Summary The P family of transposable genetic elements is thought to be a recent addition to the Drosophila melanogaster genome. New evidence suggests that the elements came from another Drosophila species, possibly carried by parasitic mites. The transposition mechanism of P elements involves DNA gap repair which may have facilitated their rapid spread through D. melanogaster worldwide. These results provide new insight into the process of a transposon’s invasion into a new species and the potential risk of extinction such an invasion might entail. Introduction Transposable genetic elements are gene-size segments of DNA with the special ability to jump from place to place on the chromosomes. They are typically found in many copies scattered about the genome, and their positions are widely variable between individuals of a given species. Since the pioneering work of McClintock on the ‘controlling elements’ of maize, transposable elements have been found to be a ubiquitous and integral part of the genome of virtually all species that have been studied genetically. Much has been learned about the DNA structures and transposition mechanisms of transposable elements(’).However, questions dealing with how they evolved, how they spread within and between species, and what roles they play - positive or negative - in the biology of their host organisms, have proven more difficult to answer. Jn Drosophila melanogaster, transposable elements are thought to constitute 10-20% of the genome(2,3). A typical individual may carry representatives of as many as SO transposon families with an average of 100 genomic copies of each family. These families of elements can be classified according to their repeat structures and transposition mechanisms. The largest group is that or the retrovirus-like elements which are characterized by direct sequence repeats of several hundred base pairs at their termini. Such elements resemble degenerate RNA viruses that cannot form infectious particles but retain the capability for making new copies of themselves via RNA intermediates. Another type, the retroposons, are also thought to employ RNA as a tr‘ansposition intermediate and utilize reverse transcriptase. They lack terminal repeats and have an AT-rich scquence at their 3’ end. A third class of transposon, the Ac-like elements, are

named after Activator, thc first transposable element identified by McClintock. These elements, which have inverted terminal repeats of less than 100 base pairs, are thought to transpose by a DNA-only mechanism. The P family of elements is the most extensively studied of the Ac-like elements in Dro~ophila(~). There are two main reasons for the special attention given to P elements: First, they have great utility for molecular biological techniques. They can be used to clone genes by a method called transposon tagging(5,@.and they can serve as vectors for introducing cloned sequenccs into the germline by transformation(’, P elements can be outfitted with reporter genes and used to identify the timing and tissue distribution of the expression of genes that happen to lie near the insertion site(”. Most recently, P elements have been used to create double-strand DNA breaks to allow experimenters to replace the flanking DNA with modified sequenced“’). A second reason for the extra attention focused on P elements is their unusual population biology. P elements were discovered in the course of investigation of the phenomenon of ‘hybrid dysgenesis’, a syndrome of abnormal germline traits, such as elevated mutability and recombination, that was seen in the hybrids from certain inter-strain crosses(]I). This phenomenon served to split the species into ‘P strains’ and ‘M strains’, depending on whether the strain contribuled paternally or maternally to the dysgenic hybrids. Genetic mapping indicated that the responsible factors were located at multiple positions on the chromosomes of the P strains, and these positions varied between unrelated P strains(l2>I3j. These observations suggested that a family of transposable elements, called P factors or P elements, was present in the P strain chromosomes but not in the M strains. The cross MO x P 6 mobilized the elements in the germline of the progeny to cause the dysgenic traits. Molecular cloning of P elements eventually confirmed this suggestion‘14).

