A hobo Transgene That Encodes the P-Element Transposase in Drosophila melanogaster: Autoregulation and Cytotype Control of Transposase Activity

Corresponding author: Michael J. Simmons, Cell Biology and Development, 250 BioScience Ctr., 1445 Gortner Ave., University of Minnesota, St. Paul, MN 55108-1095., simmo004{at}tc.umn.edu (E-mail)

Communicating editor: K. GOLIC

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry⁺, 2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

P-TRANSPOSABLE elements have proven to be valuable tools in the genetic analysis of Drosophila (ENGELS 1989 ). Natural and modified elements have been used as agents for mutagenesis and gene tagging, and appropriately marked elements have been used as vectors for genetic transformation. These applications have succeeded so richly because P-element activity can be controlled in experimental crosses. The key factors in this control are a P-encoded transposase, which catalyzes P-element excision and transposition, and a cellular state that regulates transposase activity.

P elements were discovered because of their involvement in a syndrome of germline abnormalities called hybrid dysgenesis (KIDWELL et al. 1977 ). This syndrome includes high frequencies of mutation and chromosome breakage, transmission ratio distortion, male recombination, and gonadal dysgenesis. The traits of hybrid dysgenesis are seen in the offspring of crosses between certain types of Drosophila strains. M strains have a cellular state, the M cytotype, that permits P-element activity, whereas P strains have a complementary state, the P cytotype, that represses P-element activity (ENGELS 1979A ). P elements are found in the genomes of P strains, where, for the most part, they are quiescent; they are also found in a subset of M strains that have been given the designation M' or pseudo-M (BINGHAM et al. 1982 ). True M strains do not have P elements in their genomes. Dysgenic hybrids are produced when P males are crossed to M (or M') females. The hybrids produced by the reciprocal cross typically do not show dysgenic traits or they show them with decreased frequency, because the P cytotype, which is maternally transmitted to the offspring, represses P-element activity.

P strains possess structurally complete P elements, 2.9 kilobases (kb) long, that encode a trans-acting transposase (O'HARE and RUBIN 1983 ; ENGELS 1984 ; KARESS and RUBIN 1984 ). The coding sequence in these elements is partitioned among four exons designated 0, 1, 2, and 3. In the germline, the intervening sequences of transcripts from complete P elements are removed by splicing to produce an mRNA that encodes an 87-kD polypeptide, the transposase. In the somatic tissues, the intervening sequence between exons 2 and 3 is not spliced out (LASKI et al. 1986 ). Because this sequence contains a stop codon, the incompletely spliced RNA encodes a smaller polypetide of 66 kD. P strains also have numerous incomplete P elements in their genomes (O'HARE et al. 1992 ). Most of these appear to have been derived from complete elements by deletions of internal sequences. These elements do not encode a catalytically active transposase, but they usually possess the terminal and subterminal sequences, which the transposase recognizes and attacks. Consequently, they can be excised and transposed. Several lines of evidence indicate that some incomplete P elements encode polypeptides that repress transposase activity (BLACK et al. 1987 ; JACKSON et al. 1988 ; RASMUSSON et al. 1993 ; ANDREWS and GLOOR 1995 ). The 66-kD polypeptide produced by alternate splicing of complete P-element transcripts also appears to function as a repressor of transposase activity (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; GLOOR et al. 1993 ).

P elements are mobilized when the P transposase is produced in the M cytotype. This situation occurs in the offspring of crosses between P males and M females. It also occurs when transgenes that have been engineered to produce the transposase are introduced into M cytotype strains. However, because the standard procedure for introducing transgenes into the Drosophila genome involves transformation with a P-element vector, such transgenes are inherently unstable (STELLER and PIRROTTA 1986 ; COOLEY et al. 1988 ; SIMMONS et al. 1996 ). The transposase they encode catalyzes their own excision and transposition. This article describes the properties of transgenes that are stable in the presence of the P transposase. A terminally truncated but otherwise complete P element has been introduced into the Drosophila genome by means of a transformation vector that was derived from a hobo transposable element. Like P elements, hobo elements are found in some Drosophila strains, and the largest of them, 3 kb long, encodes a trans-acting transposase that is specific for hobo elements (BLACKMAN et al. 1989 ; CALVI et al. 1991 ). Because the hobo transgenes are stable in the presence of the P transposase, they should be valuable tools for genetic analysis in Drosophila. These transgenes should also help to elucidate the complex mechanisms that control P-element activity in nature.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and husbandry:
Chromosomes and genetic markers are described in LINDSLEY and ZIMM 1992 . Fly cultures were maintained on a standard cornmeal-molasses-dried yeast medium at 25°, unless otherwise noted. Genetic manipulations of hobo transgenes were performed in stocks that lacked natural sources of the hobo transposase. In addition, these stocks lacked naturally occurring P elements, except where noted. When needed, heat shocks were administered to flies throughout their development and reproductive lives by placing cultures in a 37° cabinet incubator for 40 min once a day.

