-specific quantitative RT-PCR, expression of the Hsp70A cluster genes was typically greater than that of the Hsp70B gene cluster genes, although the latter are more numerous and, in this case, free of P element insertions.

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.

We studied the occurrence of O-type P elements in at least one species of each subgroup of the saltans group, in order to better understand the phylogenetic relationships among the elements within the saltans group and with those of species belonging to the willistoni group. We found that the O-type subfamily has a patchy distribution within the saltans group (it does not occur in D. neocordata and D. emarginata), low sequence divergence among species of the saltans group as well as in relation to species of the willistoni group, a lower rate of synonymous substitution for coding sequences compared to Adh, and phylogenetic incongruities. These findings suggest that the evolutionary history of the O-type subfamily within the saltans and willistoni groups follows the same model proposed for the canonical subfamily of P elements, i.e., events of horizontal transfer between species of the saltans and willistoni groups.

Since the maize transposon Ac can move to a new location within the genome, it has been used in removing selectable markers in transgenic plants. In this paper, we developed an inducible transposon system to truncate a selectable marker in transgenic plants. In this system, the marker gene was accompanied by the inducible transposon, but one end of the transposon was located in the intron of the marker gene. As an example of a marker gene, we isolated the rice 5-enolpyruvylshikimate-3-phosphate synthase (epsps) and modified it for glyphosate tolerance. The transposon contained Ac transposase, which fused with the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). This construct was engineered into an expression vector pCAMBIA1300, harboring a hygromycin-resistant gene. The construct was first transformed into rice calli, and transformed plants were selected on hygromycin-containing medium. The stably transformed calli underwent determination for normal transcripts and tolerance to glyphosate. The results were applied to a rice transformation with the same construct, but using glyphosate as the selective agent. By determining the transformation efficiency, T-DNA copy patterns, we demonstrate that the modified epsps could be a suitable selectable marker to create transgenic rice. Furthermore, after obtaining stable transgenic plants and inducing transposition by salicylic acid, the transposon was excised, the marker gene became truncated, and its expression was terminated. This strategy could be applicable to yield self-stabilizing transposon by locating the transposon’s end in the transposase gene’s intron.

A transposon-based random mutation library of AcMNPV, the type species of baculovirus, was constructed using a Tn5 transposon. The green fluorescence protein gene under the control of the Drosophila hsp 70 promoter was inserted into the transposon for easy tracking in insect cells. In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome. The transposed genome was then used to transfect Sf21 insect cells, and a library of mutant viruses capable of expressing green fluorescence protein was obtained. Two mutant viruses, B9F and Li6A were isolated, and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes, respectively. Both genes were determined to be nonessential in viral replication and infection. This technique will be very useful in the functional study of baculovirus genes.

The genomic frequency of crossovers, fitness components, and activity of P- and hobo-transposable elements were studied in two natural populations of Drosophila melanogaster from regions with different pesticide pressure (forest and orchard populations). A significant increase in rf (recombination frequency) in the orchard population (as compared with the forest population) has been found within segments al-b, b-cn, cn-vg (chromosome 2) and for some chromosome 3 segments, th-st, st-cu, cu-sr, sr-e. None of the populations exhibited P-properties. However, the forest population showed weak hobo activity. Both populations contained probably defective hobo copies. With respect to average fitness (as estimated using a compound-autosome strain) and fecundity, the forest population was superior to the orchard one, whereas the latter had a higher locomotor activity and resistance to etherization. The difference in rf between the populations may be due to differences in their environmental conditions. Selection for pesticide resistance may favor the propagation of genotypes with higher rf, thus, possibly, ensuring release of adaptively significant genetic variation.

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.

The P transposable element copy numbers and the KP/full-sized P element ratios were determined in eight Brazilian strains of Drosophila melanogaster. Strains from tropical regions showed lower overall P element copy numbers than did strains from temperate regions. Variable numbers of full-sized and defective elements were detected, but the full-sized P and KP elements were the predominant classes of elements in all strains. The full-sized P and KP element ratios were calculated and compared with latitude. The northernmost and southernmost Brazilian strains showed fewer full-sized elements than KP elements per genome, and the strains from less extreme latitudes had many more full-sized P than KP elements. However, no clinal variation was observed. Strains from different localities, previously classified as having P cytotype, displayed a higher or a lower proportion of KP elements than of full-sized P elements, as well as an equal number of the two element types, showing that the same phenotype may be produced by different underlying genomic components of the P-M system.

