Efficient and Dispersed Local P Element Transposition from Drosophila Females

We have investigated how Drosophila P element insertions are distributed in the chromosomal region near their starting site. A single P element residing in the euchromatin of minichromosome Dp1187 was mobilized following a cross to the Δ2-3 (99B) strain, and progeny bearing transpositions were identified with a minimum of bias by performing Southern blots on progeny. Approximately 1-2% of all progeny minichromosomes contained new insertions. Many of these ``local transpositions' landed very close to or within the starting P element; however, nearly 1% of all progeny chromosomes contained new insertions 1-180 kb from the donor element. More local insertions were observed in the progeny of females than from male parents, and most occurred in a preferred orientation relative to the starting element. These observations suggested that donor elements are frequently excised and reinserted locally without ever dissociating from a transposition complex. The high frequency and diverse distribution of local transpositions recovered from females suggested that the efficiency of insertional mutagenesis can be significantly enhanced by using a starting P element(s) located near the target of interest.     

Preferential Transposition of Drosophila P Elements to Nearby Chromosomal Sites

Two different schemes were used to demonstrate that Drosophila P elements preferentially transpose into genomic regions close to their starting sites. A starting element with weak rosy(+) marker gene expression was mobilized from its location in the subtelomeric region of the 1,300-kb Dp1187 minichromosome. Among progeny lines with altered rosy(+) expression, a much higher than expected frequency contained new insertions on Dp1187. Terminal deficiencies were also recovered frequently. In a second screen, a rosy(+)-marked element causing a lethal mutation of the cactus gene was mobilized in male and female germlines, and viable revertant chromosomes were recovered that still contained a rosy(+) gene due to an intrachromosomal transposition. New transpositions recovered using both methods were mapped between 0 and 128 kb from the starting site. Our results suggested that some mechanism elevates the frequency 43-67-fold with which a P element inserts near its starting site. Local transposition is likely to be useful for enhancing the rate of insertional mutation within predetermined regions of the genome.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these “local” insertions was actually within ～ 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.     

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.     

An Inverse Pcr Screen for the Detection of P Element Insertions in Cloned Genomic Intervals in Drosophila Melanogaster

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of ``local' P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of ~10(-4) are distributed every 40-50 kb.     

P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor.

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.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these local insertions was actually within 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Studies on the Rate and Site-Specificity of P Element Transposition

A single genetically marked P element can be efficiently mobilized to insertionally mutagenize the Drosophila genome. We have investigated how the structure of the starting element and its location along the X chromosome influenced the rate and location of mutations recovered. The structure of two P[rosy+] elements strongly affected mobilization by the autonomous "Jumpstarter-1" element. Their average transposition rates differed more than 12-fold, while their initial chromosomal location had a smaller effect. The lethal and sterile mutations induced by mobilizing a P[rosy+] element from position 1F were compared with those identified previously using a P[neoR] element at position 9C. With one possible exception, insertion hotspots for one element were frequently also targets of the other transposon. These experiments suggested that the genomic location of a P element does not usually influence its target sites on nonhomologous chromosomes. During the course of these experiments, Y-linked insertions expressing rosy+ were recovered, suggesting that marked P elements can sometimes insert and function at heterochromatic sites.     

Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En-I transposon system

Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer-Inhibitor (En-I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

The Saccharomyces retrotransposon Ty5 influences the organization of chromosome ends.

Retrotransposons are ubiquitous components of eukaryotic genomes suggesting that they have played a significant role in genome organization. In Saccharomyces cerevisiae, eight of 10 endogenous insertions of the Ty5 retrotransposon family are located within 15 kb of chromosome ends, and two are located near the subtelomeric HMR locus. This genomic organization is the consequence of targeted transposition, as 14 of 15 newly transposed Ty5 elements map to telomeric regions on 10 different chromosomes. Nine of these insertions are within 0.8 kb and three are within 1.5 kb of the autonomously replicating consensus sequence in the subtelomeric X repeat. This suggests that the X repeat plays an important role in directing Ty5 integration. Analysis of endogenous insertions from S.cerevisiae and its close relative S.paradoxus revealed that only one of 12 insertions has target site duplications, indicating that recombination occurs between elements. This is further supported by the observation that Ty5 insertions mark boundaries of sequence duplications and rearrangements in these species. These data suggest that transposable elements like Ty5 can shape the organization of chromosome ends through both transposition and recombination.     

Creation of<Emphasis Type="Italic"> EcR</Emphasis> isoform-specific mutations in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> via local P element transposition,imprecise P element excision,and male recombination

Collections of single P transposable-element insertion strains that currently inactivate more than 25% of essential Drosophila genes have proven to be a valuable tool for genome research in Drosophila melanogaster. For genes unrepresented in these collections, strategies including local P element transposition and transposase-induced imprecise excision can be used to inactivate or delete the gene of interest. Here we report our use of local P element transposition followed by imprecise P element excision and transposase-induced male recombination to generate two deficiencies specific for the EcR-A isoform of the ecdysone receptor (EcR) gene, and four larger deficiencies likely to affect multiple EcR functions. We also report here the determination of sequences flanking six EcR-B deficiencies generated in a previous imprecise excision screen. EcR-A encodes one of a family of three related nuclear receptor proteins that, together with the heterodimer partner USP, mediate ecdysone signaling during Drosophila development. Our results delineate sequences required in vivo for EcR-A function, as well as identifying EcR-A intron 1 sequences that are not essential for EcR function.Communicated by G. Reuter     

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.     

Linked and Unlinked Transposition of a Genetically Marked Dissociation Element in Transgenic Tomato

We have introduced a genetically marked Dissociation transposable element (Ds(neo)) into tomato. In the presence of Ac transposase, Ds(neo) excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Ds(neo) (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Ds(neo), we examined the genomic distribution of Ds(neo) receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Ds(neo) elements was conducted in two tomato families, derived from independent primary transformants each bearing Ds(neo) within a T-DNA at a unique position in the genome. Transposition of Ds(neo) generated clusters of insertions that were positioned on several different tomato chromosomes. Ds(neo) insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Ds(neo) transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Ds(neo). We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Ds(neo), is an effective tool for transposon tagging experiments in tomato.