P Element Transposition in Drosophila Melanogaster: An Analysis of Sister-Chromatid Pairs and the Formation of Intragenic Secondary Insertions during Meiosis

The gap-repair model proposes that P elements move via a conservative, ``cut-and-paste' mechanism followed by double-strand gap repair, using either the sister chromatid or homolog as the repair template. We have tested this model by examining meiotic purturbations of an X-linked ry(+) transposon during the meiotic cycle of males, employing the mei-S332 mutation, which induces high frequency equational nondisjunction. This system permits the capture of both sister-X chromatids in a single patroclinous daughter. In the presence of P-transposase, transpositions within the immediate proximity of the original site are quite frequent. These are readily detectible among the patroclinous daughters, thereby allowing the combined analysis of the transposed element, the donor site and the putative sister-strand template. Molecular analysis of 22 meiotic transposition events provide results that support the gap-repair model of P element transposition. Prior to this investigation, it was not known whether transposition events were exclusively or predominantly premeiotic. The results of our genetic analysis revealed that P elements mobilize at relatively high frequencies during meiosis. We estimated that approximately 4% of the dysgenic male gametes have transposon perturbations of meiotic origin; the proportion of gametes containing lesions of premeiotic origin was estimated at 32%.     

Polymorphisms on the right arm of yeast chromosome III associated with Ty transposition and recombination events.

The region of Saccharomyces cerevisiae chromosome III centromere-distal to the PGK gene is the site of frequent chromosome polymorphisms. We have sequenced this region from fragments of chromosome III isolated from three different yeast strains, GRF88, CN31C and CF4-16B. The sequence analysis demonstrates that these polymorphisms are associated with the presence of Ty and delta elements and defines a region of the chromosome which is a hot-spot for transposition events (the RAHS). The three strains can be arranged into a logical evolutionary series in which successive transposition and recombination events insert Ty elements and fuse them with consequent deletions of chromosome and of transposon sequences. The influence of such events on yeast genome evolution is discussed.     

A P Element Chimera Containing Captured Genomic Sequences Was Recovered at the Vestigial Locus in Drosophila following Targeted Transposition

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.     

P Element-Mediated in Vivo Deletion Analysis of White-Apricot: Deletions between Direct Repeats Are Strongly Favored

We have isolated and characterized deletions arising within a P transposon, P[hsw(a)], in the presence of P transposase. P[hsw(a)] carries white-apricot (w(a)) sequences, including a complete copia element, under the control of an hsp70 promoter, and resembles the original w(a) allele in eye color phenotype. In the presence of P transposase, P[hsw(a)] shows a high overall rate (approximately 3%) of germline mutations that result in increased eye pigmentation. Of 234 derivatives of P[hsw(a)] with greatly increased eye pigmentation, at least 205 carried deletions within copia. Of these, 201 were precise deletions between the directly repeated 276-nucleotide copia long terminal repeats (LTRs), and four were unique deletions. High rates of transposase-induced precise deletion were observed within another P transposon carrying unrelated 599 nucleotide repeats (yeast 2μ FLP; recombinase target sites) separated by 5.7 kb. Our observation that P element-mediated deletion formation occurs preferentially between direct repeats suggests general methods for controlling deletion formation.     

Is10 Promotes Adjacent Deletions at Low Frequency

Some transposable elements move by a replicative mechanism involving cointegrate formation. Intramolecular cointegration can generate a product called an ``adjacent deletion' in which a contiguous chromosomal segment adjacent to the transposon is deleted while the element responsible remains intact. Insertion sequence IS10 is thought to transpose by a nonreplicative mechanism. In the simplest models, nonreplicative transposition cannot give rise to an adjacent deletion because an intrinsic feature of such transposition is excision of the IS element from the donor location. We report here that IS10 can generate adjacent deletions, but at a frequency which is approximately 1/30th the frequency of transposition for the same element. We suggest that these deletions might arise either by nonreplicative transposition events that involve two IS10 elements located on sister chromosomes or by aberrant nonreplicative events involving cleavage and ligation at only one end of the element.     