A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn 5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1 x 10(-3) per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10(-3). Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.

Cuticular pheromones in Drosophila melanogaster are unsaturated hydrocarbons with at least one double bond in position 7: 7-tricosene and 7-pentacosene in males and 7,11 -heptacosadiene and 7,11 -nonacosadiene in females. We have previously shown that a desaturase gene, desat1, located in chromosome region 87 C could be involved in this process: the Desat1 enzyme preferentially leads to the synthesis of palmitoleic acid, a precursor of omega7 fatty acids and 7-unsaturated hydrocarbons. Therefore, we have searched for P-elements in the 87 region and mapped them. One was found inserted into the first intron of the desat1 gene. Flies heterozygous for this insertion showed a large decrease in the level of 7-unsaturated hydrocarbons, comparable to that observed in flies heterozygous for a deficiency overlapping desat1. Less than 1 % of flies homozygous for this insertion were viable. They were characterized by dramatic pheromone decreases. After excision of the transposon, the pheromone phenotype was reversed in 69% of the lines and the other excision lines had more or less decreased amounts of 7-unsaturated hydrocarbons. All these results implicate desat1 in the synthesis of Drosophila pheromones.

A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.

In order to study P element dynamics in natural populations of Drosophila melanogaster, 126 isofemale lines were examined from seven of the southernmost islands of Japan (the Sakishima Islands) and from Taiwan. Gonadal dysgenesis (GD) tests showed large divergences in the P-M phenotypes (P inducing and P repressing abilities) between the island populations. The P-M characteristics of each population, however, had not greatly changed in the past 15 years. Their genomic P element profiles are highly similar, consisting mostly of full-size P and of KP elements. We found no clear relationship between phenotype and genomic P element composition.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

P elements of two different subfamilies designated as M- and O-type are thought to have invaded host species in the Drosophila obscura group via horizontal transmission from external sources. Sequence comparisons with P elements isolated from other species suggested that the horizontal invasion by the O-type must have been a rather recent event, whereas the M-type invasion should have occurred in the more distant past. To trace the phylogenetic history of O-type elements, additional taxa were screened for the presence of O- and M-type elements using type-specific PCR primers. The phylogeny deduced from the sequence data of a 927-bp section (14 taxa) indicate that O-type elements have undergone longer periods of regular vertical transmission in the lineages of the saltans and willistoni groups of Drosophila. However, starting from a species of the D. willistoni group they were transmitted horizontally into other lineages. First the lineage of the D. affinis subgroup was infected, and finally, in a more recent wave of horizontal spread, species of three different genera were invaded by O-type elements from the D. affinis lineage: Scaptomyza, Lordiphosa, and the sibling species D. bifasciata/D. imaii of the Drosophila obscura subgroup. The O-type elements isolated from these taxa are almost identical (sequence divergence

We have developed a new modular vector system that facilitates the combination of various DNA fragments as functional modules for P element-mediated transformation of Drosophila melanogaster. The basic vector pP¿GS¿ contains unique sites for 17 restriction enzymes, including three 8-bp cutters, that allow one to combine various promoter elements, cDNAs and genomic DNA fragments, as well as protein tags and selectable marker genes, for a wide spectrum of transgene analyses. With this new vector system we analysed the chromosomal distribution of the Drosophila SU(VAR)3-9 protein tagged with EGFP, using hsp70-cDNA and genomic Su(var)3-9 constructs. We found preferential association of the tagged SU(VAR)3-9 with centric heterochromatin.

As part of our effort to monitor changes in the clinal pattern of P element-associated traits in eastern Australian Drosophila melanogaster, we investigated the genomic P elements of 293 isofemale lines collected in the period 1991–1994 from 45 localities. P elements were present in many copies in all genomes examined, with full-size P and KP element size classes accounting for the large majority. SR elements were not present in at least 92% of the lines tested. South of about 26° south Latitude (°SLat), the ratio of KP to full-size P elements (KP/P ratio) increased, correlating weakly with the P-M phenotypes of the populations, from moderately P populations (26–29°SLat) to M populations (37–38°SLat) North of 26°SLat, in weak P populations, the KP/P ratio was higher than between 26 and 29°Slat. The KP/P ratio appears to be higher in the northern populations than it was when previous studies were done. Overall, a high KP/P ratio among lines correlated roughly with a lack of P activity, but it also correlated with reduced repressor function. In a sample of 30 lines, a maternal effect of repressor function did not show a pattern with latitude, nor with KP/P ratio, nor with presence or absence of P activity.