Which Came First,’P Strains or M Strains? The ability of a transposori family to produce at least partial reproductive isolation between two sets o f strains was of considerable interest to population biologists and evolutionists. Especially intriguing was the fact that natural populations consisted exclusively of P element-bearing strains whereas old laboratory stocks were M strains. One possibility was that the laboratory strains somehow lose their P elcments during several thousand generations of artificial culture conditions(I5).The alternative was the ‘recent invasion’ hypothesis put forth by Kidwel1(l6, who suggested that P elements are a new addition to the D.melanogaster genome. She postulated that P elements were rare or absent in natural populations in the years 1910 to 1950 when the first experimental cultures were established. The main argument against the recent invasion hypothesis was that the addition of a new tranvposon family to thc genome would have to be an extremely rare event on any human time scale(’8). Since only about SO transposon families have accumulated in the evolution of the species, the probability that a new invasion could have taken place in Drosophila or any other well-studied species within the last

100 years is negligible. Howcver, there were also strong arguments against the recent loss of P elements from laboratory strains(16,17). First, no one had ever observed a case of a P strain switching to an M strain; by the time hybrid dysgenesis had been discovcred the two types of strains seemed to be stably established. Moreover, laboratory populations that were set up with a mixture of P and M types tendcd to evolve toward P within a few generation^""^'). Evidence for Recent Invasion Molecular studies of P element distributions eventually shifted the weight of evidence to favor Kidwell’s recent invasion hypothesis. P dements were found to be heterogeneous with the majority of them being nonautonomous, meaning that they could not produce the transposase needed for their own mobility but could jump if an autonomous element were present@ 22. 23). Such a mixture suggests that the acquisition of P elements is irreversible in a species since a population fixed for nonautonomous elements would lack transposasc and hence be stable. Moreover, the heterogeneity among P elements is almost entirely due to deletions rather than base substitutions(22,24). The rclative lack of single-base changes is consistent with a very recent bottleneck in the P element population, as would be expected if the elements had invaded the genome recently. The most powerful argument for recent invasion came from the analysis or P dements elsewhere in the genus Drosophila. The species most closely related to D. melanogaster, especially D. sirnuluns, D. sechellia and U. mauritiana, were found to lack P elements entirely, whereas several more distantly related species carried transposable elements with sequences quite similar to P (Fig. 1). Such a distribution suggests that P elements were acquired by D. m e h o g a s t e r from a more distant relative through a horizontal transfer event of some kind. The transfer must have happened during the estimated 2 million years since the divergence of the inelanogaster sibling species. The species with the best-matching P elements was D. willistoni. A P element from this species was found to match all but one of the 2907 nucleotides of the D. melanogaster P element(’”. Such a remarkable degree of conservation can only mean that the two elements have a common ancestor in the recent past - certainly much less than one million years ago, and possibly only a few decades ago, as would be required by thc recent invasion hypothesis.

Why Was the Invasion So Recent? The biggest weakness of the recent invasion model remained the unacceptably low probability that the irreversible addition of a new transposon to the genome could occur within a time frame that is infinitesimal on the evolutionary time scale. However, the identification of D. willistoni as the likely source of P elements provided an unexpected way around this dilemma. It came from the curious observation that insect collections obtained by entomologists in North America prior to the late 1800’s did not include any specimens of D. nielanogaster even though many less abundant

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dipteran species were included. Johnson(26)(see also Sturte~ a n t ( ~speculated ~)) that D. melanogaster, which is thought to have evolved in Westcrn is a newcomer to the Americas. His survey of insect collections led to the suggestion that the species was first introduced in the West Indies in the early 18OO’s, probably via slave ships. From there it spread to North and South America, and is now a predominant species throughout both continents. Meanwhile, D. willistoni is thought to have evolved in South and Central America, and is still endemic to those regions plus parts of Florida. If Johnson’s sccnario is correct, it would imply that D. willistoni and D. melanogaster would never have come into contact with each other until just prior to the time P elements are presumed to have been introduced into the D. melanogasler genome. Thus, the opportunity for P elements to invade the D. melanogaster genome was actually created by humm activity, and its occurrence in recent years need no longer be considered an unlikely event. With plausible explanations for where P elements came from and when they were introduced, there are still two important questions to be addressed: (1j How did P elements get from D. Miillistoni genome to D. melanogaster?; (2) Once

there, how did P elements spread through the global population of D. melanogaster?