Interaction of mobile elements P and mdg3 in Drosophila melanogaster: genetic aspects

It was found earlier that two unstable sn mutants isolated from natural populations are connected with insertion of mobile element mdg3 into the 7D1-2 region where singed gene (1-21.0) is localised. From two original sn mutants, a series of unstable sn alleles, both mutant and normal for phenotype, was extracted. Then we studied, how they change the mutation rate in germinal and somatic cells of different hybrids with pi 2 stock having P cytotype and active P elements in the chromosomes. Addition of P chromosomes, independently of the background of cytoplasm, proved to reduce the sn instability. The level of sn mutability was decreased with increasing the dose of P chromosomes. It is suggested that mutation events are caused by transposition of mdg3 and that both mdg3 and P elements compete for the same cellular factor, capable of activation of transposition process.     

Deletions and Amplifications of Tandemly Arranged Ribosomal 5s Genes Internal to a P Element Occur at a High Rate in a Dysgenic Context

We observed unusual kinds of rearrangements within tandemly clustered 5S genes internal to a P element in dysgenic context. Rearranged P transposons, initially containing eight 5S genes, were found to display discrete numbers of 5S genes, from 4 up to 17 units. Precise deletions and amplifications occurred at a high rate (40%), at both original and new insertion sites. These events can be explained by a ``cut and paste' transposition model. Possible links between rearrangements due to dysgenic-like processes and concerted evolution are discussed.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site.

A general approach for isolating large nested deletions in P1 artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) by retrofitting with a loxP site-containing Tn10 mini-transposon is described. Cre-mediated recombination between the loxP site existing in these clones and one introduced by transposition leads to deletions and inversions of the DNA between these sites. Large deletions are selectively recovered by transducing the retrofitted PAC or BAC clones with P1 phage. The requirement that both loxP sites in the cointegrate be packaged into a P1 head ensures that only large deletions are rescued. PCR analyses identified these deletions as products of legitimate recombination between loxP sites mediated by Cre protein. BACs produce deletions much more efficiently than PACs although the former cannot be induced to greater than unit copy in cells. Mammalian cell-responsive antibiotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis. Most importantly, the loxP site retrofitting and P1 transduction can be performed in the same bacterial host containing these clones directly isolated from PAC or BAC libraries. These procedures should facilitate physical and functional mapping of genes and regulatory elements in these large plasmids.     

Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster

Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system.     

Distribution and structure of cloned P elements from the Drosophila melanogaster P strain pi 2.

P transposable elements of Drosophila melanogaster cloned from the strong P strain pi 2 have been analysed. The structures and chromosomal locations of 26 of the 30-50 elements estimated to be present in pi 2 have been determined. At one location two elements are inserted 100 base pairs (bp) apart, and in a second location two elements are only separated by the 8 bp duplicated upon P-element insertion. In addition to 2.9 kilobase-pair (kbp) elements, elements with 14 different internal deletions from 1.3 to 2.3 kbp in size have been isolated. There are 7 copies of the 2.9 kbp element, 2 copies each of 5 internally deleted elements and a single copy of 9 internally deleted elements. One of the elements found twice is the KP element, which may play a role in the regulation of hybrid dysgenesis in strains which contain many copies of this element. Apart from internal deletions the elements are extremely homogeneous in DNA sequence, with only 2 single base polymorphisms detected twice each in over 16 kbp of P-element sequence. Although transpositions are infrequent in an inbred P cytotype strain such as pi 2, the distribution of these cloned elements indicates that when the genomic library was made, the strain was polymorphic with respect to element location. The distribution and structures of the element are discussed with respect to models for regulation of P-element transposition.     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.     

Potential Role of Transposable Elements in the Rapid Reorganization of the Fusarium oxysporum Genome

The activity of several families of transposable elements (TEs) in the genome of Fusarium oxysporum represents a potential source of karyotypic instability. We investigated transposon-mediated chromosome rearrangements by analyzing the karyotypes of a set of strains in which transposition events had occurred. We uncovered exceptional electrophoretic karyotype (EK) variability, in both number and size of chromosomal bands. We showed that EK differences result from chromosomal translocations, large deletions, and even more complex rearrangements. We also revealed many duplicated chromosomal regions. By following transposition of two elements and analyzing the distribution of different families of TEs on whole chromosomes, we find (i) no evidence of chromosomal breakages induced by transposition, (ii) a clustering of TEs in some regions, and (iii) a correlation between the high level of chromosomal polymorphism and the concentration of TEs. These results suggest that chromosome length polymorphisms likely result from ectopic recombination between TEs that can serve as substrates for these changes.     

