Stephen B. Daniels,*' Arthur Chovnick? and Margaret G. Kidwell* .... and DICKINSON 1984). Despite ... 11 P-transformed lines originally described by DANIELS,.
Copyright 0 1989 by the Genetics Society of America
Hybrid Dysgenesis in Drosophila simulans Lines Transformed With Autonomous P Elements Stephen B. Daniels,*' Arthur Chovnick? and Margaret G. Kidwell* *Department
of Ecology and
Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, and tDepartment of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06268
Manuscript received February 19, 1988 Accepted for publication October 25, 1988 ABSTRACT The molecular and phenotypic analysis ofseveral previously described P element-transformed lines of Drosophila simulans was extended in order to determine whether they had the potential to produce a syndromeof P-M hybrid dysgenesis analogous to the onein Drosophila melanogaster. The transformed line with the highest number of P elements at the beginning of the analysis, DsPr-5C, developed strong P activity potential and P element regulation, properties characteristic of D. melanogaster P strains. The subsequent analysis of sublines derived from 34 single pair matings of DsPr-5C revealed that they were heterogeneous with respect to both their P element complements and P activity potentials, but similar with respect to their regulatory capabilities. The subline with the highest P activity, DsPr-5C-27, was subsequently used as a reference P strain in the genetic analysis of the D. simulans transformants. In these experiments, the reciprocal cross effect was observed with respect to both gonadal sterility and male recombination. As in D. melanogaster, the induction of gonadal sterility in D. simulans was shown to be temperature-dependent. Molecular analysis of DsPr-5C-27 revealed that it has approximately 30 P elements per genome, at least some of which are defective. The number of potentially complete P elements in its genome is similar to the number in the D. melanogaster P strain, Harwich-77. Overall our analysis indicates that P-transformed lines of D. simulans are capable of expressing the major features of P-M hybrid dysgenesis previously demonstrated in D. melanogaster and that P elements appear to behave in a similar way in the two sibling species.
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HE phenomenon of hybrid dysgenesis was first reported in Drosophila melanogaster (KIDWELL, KIDWELLand SVED 1977)and has been defined as a syndrome of aberrant genetic traits that is induced in the hybrid progeny of certain intraspecific crosses, usually in one direction only (KIDWELL1979). In this species, the P-M (KIDWELL,KIDWELLand SVED1977; ENGELS1988) and I-R (BREGLIANO et al. 1980; FINNEGAN 1988) systems have been described in considerable detail. In each of these systems, the manifestations of hybrid dysgenesis have been correlated with the destabilization of a specific transposable element family. In theP-M system, the activity of the P element is responsible for a numberof associated traits includingtemperature-dependent sterility, male recombination, chromosomal aberrations, segregation distortion, elevated rates of mutation, nondisjunction and female recombination. In the I-R system, the activation of the I element also produces a group of traits which includes mutation,chromosomalaberrations and a distinctive type of temperature-dependent sterility. A third system associated with the hobo element has been reported recently (BLACKMAN et al. 1987; YANNOPOULOS et al. 1987), but, currently, many ques-
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Present address: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06268. Genetics 121: 281-291 (February, 1989)
tions remain about the phenotypic properties of hobo and its contribution to the phenomenon of hybrid dysgenesis (BLACKMAN and GELBART 1988). P-homologous sequences are widely distributed among all the species groupsthat comprise the subgenus Sophophora (DANIELS et al. 1984; DANIELS and STRAUSBAUGH 1986; STACEY et al. 1986; ANXOL A B ~ H ~ R ENOUAUD , and P~RIQUET 1987; S. B. DANIELS, unpublished results), although, interestingly, they are not found in the species most closely related to D . melanogaster, including its sibling species D. simulans (BROOKFIELD, MONTGOMERYand LANGLEY 1984). The explanation for this disjunct distribution is not completely clear, but there is a growing body of evidence that supports the hypothesis that P elements have only recently been introduced into theD . melanogaster genome (within the past 40 yr) presumably by horizontal transmission from some as yet unknown source (KIDWELL 1983; A N X O L A B ~ H ~ R E , KIDWELLand P~RIQUET 1988). T h e other P-bearing species in the genus have apparently harbored P element sequences for a much longer evolutinary period (DANIELS and STRAUSBAUGH 1986;LANSMANet al. 1987; S. B.DANIELS, unpublished results), but it is not presently known whether any of these species contain the active element found in D. melanogaster.