Horizontal Transfer Fertile hybrids between D. rnelanogasrer and D. willisfoni have never been observed and are probably not possible. Therefore, we must assume that P elements got from one species to the other by a horizontal transmission event of some kind. Moving P elements between species can readily be achieved in the laboratory by microinjection of P elementbearing plasmid DNA into embryos prior to the cellular blastoderm stage(”). In the presence of transposase, P elements jump from the injected plasmid to the chromosomes of germline cells. Progeny with P elements are then recovered in the next generation. But could a similar process occur in nature? One possibility is that viruses might serve to shuttle transposons between species. When a virus infects a transposonbearing organism, the transposable element might enter the genome of the virus, which could then carry the element into cells of another species. The first step of this process has been documented in insects. Several cases in which a transposable element jumped into the genome of the lepidopteran baculovirus, Autographu culfornica, have been observed(”’). The virus was able to remain infectious with the transposon in its genome. However, the second step, in which the transposable element moves from the viral genome to a chromosome of a new host, has not been observed to date. More recently, parasitic mites have been implicated as potential carriers of P elements. Houck el aE.(31) grew a population of the mite Proctolaelaps regalis in a P strain culture. When they extracted DNA from these mites they found evidence of P element sequences. Controls consisting of P. regalis grown in an M strain culture were negative, as were tests with a different, non-parasitic, mite species grown in a P strain culture. These controls demonstrate that the mites acquired their P sequences from the flies. The experiments did not determine whether the P DNA was actually incorporated in the mite genome or merely present as undigested Drosophila tissue. It is also possible that the P sequences were present in a virus that had been acquired by the mite during feeding. In any casc, the authors point out that the mite’s mouth parts could niiinic a laboratory microinjection needle for intracellular transfer, Other features of P. regalis, such as its tendency to feed on multiple hosts in rapid succession and its presence in geographical locations shared by D. melarzoguster and D. willistoni, enhance its potential as a P element vector. In future experiments Houck et al. hope to detect actual mite-mediated interspecific transfer of P elements. It is possible, however, that the frequency of such events is too low to detect experimentally yet high enough to be important in nature. The Spread of P Elements Within the Species The horizontal transfer event presumably happened only once or a very small number of times. After that, the P elements had to establish a high copy number in the genome and spread throughout D. melanogaster, which was by that time a

cosmopolitan species. According to the reccnt invasion hypothesis, this spread must have occurred within the span of 30-40 years. In the standard theory of population genetics, the primary ways a gene can spread through a population are random drift and natural selection. Drift can be ruled out in this case because of the short time involved relative to the population number. Natural selection is also unlikely for several reasons. The only known phenotypic effects of P elements are deleterious; their mobilization results in sterility and increased mortality (32-34), and the resulting insertion mutations are usually harmful(3s).Furthermore, M strains suffer no detectable fitness disadvantage despite not having P elements. Therefore, the key t o the rapid spread of P elements probably lies in their transposition mechanism. One way a transposon might spread is by a replicative transposition mechanism. If the donor site is retained when a new copy is made a1 a recipicnt site, there is a net gain of one copy. However. the only eukaryotic dements known to transpose replicatively are those with RNA intermediates, which leaves out P elements. Some DNA-only elements in plants are thought to transpose non-replicatively during the DNA synthesis stage of the cell cycle. They tend to jump from chromosomal sites that have already replicated and land at unreplicated sites. Thus, when DNA synthesis is complete, therc is a gain of one copy of the element (reviewed in ref. 36). Recent work indicates that P elements use a different approach to increase their copy number. Analysis of reversion of insertion mutations showed that the precise loss of P elements was greatly enhanced by the presence of a homologous site lacking the e l e m c ~ ~ tThese ( ~ ~ J observations . led to a model of P element mobility in which non-replicative transposition leaves behind a double-strand DNA break at the donor site (Fig. 2). According to this model, a sister chromatid with a P element still at the donor site is available for use as a template to repair the break. In the process of this repair, the P element from the sister strand is copied hack to the donor site, leaving a net gain of one P element copy. The homolog-dependent loss is explained by cases in which the homologous chromosome must serve as the template because the sister strand is not used or not yet synthesized. Subsequent work has provided strong support for this model. Gloor et showed that an in vitro modified sequence could be used as the template, and that marker sites flanking the P insertion site were copied in to repair the P-induced gap. The authors concluded that P elements, and perhaps other Ac-like transposons, can make use of the cell’s own DNA repair machinery to increase their copy number without the aid of natural selection. With such an efficient mechanism, the spread of P elements through a spccies within a few decades is not unreasonable. Species that are essentially panmictic and have biparental inheritance are most susceptible to rapid transposon invasion(38), and L). melanoguster has both of these characteristics.