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase.

The effects of interactions between P element and yellow regulatory sequences on the control of yellow expression were studied. The y mutations used in the analysis lack a segment of upstream sequence that extends from position -146 bp to -70 bp, relative to the transcription start site of the yellow gene. This sequence has been found to be necessary for the function of the yellow promoter. The insertion of one or two P element copies at position -69 bp compensates for the deletion in the regulatory region and restores yellow expression. After mobilization of the P element, new phenotypes were selected and molecularly characterized. Two regions in the 5' part of the P element, from 23 bp to 71 bp and from 82 bp to 108 bp, can each partially compensate for the yellow deletion. In addition, deletion derivatives of the P element were themselves able to activate yellow transcription. All such P elements retain at least 108 bp of sequence at the 5' end and 15-17 bp at the 3' end. Thus, the region of the P element from 23 bp to 108 bp contains cis-regulatory elements that can influence the transcription of neighboring genes.

The latitudinal cline in P transposable element-associated characteristics in eastern Australian populations of Drosophila melanogaster has changed between 1986 and 1991–1994. New collections were made in 1991–1994 from localities along the eastern coast of Australia. P element-associated properties of 256 isofemale lines from 43 localities were evaluated using gonadal dysgenesis and/or singed-weak hypermutability assays. The overall results indicate that both P activity and P susceptibility have declined, with all populations showing a tendency towards a state with little P activity potential but with P repressor function (neutral or ‘Q’). P repressor function is strong in all populations except some of the most southerly. P activity potential peaks at about 27° SLat, and drops off to the south (as in 1983–1986 collections) and to the north (in contrast to 1983–1986 collections); thus the cline is no longer a simple P-to-Q-to-M pattern from north to south, but is now Q-P-Q-M. A mtDNA RFLP that putatively distinguishes North American and European populations varies in frequency among the populations but the frequency does not vary clinally with latitude, ruling out massive introductions from North America and Europe as causing the cline.

Molecular analysis suggests that the pomace fly Drosophila melanogaster acquired the P family of transposable elements from another Drosophila species, D. willistoni. Since the two species are distantly related, it has been assumed that transmission of P element DNA from D. willistoni to D. melanogaster was mediated by a vector. The possibility of an alternative mode of transmission was assessed by characterizing the sexual behaviors of D. willistoni males and females, then observing D. willistoni and D. melanogaster males and females to see whether males from one species interacted sexually with females from the other species in a laboratory setting. We observed that D. melanogaster males court D. willistoni females vigorously and, in some cases, stimulate the females to be receptive to copulation. However, D. willistoni males perform relatively little courtship in response to D. melanogaster females and do not attempt to copulate. Thus, it is unlikely that sexual interactions effected the transmission of P element DNA from D. willistoni to D. melanogaster in the flies' natural habitat.

A set of 340 P insert lines on the X chromosome and the autosomes were screened for altered responses of the labellar chemoreceptors to salts and sucrose. A mutant linegustJ was isolated in which the electrophysiological response of the salt-sensitive neuron to Na+ in the sensilla of the proboscis is reduced. The responses to KC1 and sucrose are unaffected. In feeding tests,gustJ flies have Na+-specific defects. Heterozygotes ofgustJ with two other salt mutantsgustE andBE1323 are normal. Multiple alleles ofgustJ have been obtained by excision of the original P element. All mutants have defects in Na+ sensing specifically, thus defining a new gene that affects Na+ response of the fly.

Transposable elements are ubiquitous in all organisms and represent a dynamic component of their genomes, causing mutations and thereby genetic variation. Because of their independent and expansive replication strategy, these elements are called selfish and were thought to have no impact on the adaptive evolution of their host organisms. Although most TE-induced mutations seem to exert only negative effects on the fitness of their carrier, recent evidence indicates that in the course of evolution at least some TE-mediated changes have become established features of the host genome. For example, the insertion of TEs may provide novel cis-regulatory regions to preexisting host genes or TE-derived trans-acting factors may undergo a molecular transition into novel host genes through a process described as molecular domestication. The stationary P element related gene clusters of D. guanche, D. madeirensis and D. subobscura provide an excellent model system to study the evolutionary impact of TEs on genome evolution. Each cluster unit consists of a cis-regulating section composed of different insertion sequences followed by the first three exons of a P element that are coding for a 66 kDa ‘repressor-like’ protein.