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Evidence for P-M hybrid dysgenesis has not been documented in P-bearing sophophoransother thanD. melanogaster. With the adventof an efficient method to transduce cloned P elements, or their derivatives, into Drosophila germlinechromosomes (RUBIN and SPRADLING 1982), it is now possible to introduce autonomous P elementsinto the genomes of species thatdonot naturally carry them. Such transformants would provide the material to determine whether P elements behave in other species as they do in D . melanogaster. There have been several studies in which successful interspecific genetransfer has been achieved. For example, SCAVARDA and HARTL(1 984) transformed a ry- strain of D. simulanswith a selectable ry+-bearing P element vector and BRENNAN,ROWANand DICKINSON (1984) introduced an autonomous P element into the germline of Drosophila hawaiiensis, suggesting that, at least within the genus, there are no species barriers to prevent the initial insertion event.In other experiments, DANIELS, STRAUSBAUGH and ARMSTRONG (1985) focused on a molecular analysis of P element behavior in a number of D . simulans transformed lines during thefirst 12 generations following germline integration. They observed that P element copy number increased over timein some of the lines and that this increase was accompanied by transposition of elements to new genomic sites. Similar results were obtained with the D . hawaiiensis P transformants (BRENNAN, ROWAN and DICKINSON 1984). Despite the molecular evidence thatP elements can integrate andmultiply in the genomes of species other than D.melanogaster, evidence forthephenotypic manifestations of hybrid dysgenesis has been sketchy and largely unsubstantiated. In this paper, we have continued the analysis of the D . simulans P transformants generated by DANIELS, STRAUSBAUGH and ARMSTRONG (1985), focusing particularly on whether the features characteristic of P-M hybrid dysgenesis arise in a newly invaded species. THE EXPERIMENTAL SYSTEM
T h e P family of transposable elements consists of members that are homologous in sequence but heterogeneous in size (O’HAREand RUBIN1983). P elements can be divided into two types, complete or autonomous elements(sometimes called P factors) and defective,nonautonomouselements.Complete elements are 2.9 kb in length. Nonautonomous ones are smaller and variable insize and are derivedfrom autonomous elements by internal deletions. The frequency of such deletions is high under conditions of active transposition (VOELKER et al. 1984; DANIELSet al. 1985; SEARLES et al. 1986; TSUBOTA and SCHEDL 1986). Autonomous elements are able to catalyze their own transposition and can also act in transto promote
the transposition of nonautonomous ones. In theP-M system of hybrid dysgenesis, most strains of D . melanogaster can be classified as either P, M or Q. Classification is based on the phenotypic properties of the F1 progeny that are produced from specific reference crosses (see MATERIALS AND METHODS for details). Gonadal (GD) sterility is one dysgenic trait that is frequently used to measure the levelof P activity. It is characterized by the failure of one or both gonads to develop beyond a rudimentary stage (ENGELS and PRESTON1979; SCHAEFER, KIDWELLand FAUSTO-STERLING 1979). P strains exhibit P activity potential (i.e., they produce hybrid dysgenesis when males are crossed to M strain females). They also possess a strong ability to regulate P element expression which is often referred to as the P cytotype (ENGELSand PRESTON1979). M strains, on the other hand,rarely have any significant level of P activity potential, but have high P susceptibility (M cytotype), i.e., they are not able to regulate completely the activity of P elements. Q, or neutral, strains show little or no dysgenesis when crossed to either M or P strains, irrespective of direction. At present, the regulation of P element movement is not well understood. The nonreciprocal nature of the dysgenic response implicates certain maternally transmitted cytoplasmic “factors” as being important components in the regulatory process involving cytotype (ENGELS 1979; ENGELSand PRESTON1979). Other regulatory systems apparently lack these cytoplasmic determinants (KIDWELL1985; BLACKet al. 1987). For more detailed information, see ENGELS’ (1 988) recent review of P elements. MATERIALS AND METHODS