Summary and Further Questions The evidence reviewed above suggests the following

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Fig. 2. Gap repair model of P element transposition(", 37). A. Following the DNA synthesis phase of the cell cycle, a P element jumps non-replicatively, leaving a double-strand gap at the donor site. This gap might be widened by exonuclease ac(ivily. B . Repair of the gap is begun with the sister chromatid serving as a template. An intermediate structure is formed with two Holliday junctions. C. DNA synthesis and resolution of the Holliday junctions produces a new P clement copy at the donor site.

sequence of events: P elements were well established throughout the willistoni group species long before D. rnelanogaster made its appearance in the Americas via human activity. A horizontal transfer event, possibly facilitated by parasitic mites, moved one or more copies of the element to the D. melunogaster genome. This happened sometime in the late 19thor early 20" century. P elements, aided by their transposition mechanism coupled with DNA gap repair, then became established in the species worldwide. The only strains that failed to acquire P elements were the reproductively isolated laboratory cultures taken before the invasion was complete. This scenario, though plausible and consistent with all present data, still leaves several open questions. First, having concluded that P elements came to D. melanoguster from D. willistoni. we only move the question of the ultimate origin of the elements back one step. P elements probably pre-date the evolution of Drosophila, since P-like transposons have been found in other genera(39) and even outside the

Drosophilidae family(40).One clue concerning how transposons could have evolved is the finding that P elements can sometimes capture and mobilize genomic sequenced4'). Another open question is how important and frequent is the horizontal transfer of genetic material. The absence of P elements in D. simulans can be taken as an indication that horizontal transfer is a rare occurrcnce. D. sirnuluns, like D. melanogaster, is a cosmopolitan species which currently shares habitats with D. willisroni. Furthermore, microinjection experiments have shown that P elements can function in Lf. s i r n u l a n ~ ( ~Therefore, ~ - ~ ~ ) . the failure of P elements to invade D. simulans genome can best be attributed to the lack of horizontal transfer. Several other transposable element families have species distributions suggestive of horizonlal t r a n ~ f e r c ~ but ~ , ~the ~ )evidence , in these cases is less convincing than the case for P elements. Could ordinary, non transposable, DNA sequences be moved between species the same way'? Such an occurrcncc would be much less likely than the horizontal transfer of transposons. Note that in the above scenario, the P element's mobility was crucial not only for spreading the element through the species, but also for the horizontal transFer cvent itself. The DNA introduced into a germ cell by a parasitic mite still had to be integrated into the chromosomes somehow. Presumably, that integration took place by the P element transposition mechanism. Finally, what are the consequences for a species when its genome is invaded by a transposable element? One suggestion is that transposable elements could facilitate the evolution of the species by causing beneficial insertion niutat i ~ n s ( ~However, ~J. such mutations, which should be apparent as high-frequency insertion sites, have not been observed for P or other Drosophila transposons. Instead, the observed distributions of sites fit models in which transposition is balanced by opposing selection forces directed against deleterious element-induced mutations and chromosome [email protected] to this view, the number of elements reaches an equilibrium at which the avcrage fitness of the species is lower than it was prior to the invasion. The notion that transposons act as genetic parasites implies that the invasion of a new element places a species at an elevated risk for extinction. The extent of the risk would depend upon an array of parameters, such as the element's transposition rate, its insertional site specificity, and the demographics of the host species. Experimental manipulation of some of these parameters in laboratory Drosophila populations shows that conditions favoring extinction are readily obtained. Figure 3 shows an example in which P elements were introduced into a sib mating line whcre inbreeding would make the elimination of harmful insertion mutations much less efficient. Proliferation of the elements led to loss of the stock after 8 generations. Several repetitions of this experiment yielded a similar outcome, although the number of generations before extinction was variable(21). Regulation of the transposition rate is another important factor. P elements possess a complex regulation system, encoded by the elements themselves, which keeps their transposition rate low within P strains (reviewed in ref. 4). Without such a mechanism, the equilibrium copy number

