Structure and associated mutational effects of the cysteine proteinase (CP1) gene of Drosophila melanogaster.

The complete structure of the cysteine proteinase (CP1) gene reveals two large 5' introns as well as a small third intron. Deletion studies have shown that null mutations for the locus are female sterile with partial male sterility as well as wing and pigmentation effects. Null alleles can be produced by either deletions to the left or deletions to the right of a P element insertion in the long second intron of the gene. A nearby phenylalanyl tRNA synthetase gene (Pts) was also identified.

P-element-induced male recombination and gene conversion in Drosophila.

A P-element insertion flanked by 13 restriction fragment length polymorphism (RFLP) marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the nonrearranged male recombinants. Most recombination events can be explained by the "hybrid element insertion" model, but, for those lacking a duplication or deletion, a second step involving double-strand gap repair must be postulated to explain the distribution of crossover points.

Flanking duplications and deletions associated with P-induced male recombination in Drosophila.

We studied P element-induced recombination in germline mitotic cells by examining the structure of the recombinant chromosomes. We found that most recombinants retain a mobile P element at the site of the recombination, usually with either a deletion or a duplication immediately adjacent to the P end at which the crossover occurred. The sizes of these deletions and duplications ranged from a few base pairs to well over 100 kb. These structures fit the "hybrid element insertion" (HEI) model of male recombination in which the two P-element copies on sister chromatids combine to form a "hybrid element" whose termini insert into a nearby position on the homologue. The data suggest that P-induced recombination can be used as an efficient means of generating flanking deletions in the vicinity of existing P elements. These deletions are easily screened using distant flanking markers, and they can be chosen to extend in a given direction depending on which reciprocal recombinant type is selected. Furthermore, the retention of a mobile P element allows one to extend the deletion or generate additional variability at the site by subsequent rounds of recombination.

Long-range cis preference in DNA homology search over the length of a Drosophila chromosome.

P element-induced chromosome breakage on the X chromosome of Drosophila melanogaster was repaired six times more frequently when a homologous template was located anywhere on the X chromosome rather than on an autosome. Cis-trans comparisons confirmed that recombinational repair was more frequent when the interacting sequences were physically connected. These results suggest that the search for homology between the broken ends and a matching template sequence occurs preferentially in the cis configuration. This cis advantage operates over more than 15 megabases of DNA.

Type I repressors of P element mobility.

We describe here a family of P elements that we refer to as type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the canonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects were very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3' boundary of a functional type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the type I repressors form a structurally distinct group that does not include more extensively deleted repressor elements such as the KP element described previously.

Targeted gene replacement in Drosophila via P element-induced gap repair.

Transposable elements of the P family in Drosophila are thought to transpose by a cut-and-paste process that leaves a double-strand gap. The repair of such gaps resulted in the transfer of up to several kilobase pairs of information from a homologous template sequence to the site of P element excision by a process similar to gene conversion. The template was an in vitro-modified sequence that was tested at various genomic positions. Characterization of 123 conversion tracts provided a detailed description of their length and distribution. Most events were continuous conversion tracts that overlapped the P insertion site without concomitant conversion of the template. The average conversion tract was 1379 base pairs, and the distribution of tract lengths fit a simple model of gap enlargement. The conversion events occurred at sufficiently high frequencies to form the basis of an efficient means of directed gene replacement.

A stable genomic source of P element transposase in Drosophila melanogaster.

A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable. Its transposase activity is higher than that of an entire P strain, but it rarely undergoes internal deletion, excision or transposition. This element was constructed by F. Laski, D. Rio and G. Rubin for other purposes, but we have found it to be useful for experiments involving P elements. We demonstrate that together with a chromosome bearing numerous nonautonomous elements it can be used for P element mutagenesis. It can also substitute efficiently for "helper" plasmids in P element mediated transformation, and can be used to move transformed elements around the genome.

Somatic effects of P element activity in Drosophila melanogaster: pupal lethality.

Nonautonomous P elements normally excise and transpose only when a source of transposase is supplied, and only in the germline. The germline specificity depends on one of the introns of the transposase gene which is not spliced in somatic cells. To study the effects of somatic P activity, a modified P element (delta 2-3) lacking this intron was used as a source of transposase. Nonautonomous P elements from a strain called Birmingham, when mobilized in somatic cells by delta 2-3, were found to cause lethality, although neither component was lethal by itself. The three major Birmingham chromosomes acted approximately independently in producing the lethal effect. This lethality showed a strong dependence on temperature. Although temperature sensitivity was limited to larval stages, the actual deaths occurred at the pupal stage. Survivors, which could be recovered by decreasing the temperature or by reducing the proportion of the Birmingham genome present, often showed multiple developmental anomalies and reduced longevity reminiscent of the effects of cell death from radiation damage. Although the genetic damage occurred in dividing imaginal disc cells, the phenotypic manifestations--death and abnormalities--are not observed until later. The survivors also showed gonadal dysgenic (GD) sterility, a well-known characteristic of P-M hybrid dysgenesis. To explain these findings, we suggest that pupal lethality and GD sterility are both caused by massive chromosome breakage in larval cells, resulting from excision and transposition of genomic P elements acting as substrate for the transposase.

Formation of chromosome rearrangements by P factors in Drosophila.