D. simulans strains employed:All nontransformed stocks used in this study were found to be devoid of P element sequences by Southern blot analysis. 8DS is a laboratory strain provided by H. KRIDER at the University of Connecticut; it was used as the injection recipient in the original transformation experiments (DANIELS, STRAUSBAUGH and ARMSTRONG 1985). 14021-0251.2, -0251.3, -0251.5 and -0251.8 are wildtype lines obtained from the National Drosophila Species Resource Center at Bowling Green State University. DsTuc2, DsTuc4, DsTuc6, and DsTuc8 are lines established from single gravid females captured from the wild in Tucson, Arizona, in 1985. f ;p m net; st e is a multiply marked strain homozygous for the following recessive markers: forked c f ; I, 56); plum (pm; 11, 103); net (net; 11, 0); scarlet ( s t ; 111,42); ebony (e, 111, 61) personal communica(STURTEVANT 1929; J. S. F. BARKER, tion). DsPr-1, -3, -4, -5, -9, -14, -15, -22, -25, -42, -46 are the 1 1 P-transformed lines originally described by DANIELS, STRAUSBAUGH and ARMSTRONG (1985). DsPT-~A,-5B, -5C, -25A,-25B,-25C are single pair sublines derived from two of the original transformed lines, DsPr-5and DsPr-25, at the third generation following transformation (DANIELS, STRAUSBAUCH and ARMSTRONG 1985).
Hybrid in Dysgenesis DsPr-5C-1 through -34 are single pair sublines that were established from theDsPr-5C line 16 months following transformation. D. melanogaster strainsemployed: Canton-S and rySo6 are M strains that do not carry any P elements. Harwich-77 is an inbred, strong P strain obtained in 1977 as a subculture from the original Harwich line. See DANIELS et al. (1987b) for details concerning the molecular characterization of the P element composition of this strain. Agana is an inbred line collected at Agana, Guam, in the 1960s. It is classified as a moderate P strain on the basis of standard phenotypic tests. Plasmids pr25.1 contains an autonomous P element that is capable of catalyzing its own transposition from plasmid to germline DNA and is flanked by genomic sequences from polytene chromosome region 17C; for further details see SPRADLING and RUBIN(1982)and O’HAREand RUBIN (1983). pr25.7BWC (“both wings clipped”) contains a P element that lacks 39 bp from its 5’ end and 23 bp from its 3’ end. This plasmid was constructed by K. O’HARE anddoes not contain any flanking genomic DNA. Generation and culturing of transformants. All transformants were generated in February of 1984 by embryo microinjection (SPRADLING and RUBIN1982), and separate a line was established from each (DANIELS, STRAUSBAUCH and ARMSTRONG 1985). For the first 9 months, the original lines were cultured in half-pint bottles; thereafter, theywere maintained in duplicate vial cultures. The single pair sublines that were derived from DsPr-5andDsPr-25 (see above) were propagated in essentiallythe same manner. All cultures were grown on a cornmeal-sucrose-agar-Brewer’s yeast medium seeded with live yeast and were maintained at room temperature (21-25 ”). Southern blotting analysis: Samples for mass extraction of genomic DNA were routinely prepared from approximately 100 adult flies by the method described by DANIELS and STRAUSBAUGH (1986). Procedures for restriction enzyme digestion, agarose gel electrophoresis, gel blotting, preparation of nick-translated probes and filter hybridization were essentially asdescribed in RUSHLOW, BENDER and CHOVNICK (1984). In situ hybridization: Salivary gland chromosomes were hybridized and labeled with biotinylated BWC DNA (ENGELS et al. 1986). Gonadal sterility tests: The specific reference crosses that were used to characterize the P-M phenotypic properties of the transformed D. simulans lines are analogous to those used to classify strains of D.melanogaster (e.g., KIDWELL and NOVY1979; ENGELS and PRESTON 1979; DANIELS et al. 1987b). Pactivity potential was measured by crossing males from the line in question to females from a strain free of P elements (cross A). In D.melanogaster, the standard tests to measure P element regulation @e., crosses of the A* type) employmales from reference strains having known hlgh levels of P activity. At the beginning of our experiments with D.simulans, a strong Pstrain was obviously not available and the Cross A* analysiswas only made possible after the DsPr-5C-27 strain had developed a strong ability to induce GD sterility (see RESULTS). Therefore, in the interim, other ways to assess P element regulation had to be devised. One measure of regulation was provided by frequencies of intrastrain sterility at high temperature. Also, cross B, the reciprocal of cross A, provided an indication of regulatory potential. If there is strong regulation present in the tested strain, progeny from both intrastrain and cross B matings are expected to show much lower sterility than do those from cross A. Conversely, inthe absence of regulation,