Independence of excision frequency and transposition frequency of P element in Drosophila melanogaster.

Although a family of transposon, P elements, are used as tools for molecular genetics in Drosophila melanogaster, the molecular details and mechanism of their mobilization process have not been studied extensively. In particular, the relationship between excision and transposition is little understood. We have previously produced a transgenic fly with a P element insertion that is nonautonomous (stable without transposase) and is highly-transposable in the presence of transposase. Using this insertion, we traced its mobilizations following introduction of a stable transposase source. We found a strain that has a 26-bp tandem repeat at the end of the original P element insertion. The 26-bp repeat reduced the frequency of excision although the frequency of transposition was not altered. Our results indicate independence of transposition from excision and importance of terminal repeat in excision.     

Developmental profile of P element transposition inDrosophila somatic cells

Transposition of the P element duringDrosophila ontogenesis was monitored. A modified P element was transposed by the PΔ2-3 transposase source. P elements inserted into the genome were cloned by the plasmid rescue at various developmental stages of the G1 hybrid to trace events in somatic cells. The transposed elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P elements. Transposition began from the late embryonic stage, but occurred rarely. Frequent transposition was observed from the late third instar to early pupal stage. From these results, transposition of the P element would appear to be affected by the developmental state of somatic host cells.     

ModuleOrganizer: detecting modules in families of transposable elements

Background  

Most known eukaryotic genomes contain mobile copied elements called transposable elements. In some species, these elements account for the majority of the genome sequence. They have been subject to many mutations and other genomic events (copies, deletions, captures) during transposition. The identification of these transformations remains a difficult issue. The study of families of transposable elements is generally founded on a multiple alignment of their sequences, a critical step that is adapted to transposons containing mostly localized nucleotide mutations. Many transposons that have lost their protein-coding capacity have undergone more complex rearrangements, needing the development of more complex methods in order to characterize the architecture of sequence variations.     

Targeted Transposition at the Vestigial Locus of Drosophila Melanogaster

Targeted transposition is the replacement of one P element with another. We are exploiting this unique property of P elements to study the complex regulatory domain of the Dopa decarboxylase (Ddc) gene in Drosophila melanogaster. P element constructs targeted to the same site in the genome will be subjected to the same position effect. This allows the subtle effects typical of most mutations in the Ddc regulatory region to be measured in the absence of the variable influences of position effects which are associated with the current method of germline transformation. We have investigated some of the parameters affecting targeted transposition of a Ddc transposon, P[Ddc], into a P element allele at the vestigial locus. These events were detected by an increased mutant vg phenotype. The location of the donor transposon in cis or in trans to the target had little effect on the frequency of targeting. Likewise, the mobility of different donor elements, as measured by their rate of transposition to a different chromosome, varied nearly 20-fold, while the rate of targeted transposition was very similar between them. All targeted alleles were precise replacements of the target P element by P[Ddc], but in several cases the donor was inserted in the opposite orientation. The targeted alleles could be described as the result of a replicative, conversion-like event.     

Evidence for a role of translational frameshifting in the expression of transposition activity of the bacterial insertion element IS1

The bacterial insertion element IS1 contains two essential open reading frames, insA and insB, arranged in tandem. We have introduced a number of site-specific mutations into the region including the 3'-terminal region of insA, the region between insA and insB, and the ATG codon at the start of insB. Relative transposition activities of mutant and wild-type elements were determined using a modified in vivo cointegration assay. The results support the hypothesis that a translational (-1) frameshift occurring in the 3'-terminal region of insA and linking insB to insA is responsible for the synthesis of the active IS1 transposition enzyme. Further results with IS1 elements containing internal deletions are in agreement with a role of the normally terminated insA product as an inhibitor of transposition.