The use of transposable elements (TEs) as genetic drive mechanisms was explored using Drosophila melanogaster as a model system. Alternative strategies, employing autonomous and nonautonomous P element constructs were compared for their efficiency in driving the ry+ allele into populations homozygous for a ry- allele at the genomic rosy locus. Transformed flies were introduced at 1%, 5%, and 10% starting frequencies to establish a series of populations that were monitored over the course of 40 generations, using both phenotypic and molecular assays. The transposon-borne ry+ marker allele spread rapidly in almost all populations when introduced at 5% and 10% seed frequencies, but 1% introductions frequently failed to become established. A similar initial rapid increase in frequency of the ry+ transposon occurred in several control populations lacking a source of transposase. Constructs carrying ry+ markers also increased to moderate frequencies in the absence of selection on the marker. The results of Southern and in situ hybridization studies indicated a strong inverse relationship between the degree of conservation of construct integrity and transposition frequency. These finding have relevance to possible future applications of transposons as genetic drive mechanisms.

The phylogenetic distribution of transposable families, P, gypsy, hobo, I, and mariner has been analyzed in 33 species of 11 groups of neotropical Drosophila and a Drosophilidae species Zygotrica vittimaculosa, using squash blot and dot blot. Genomic DNA of almost all neotropical species tested hybridized with gypsy probe and some species showed a particularly strong hybridization signal, as D. gaucha, D. virilis, and species of flavopilosa group. The hobo element was restricted to melanogaster group and some strains of D. willistoni. Only D. simulans DNA showed hybridization to mariner probe in all species tested and D. simulans and D. melanogaster showed hybridization with I element probe. P element homologous sequence was present in D. melanogaster and all species and strains of the willistoni and saltans groups tested. The presence of at least one P-homologous sequence was detected in Drosophila mediopunctata. This one was the only P-bearing species of all six tested from the tripunctata group. Four different pairs of primers homologous to segments of the canonical sequence of D. melanogaster's P were used to amplify specific sequences from D. mediopunctata DNA, showing the occurrence of seemingly well-conserved P-homologous sequences.

The P family of transposable elements in Drosophila melanogaster has, since its introduction into D. melanogaster populations in this century, diversified into a number of internally deleted forms. One of these, the KP element, is abundant in the genomes of flies from populations from Asia, Europe and Africa. There is some evidence that this sequence can act as a repressor of transposition. We have studied a mutation, signed(very weak) (snVW), in which a KP element is one of two P elements inserted into the 5' exon of the X-linked singed gene. These elements can be mobilized by a trans-acting dominant mutation, Mo, with a maternal effect, on the second chromosome. The rate of somatic reversion of snvw induced by Mo is reduced threefold if the mother herself possesses snvw on her X chromosomes. This implies that snvw may be responsible for a form of repression of P element movement. The cause of this effect may be related to transcription of the KP element in snvw. However, an effect of other genomic P elements in the repression of somatic reversion of snvw cannot be entirely excluded.

Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformed Drosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatic hsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatic hsp26 gene. In contrast, the heterochromatic hsp26 transgenes are present in approximately the same copy number as the endogenous euchromatic hsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV.

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10-20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.

The effect of P-M hybrid dysgenesis on life span was investigated by using three strains of D. melanogaster (Canton-S, Harwich-w and Malatya w.t.). F1 hybrids of dysgenic crosses developed on 29°C and lived as adults on 25°C produced unexpected white eye color in both sexes. In one type of cross, which has strong P element activity, it was found that hybrid dysgenesis affects longevity distinctly and shortens it, especially in the F1 produced at higher developmental temperature.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

In order to clarify the cause of null mutations in enzyme loci, the molecular structure of six null mutations in the Gpdh locus (encoding alpha GPDH: alpha glycerol-3-phosphate dehydrogenase (NAD+), E.C. 1.1.1.8; map position at 2-17.8) that arose in mutation accumulation experiments was examined. A restriction map analysis showed that five of the mutations are insertional mutations whereas the sixth is a deletion. The Gpdh regions of these null mutations were then cloned and sequenced. The inserted DNA fragments are all internally deleted P elements measuring 1.1 kb in length. Two are a KP element and two others are a HP element. All the insertions occur in the region near the initiation signal of transcription. The deletion encompasses the seventh and eighth exons over a length of 1.1 kb. These results therefore indicate that the null mutation rate at the Gpdh locus is largely influenced by P elements.