generation

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Fig. 3. Spread of Pelemcnts in a sib-mating line(21).The data represcnl one of four independent cases in which rapid proliferation of P elements ended with extinction of the line. The number and chromosomal locations of 1' elements are shown for each generation of a line started with a single autonomous P element introduced by microinjection at cytological position 17B. Cytological divisions 1 to 100 are arrayed in numerical order along the horizontal lines. Each generation was started by a single pair mating, with a third individual selected at the larval stage for in situ hybridization of labeled P sequences to the polytene chromosomes. By generation 8 there were no larvae to spare for in situ hybridization, and no fertile adults were obtained for generation 9. The 5-generation time lag betwcen the initiation of the line and the appearance of the first new site might be the result of an abnormally low transposition rate of the 17B insertion site.

would be much higher, and the risk of extinction correspondingly greater. A second regulatory mechanism. which acts on the level of' RNA processing, prevents P mobility in somatic cells(49),thus minimizing the detrimental effects of transposition. Note that these regulatory mechanisms are presumed to have evolved in another species long before P elements were introduced into D. melunogaster, Perhaps it was fortunate for D. melunogu.ster, and for those of us who study the species, that the regulatory systems remained functional when P elements found themselves in their new host species.

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Possible horizontal tranrfer of Drosophila genes by the mite Proctolaelaps r e g o h . Science 253. 1125-1128. 32 Engels, W. R. and Preston, C. R. (1979). Hybrid dy5genesis in Drosophilu rnelanogusfer:The biology ofmale and remale sterility. Genetics 92, 161-175. 33 Ychaefer, R. E., Kidwell, M. G . and Fausto-Sterling, A. (1979). Hyhrid Ilysgcnesis in Drosophila ineIanogusuJlur.: morphological and cytological studies of ovarian dysgcncsis. Genctics92,1141-1152. 34 Engels, W. R., Benz, W. K., Preston, C. R., Graham, P. L., Phillis, R. W. and Kohertson, H. M. (1987). Somalic effects of P element activity in Drosophilu melanogasier: Pupal lethalily. Genetics 117.745-757. 35 Fitzpatrick, B. and Svcd, J. A. (1986). High levels of fitness niodifiets induced by hybrid dysgenesis in Drosophila rnelfinugasrer. Genet. Kf% 48, 89-94. 36 Fedoroff, N. (1989). Maire transposable elemcnts. In Mohile DNA (ed. D. Berg and M. Howe), pp. 375-412 American Society ofhlicrohiology,Washington, D.C. 37 Engels, W. R., Johnson-Schlitz, U. %I.,Eggleston, a'.B. and Sved, J. (1990). High-frequency'l element loss in Drosophila is homolog-dependent. Cel/ 62.5 15-525. 38 Crow, J. F. (1984). The P factor: a tranyposable elemenl in Drosophila. In hfutatioiz, Cancer and Ma@vmation (ed. E. H. Y. Chu arid W. M. Generoso). pp. 257-273. Plenum, 3 9 S i o n e l i g , M. andAnxdabeherc,D.(1991).APele~nentof,Scaptomyzapalli