Search

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

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

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coli , corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function. Furthermore, D-elp1 depletion results in increased steady state levels of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast, although Dcr-2 depletion results in increased transposon RNA levels and a reduction in the corresponding endo siRNAs, there is no change in the transposon antisense RNA levels. In D-elp1 null third instar larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly with Dcr-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. These results identify an aspect of the RNAi pathway in Drosophila that suggest transposon derived endo siRNAs, critical for transposon suppression, are produced, in part, in a D-elp1 dependent step that converts transposon RNA into dsRNA that is subsequently processed by Dcr-2. The generality of this mechanism in genome defense and RNA silencing in higher eukaryotes is suggested.

Hybrid dysgenesis in Drosophila is a syndrome of gonadal atrophy, sterility, and male recombination, and it occurs in the progeny of crosses between males that harbor certain transposable elements (TEs) and females that lack them. Known examples of hybrid dysgenesis in Drosophila melanogaster result from mobilization of individual families of TEs, such as the P element, the I element, or hobo . An example of hybrid dysgenesis in Drosophila virilis is unique in that multiple, unrelated families of TEs become mobilized, but a TE designated Penelope appears to play a major role. In all known examples of hybrid dysgenesis, the paternal germ line transmits the TEs in an active state, whereas the female germ line maintains repression of the TEs. The mechanism of maternal maintenance of repression is not known. Recent evidence suggests that the molecular machinery of RNA interference may function as an important host defense against TEs. This protection is mediated by the action of endogenous small interfering RNAs (siRNAs) composed of dsRNA molecules of 21-25 nt that can target complementary transcripts for destruction. In this paper, we demonstrate that endogenous siRNA derived from the Penelope element is maternally loaded in embryos through the female germ line in D. virilis . We also present evidence that the maternal inheritance of these endogenous siRNAs may contribute to maternal repression of Penelope .

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals P elements in Drosophila.

With the available eukaryotic genome sequences, there are predictions of thousands of previously uncharacterized genes without known function or available mutational variant. Thus, there is an urgent need for efficient genetic tools for genomewide phenotypic analysis. Here we describe such a tool: a deletion-generator technology that exploits properties of a double transposable element to produce molecularly defined deletions at high density and with high efficiency. This double element, called P { wHy }, is composed of a “deleter” element hobo , bracketed by two genetic markers and inserted into a “carrier” P element. We have used this P { wHy } element in Drosophila melanogaster to generate sets of nested deletions of sufficient coverage to discriminate among every transcription unit within 60 kb of the starting insertion site. Because these two types of mobile elements, carrier and deleter, can be found in other species, our strategy should be applicable to phenotypic analysis in a variety of model organisms.

Insulator DNAs and promoter competition regulate enhancer–promoter interactions within complex genetic loci. Here we provide evidence for a third mechanism: promoter-proximal tethering elements. The Scr - ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3′ of the ftz gene and ≈25 kb 5′ of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1- ftz and T1- Scr interactions. A 450-bp DNA fragment located ≈100 bp 5′ of the Scr transcription start site is essential for T1- Scr interactions and can mediate long-range activation of a ftz / lacZ reporter gene when placed 5′ of the ftz promoter. We suggest that the Scr 450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer–promoter interactions at other complex genetic loci.

Maternal transmission of RNAs or proteins through the egg cytoplasm plays an important role in eukaryotic development. We show that the transposase activity encoded by the P transposable element of Drosophila melanogaster is transmitted through the oocytes of females heterozygous for this element even when these oocytes do not carry the element itself. However, this maternal transmission is abolished when the last of three introns is removed from the P element. These facts imply that maternal transmission of transposase activity involves the RNA transcribed from the P element rather than the polypeptide it encodes, and that to be transmitted maternally, this RNA must possess the last intron. Examination of the intron's sequence reveals that it contains a motif of nine nucleotides that has been implicated in the maternal transmission of developmentally significant RNAs. This same intron limits expression of the P transposase to the germ line of Drosophila . Thus, the last P intron has two important biological functions.