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5′ end and 14 by at the 3′ end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.

In Drosophila melanogaster, transposition of the P element is under the control of a cellular state known as cytotype. The P cytotype represses P transposition whereas the M cytotype is permissive for transposition. In the long-term, the P cytotype is determined by chromosomal P elements but over a small number of generations it is maternally inherited. In order to analyse the nature of this maternal inheritance, we tested whether a maternal component can be transmitted without chromosomal P elements. We used a stable determinant of P cytotype, linked to the presence of two P elements at the tip of the X chromosome (1A site) in a genome devoid of other P elements. We measured P repression capacity using two different assays: gonadal dysgenic sterility (GD) and P-lacZ transgene repression. We show that zygotes derived from a P cytotype female (heterozygous for P (1A)/balancer devoid of P copies) and which inherit no chromosomal P elements from the mother, have, however, maternally received a P-type extra-chromosomal component: this component is insufficient to specify the P cytotype if the zygote formed does not carry chromosomal P elements but can promote P cytotype determination if regulatory P elements have been introduced paternally. We refer to this strictly extra-chromosomally inherited state as the "pre-P cytotype". In addition, we show that a zygote that has the pre-P cytotype but which has not inherited any chromosomal P elements, does not transmit the pre-P cytotype to the following generation. The nature of the molecular determinants of the pre-P cytotype is discussed.

Large inserts have been found to appear within the yellow (y) locus of several unstable mutant lines derived from a parental strain (y2 sc+ w(aG)) of Drosophila melanogaster. The inserts of the individual mutant lines correspond roughly to a series of multimers (4.1, 7.1, 14.3 and 28.9 kbp, respectively) and are always located at the same position, somewhat upstream of the y gene transcription start site. Each of them begins and ends with a 1-kbp sequence which shares restriction sites with P elements. One of these inserts (14.3 kbp) has been partially sequenced (1270 bp from the 5' end). The first 1185 bp match exactly the sequence of an internally deleted P element. The remaining part of the sequence, on the other hand, is unrelated to P elements. It is assumed that large DNA sequences may be captured by the flanking P-element fragments and inserted into new chromosomal sites.

A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phs pi) effectively repressed P excision in a dose-dependent manner at very low concentrations relative to somatically active transposase (encoded by the hs pi delta 2-3 gene). Maximum repression required transcription of the complete transposase gene. Dose-dependent repression of P excision was also observed in the presence of a vector plasmid (pCarnegie4) having only the terminal sequences, including transposase binding sites, of the P element. However, repression required considerably higher concentrations of pCarnegie4 than phs pi, and elimination of P excision was not observed.

The P-M system of hybrid dysgenesis in Drosophila melanogaster was investigated on the basis of gonadal dysgenesis, using 1,590 strains from 28 natural populations in Japan, and 20 populations from Southeast Asia, the Pacific area and Africa. Strong P strains were found sporadically in several populations in Japan. Few strong M strains were observed. Q strains were present at a high frequency in most populations. Thus, most populations in these areas were regarded as Q populations. The distribution of the P element and the evolution of P, Q and M populations are also discussed.

Twenty transformed lines have been isolated as a result of the germ line insertion of a 3.2 kb alcohol dehydrogenase (Adh) gene fragment into an Adh negative strain of Drosophila melanogaster by P element-mediated transformation. More than half of these lines exhibited abnormal ADH expression. The level of ADH expression ranges from zero in some lines to near normal levels in others, and the pattern of ADH expression in the larval gut is also abnormal in many of these lines. Each of the abnormal tissue-specific patterns is stable and characterized by the absence or reduction of ADH expression in certain tissues. High levels of ectopic expression were not observed. In two of these lines, the pattern of ADH staining is highly restricted: it is limited to the medial midgut in line MM-50, and to the gastric caecae and the proventriculus in line GC-1. In heterozygotes between these two lines ADH is expressed in both of these tissues. To test the hypothesis that this abnormal expression is due to position effects, inserts were mobilized to new locations. The mobilized inserts exhibited new patterns of tissue-specific expression associated with new cytological insert locations, showing that the abnormal expression in lines MM-50 and GC-1 is due to tissue-specific position effects and not to mutations. The results are discussed in the context of chromatin structure as a possible cause of these position effects.