We studied a collection of 746 chromosome rearrangements all induced by the activity of members of the P family of transposable elements in Drosophila melanogaster. The chromosomes ranged from simple inversions to complex rearrangements. The distribution of complex rearrangement classes was of the kind expected if each rearrangement came about from a single multibreak event followed by random rejoining of chromosome segments, as opposed to a series of two-break events. Most breakpoints occurred at or very near (within a few hundred nucleotide pairs) the sites of preexisting P elements, but these elements were often lost during the rearrangement event. There were also a few cases of apparent gain of P elements. In cases in which both breakpoints of an inversion retained P elements, that inversion was capable of reverting at high frequencies to the original sequence or something close to it. This reversion occurred with sufficient precision to restore the function of a gene, held-up-b, which had been mutated by the breakpoint. However, some of the reversions had acquired irregularities at the former breakpoints that were detectable either by standard cytology or by molecular methods. The revertants themselves retained the ability to undergo further rearrangements depending on the presence of P elements. We interpret these results to rule out the simplest hypotheses of rearrangement formation that involve cointegrate structures or homologous recombination. The data provide a general picture of the rearrangement process and its possible relationship to transposition.

Identifying P factors in Drosophila by means of chromosome breakage hotspots.

A syndrome of germline abnormalities in Drosophila melanogaster called hybrid dysgenesis is thought to be caused by transposable genetic elements known as P factors. Several lines of evidence presented here show that the chromosomal positions of at least some P factors can be identified as points of frequent chromosome breakage (hotspots). Starting with a strain (pi 2) in which four hotspots had been identified on the X chromosome, we found individual hotspots vanished when their part of the chromosome was replaced by the homologous part from a strain known to lack P factors. All hotspots in the non-substituted parts of the chromosome remained functional, indicating that they can act autonomously. We also observed a new breakage site coinciding with the appearance of an unstable mutation at the singed bristle locus (snW). This mutation was dysgenesis-induced, and previous genetic evidence suggested that it was caused by the insertion of a P factor at that locus. We also present preliminary evidence for rapid scrambling of the positions of hotspots under certain conditions, and we describe a new procedure for efficiently determining the positions of hotspots on a given chromosome.

Components of hybrid dysgenesis in a wild population of Drosophila melanogaster.

Hybrid dysgenesis is a condition found in certain interstrain hybrids of Drosophila melanogaster caused by the interaction of chromosomal and cytoplasmic factors. Germ-line abnormalities, including sterility, high mutability and male recombination, appear in the affected individuals. There are at lest two distinct systems of hybrid dysgenesis. We examined a Wisconsin wild population in two consecutive years to determine the distribution of the chromosomal P factor and the extrachromosomal M cytotype that together cause one kind of hybrid dysgenic sterility. The P factor was found to be very common in the population, with all three major chromosomes being polymorphic for it. This polymorphism was strongly correlated with variability for male recombination elements, suggesting that these two traits are part of the same system of hybrid dysgenesis. There was a slight tendency for the P factor to be lost in lines taken from this population and inbred in the laboratory for many generations. A large-scale search for the M cytotype, which causes susceptibility to the P factor, showed that it is present in the population at only very low frequencies. Further evidence that the population is mostly immune to the action of the P factor was our finding of a general lack of dysgenic sterility in the wild flies themselves. However, we were able to isolate several wild strains that consistently showed the M cytotype. In some cases, the frequency of the M cytotype could be maintained in these lines, but it could not usually be increased by artificial selection. Some possible consequences of hybrid dysgenesis for the evolutionary biology of Drosophila are suggested.

Pleiotropic Effects on Fitness of Mutations Affecting Viability in DROSOPHILA MELANOGASTER.

The relative viabilities and fitnesses of wild-type second chromosomes in heterozygous condition were determined. Joint analysis of these permitted an estimation of a parameter that relates the viability effect of a mutation to its effect on fitness as a whole. For newly arisen mutations, the estimate was slightly greater than one, indicating that the reductions in viability caused by these mutations are associated with reductions in other components of fitness. For mutations from an equilibrium population, the estimate of the parameter was near zero, implying that the deleterious viability effects of these mutations are compensated by improvements in other aspects of fitness.

Hybrid dysgenesis in Drosophila melanogaster: the biology of female and male sterility.

High levels of female and male sterility were observed among the hybrids from one of the two reciprocal crosses between a wild strain of D. melanogaster known as pi2 and laboratory strains. The sterility, which is part of a common syndrome called hybrid dysgenesis, was found to be associated with the rudimentary condition of one or both of the ovaries or testes. All other tissues, including those of the reproductive system were normal, as were longevity and mating behavior. The morphological details of the sterility closely mimic the agametic condition occurring when germ cells are destroyed by irradiation or by the maternal-effect mutation, grandchildless. We suggest that sterility in hybrid dysgenesis is also caused by failure in the early development of germ cells. There is a thermo-sensitive period beginning at approximately the time of initiation of mitosis among primordial germ cells a few hours before the egg hatches and ending during the early larval stages. Our results suggest that hybrid dysgenesis, which also includes male recombination, mutation and other traits, may be limited to the germ line, and that each of the primordial germ cells develops, or fails to develop, independently of the others. This hypothesis is consistent with the observed frequencies of unilateral and bilateral sterility, with the shape of the thermosensitivity curves and with the fact that males are less often sterile than females. The features of this intraspecific hybrid sterility are found to resemble those seen in some interspecific Drosophila hybrids, especially those from the cross D. melanogaster X D. simulans.