Transposable elements have been used widely in the past 20 years for gene transfer and insertional mutagenesis in Drosophila . Transposon-based technology for gene manipulation and genomic analysis currently is being adopted for vertebrates. We tested the ability of Minos , a DNA transposon from Drosophila hydei , to transpose in mouse tissues. Two transgenic mouse lines were crossed, one expressing Minos transposase in lymphocytes under the control of the CD2 promoter/locus control region and another carrying a nonautonomous Minos transposon. Only mice containing both transgenes show excision of the transposon and transposition into new chromosomal sites in thymus and spleen cells. In addition, expression of Minos transposase in embryonic fibroblast cell lines derived from a transposon-carrying transgenic mouse resulted in excision of the transposon. These results are a first step toward a reversible insertional mutagenesis system in the mouse, opening the way to develop powerful technologies for functional genomic analysis in mammals.

We determined the genomic sequence at the site of insertion in 2,266 unselected P element insertion events. Estimating physical properties of the genomic DNA at these insertion sites—such as base composition, bendability, A-philicity, protein-induced deformability, and B-DNA twist—revealed that they differ significantly from average chromosomal DNA. By examining potential hydrogen bonding sites in the major groove, we identified a 14-bp palindromic pattern centered on the 8-bp target site duplication that is generated by P element insertion. Our results suggest that the P-element transposition mechanism has a two-fold dyad symmetry and recognizes a structural feature at insertion sites, rather than a specific sequence motif.

P element insertion is essentially random at the scale of the genome. However, P elements containing regulatory sequences from Drosophila engrailed and polyhomeotic genes and from the Bithorax and Antennapedia complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing Polycomb group response elements (preferential insertion). This phenomenon is thought to be mediated by Polycomb group proteins. In this report, we describe a case of homing of P elements containing regulatory sequences of the linotte gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of Polycomb group proteins but by a new, as yet unknown, mechanism. We also suggest that P element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of Drosophila reverse genetics, for which there is no other described gene targeting technique.

Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Drosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp70-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp70-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three of the insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp70-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.

The demonstration by Zhang and Spradling (1) of efficient P element transposition into heterochromatic regions will aid ongoing studies of heterochromatin structure and function. P element insertions will provide entry points for further molecular analysis of heterochromatin and will allow the isolation of small and large heterochromatic deficiencies. The generation of heterochromatic P insertions also will aid the study of heterochromatic genes. Of the heterochromatic insertions isolated by Zhang and Spradling, five were homozygous lethal, and one of these defined a lethal locus not previously uncovered by heterochromatic deficiencies. P elements have previously been used to mutagenize and clone specific heterochromatic genes (14, 19, 26). New methods, like those described here (1, 32), should allow the efficient identification and molecular isolation of other single-copy heterochromatic genes. Furthermore, since position-effect suppression allowed the recovery of heterochromatic P insertions, it may also allow the recovery of insertions in euchromatic regions previously refractory to P mutagenesis. Studies of position-effect variegation show that genes normally found in heterochromatin require a heterochromatic context for normal expression and that heterochromatin is inhibitory to euchromatic gene expression (16). The physical basis of these related phenomena--chromatin assembly, nuclear positioning, and/or heterochromatin elimination--can be resolved only with a more thorough understanding of heterochromatin structure and functions. Analyzing heterochromatin also will help define the chromosomal components responsible for inheritance processes such as chromosome pairing, sister chromatid adhesion, and centromere function. These efforts will be facilitated by the effective use of P elements combined with other current molecular-genetic approaches.

We describe a method of screening for transposon insertions in or near Drosophila loci that correspond to cloned DNA sequences. We mobilize a modified P element transposon that carries a bacterial plasmid origin of replication and a drug-resistance marker. The genomic sequences flanking each transposon insertion site can then be rescued as a plasmid in Escherichia coli. Libraries of such plasmids, representing pools of transposon-mutagenized individuals, are used as hybridization probes against cloned sequences to determine whether a transposon has inserted next to a particular site in the genome. The number of loci that can be screened simultaneously by this procedure is quite large. We have screened an array of cDNA clones representing almost 700 distinct loci against libraries representing 760 mutagenized flies, and we obtained hybridization signals to 7 different cDNAs. Three of these events have been analyzed in detail and represent genuine insertions near genomic sequences that correspond to the cDNAs.

Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.

An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.