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progeny from these matings are expected to show relatively high sterility. Thus, like cross A*, cross B and theintrastrain mating serve asindicesof a line’sability to suppress P element activity. Cross B*, which is the reciprocal of cross A*, was employed as a control cross to detect the action of any confounding factors, unrelated to P , that might cause a condition similar to GD sterility. For each reference cross, 20-30 pairs of flieswere mated en masse in half-pint bottles and immediately placed at 29”. Two to three days later, parents were removed from the culture. Approximately 2 days following the onset of eclosion, F1 progeny were collected and allowed to mature for 3-5 days at room temperature. In mostof these crosses, 25-50 F1 femaleswere then taken at random fordissection. In some cases multiple tests were performed, enabling the calculation ofstandard errors.Dissected gonads were scored as either normal or dysgenic (unilateral or bilateral). Ovaries with at least one developed ovariole were scored as nondysgenic. The frequency of gonadal sterility was calculated by dividing the numberof dysgenic gonads by the total number of gonads scored. In several experiments, phenotypic tests were performed on D.melanoguster strains for comparison. These tests were conducted essentially as outlined above with the exception that 10-1 5 pairs offlies were used to establish theFI cultures. Male recombination tests: Male recombination was measured under dysgenic, nondysgenic and control conditions. In order toestablish the dysgenic state, a cross ofthe Atype was made (see above), in which P-transformed males were mated to virgin femalesfrom the multiply marked f ; pm net; st e stock. For the nondysgenic condition, a cross of the B type was made, in whichf; pm net;st e males were mated to P-transformed females. For both types of crosses, two separate experimental series were established: transformants from DsPr-5C were used in one, transformants from DsPr5C-27in the other. As a control, nontransformed males from the parental 8DS stock were crossed tof; pm net; st e females. In all cases, crosseswere made by mass mating 1520 pairs in half-pint bottles at 23”. The resulting F, males were mated singly in vials to approximately sevenf; pm net; st e females, and 3-4 broods were established by turning parents onto new food every 2-3 days. All of the progeny from each male were screend for exceptional (recombinant) phenotypes. Two different, but related, estimates of male recombination were computed: the mean frequency of male recombination, and themean minimum frequency of independent events (KIDWELLand KIDWELL 1976). The former expresses the total number of recombinants observed as a percentage of the total progeny count. The latter adjusts for clusters of recombinants and is an estimate of the minimum number of recombinant events, also expressed as a percentage of the total progeny count. For this calculation, only one event is scored when more than one recombinant chromosome within a given interval is recovered. RESULTS
Derivation and preliminary molecular analysis of D. simulans P element transformants: D.simulans P element transformants were generated by microinjecting ~ ~ 2 5plasmid .1 DNA into the posterior cytoplasm of embryos from the D. simulans strain, 8DS. Eleven independent transformed lines were initially established; three generations later, three single-pair
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FIGURE I.-Sotlthern blots of D. simulans transformed lines at two points in time. Genomic D N A was digested with PvuII and probed with the Xhol/Sall fragment from px25.1. T h e arrowhead indicates the 0.9-kb internally derived P element fragment. A, Analysis of the I I original transformed lines and six isopair sublines at month 12. Lanes 1 and 2, dilutions of DNA from a D. melanogaster P strain (Harwich77) with, respectively, I/> and ‘14 of the amount shown in the other lanes; lanes 3-1 3, the original tranformed lines in the order DsPa-5, -9, 14. -22, -25. -42, -46, - 1 . -3, -4 and -15; lanes 14-19. single pair sublines derived from two of the original lines in the order D s P d A , -5B. 5