The demonstration by Zhang and Spradling (1) of efficient P element transposition into heterochromatic regions will aid ongoing studies of heterochromatin structure and function. P element insertions will provide entry points for further molecular analysis of heterochromatin and will allow the isolation of small and large heterochromatic deficiencies. The generation of heterochromatic P insertions also will aid the study of heterochromatic genes. Of the heterochromatic insertions isolated by Zhang and Spradling, five were homozygous lethal, and one of these defined a lethal locus not previously uncovered by heterochromatic deficiencies. P elements have previously been used to mutagenize and clone specific heterochromatic genes (14, 19, 26). New methods, like those described here (1, 32), should allow the efficient identification and molecular isolation of other single-copy heterochromatic genes. Furthermore, since position-effect suppression allowed the recovery of heterochromatic P insertions, it may also allow the recovery of insertions in euchromatic regions previously refractory to P mutagenesis. Studies of position-effect variegation show that genes normally found in heterochromatin require a heterochromatic context for normal expression and that heterochromatin is inhibitory to euchromatic gene expression (16). The physical basis of these related phenomena--chromatin assembly, nuclear positioning, and/or heterochromatin elimination--can be resolved only with a more thorough understanding of heterochromatin structure and functions. Analyzing heterochromatin also will help define the chromosomal components responsible for inheritance processes such as chromosome pairing, sister chromatid adhesion, and centromere function. These efforts will be facilitated by the effective use of P elements combined with other current molecular-genetic approaches.

We have used DNase I footprinting and partially fractionated nuclear extracts from Drosophila Kc tissue culture cells to identify DNA-binding proteins that interact with the terminal repeats of P transposable elements. We have identified a binding activity that interacts specifically with a region of the 31-base-pair terminal inverted repeats that is directly adjacent to the duplication of target site DNA. Binding occurs to both the 5' and 3' inverted terminal repeats irrespective of the sequence of the duplicated target DNA. UV photochemical crosslinking studies suggest that the binding activity resides in a polypeptide of 65-70 kDa. Biochemical fractionation and oligonucleotide affinity chromatography have been used to purify the binding activity to near homogeneity and identify a polypeptide of 66 kDa in the highly purified preparations. The site to which binding occurs is included in a region absolutely required for P element transposition, suggesting that this binding protein may be a cellular factor involved in P element transposition.

Hybrid dysgenesis is manifested as a group of correlated aberrant genetic traits such as sterility, increased mutation rate, and male recombination. Previous work has shown that it appears when males of strains carrying either of two independent families of transposable elements called I and P factors are hybridized with females of susceptible strains called R and M , respectively. Here the results of an extensive survey for dysgenic potential in Drosophila melanogaster strains are reported. Striking temporal trends in the distribution of strains were observed with respect to the two transposable element systems; in particular, the frequency of R and M strains is positively correlated with laboratory age. In recent tests of strain samples, those collected from nature about 50 years ago were the earliest observed to possess I characteristics. The I type was increasingly frequent in samples from strains more recently originating in the wild. This type is apparently ubiquitous in present day natural populations. the P type was not found in strain samples collected before 1950, and collections made subsequently showed increasing frequencies of P -factor activity with decreasing laboratory age. Marked geographical patterns are documented in the contemporary worldwide distribution of variant strains within the P - M system. M strains are currently fairly common in natural populations from various parts of the world, except on the American continent where they are rare. The degree and distribution of quantitative variation within M and P strain categories is related to their time of origin in the wild. The implications of these results are discussed in relation to the hypothesis that hybrid dysgenesis determinants have evolved recently in natural populations and to an alternative hypothesis of laboratory evolution.

We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method.

P elements, a class of mobile genetic elements that cause hybrid dysgenesis in Drosophila melanogaster, have thus far been shown to occur only in this species. Using whole genome blot analysis, we present evidence which indicates that sequences homologous to D. melanogaster P elements also occur in Drosophila paulistorum, a distant relative. D. paulistorum is considered a species complex, consisting of six known incipient species or semispecies. All six semispecies possess P element homologous sequences. We further show that there is conservation of restriction enzyme recognition sites between the D. melanogaster and D. paulistorum P element sequences. The presence of P elements in these two species may clarify the roles of recent invasion and rapid loss in the temporal distribution of P elements in Drosophila.

In the P-M system of hybrid dysgenesis of Drosophila melanogaster, some M strains possess chromosomal P elements. The chromosomes from one of these pseudo-M strains reduced the instability of a P element-insertion mutation of the singed bristle locus. We suggest that this reduction is an indication of competition for a transposase that binds to the P elements on the chromosomes from the pseudo-M strain as well as to the P elements at the singed locus; the pseudo-M strain's P elements might therefore be said to titrate the transposase, reducing its availability to interact with the P elements at the singed locus. We hypothesize that a similar mechanism regulates the movement of P elements in the P strains of D. melanogaster, although in this case we propose that the titrating elements are extrachromosomal and that they are generated by the action of the transposase itself.