Mutability assays for P-transposase activity in male and female germlines:
Measurements of transposase activity employed a genetic assay that detects excisions of either of two incomplete P elements that are responsible for a mutant allele of the X-linked singed (sn) gene (ENGELS 1979B ; ROIHA et al. 1988 ). This allele, called weak singed (sn^w), causes a mild malformation of the bristles in the adult cuticle. When one or the other of the P elements in sn^w is excised by transposase activity in the germline, the sn^w allele changes to either an extreme singed (sn^e) or a pseudo-wild (sn⁺) allele, with corresponding visible changes in the bristle phenotype the next generation.

To measure transposase activity in the male germline, sn^w males carrying one or more autosomal sources of the P transposase were crossed individually to three or four C(1)DX, y f females. Because these females carry attached-X chromosomes, the singed allele from the father is transmitted patroclinously to his sons. These sons were classified for bristle phenotype and counted, and the proportion showing either the extreme or the pseudo-wild singed phenotypes was used as the index of transposase activity in the father's germline. When the modified P transgene P(ry⁺, 2-3)99B (ROBERTSON et al. 1988 ; ROBERTSON 1996 ) was used as the source of the P transposase, the C(1)DX, y f females used in the test crosses had the genetic background of the ₂ P strain. This genetic background represses the somatic instability of sn^w induced in the offspring by P(ry⁺, 2-3)99B and thereby permits these offspring to be classified unambiguously for bristle phenotype (ROBERTSON and ENGELS 1989 ).

To measure transposase activity in the female germline, sn^w/sn⁺ females carrying one or more autosomal sources of the P transposase were crossed individually to three or four y sn³ v car males. In heterozygous combination with a sn^e derivative of sn^w, sn³ causes an extreme singed phenotype; however, in heterozygous combination with sn^w, the phenotype is weak singed. The weak singed and extreme singed progeny from the crosses were counted and the proportion that were extreme singed was used as the index of transposase activity in the mother's germline. In these tests the phenotypically wild-type class was ignored because of the preexisting sn⁺ allele in the mother's genotype.

The progeny emerging in the test crosses to measure P-transposase activity in the germlines of either males or females were counted until the 17th day after the crosses were established, unless otherwise noted. Statistical differences were evaluated by z-tests.

Gonadal dysgenesis assay for P-transposase activity in the female germline:
To test for the induction or repression of gonadal dysgenesis, flies from the strains to be tested were mass mated at 21° for 2 days; the mated females were then separated into individual culture vials, which were reared at 29° for 12 days. The progeny of these cultures were transferred to fresh culture vials, and after 2–3 days of maturation at 21°, the females among them were examined for egg production by squashing them between two glass slides. A solution of blue dye was placed between the slides to help visualize the eggs. Females lacking eggs were considered to have gonadal dysgenesis. Statistical differences were evaluated by the Mann-Whitney rank sum test.

Molecular techniques:
Standard procedures were used to extract and manipulate DNA. A hobo transformation vector that contained a terminally truncated but otherwise complete P element fused to the hsp70 promoter was constructed by cloning the EcoRV/XbaI fragment from pCaSpeR/hsp70/CP (SIMMONS et al. 1996 ) into the polycloning region of pMartini, a plasmid derived from pBS-SK (B. CALVI, personal communication). The cloned fragment contained the hsp70 promoter fused to the complete P element (abbreviated CP) sequence from base pair 39 to base pair 2871 in the canonical P sequence (O'HARE and RUBIN 1983 ). The resulting plasmid was digested with NotI, which cleaves on either side of the polycloning region of pMartini, and the liberated fragment, which contained the hsp/CP fusion, was cloned into the unique NotI site of pHawN, a hobo transformation vector marked with the mini-white gene (B. CALVI, personal communication). The structure of the P element in the resulting plasmid, denoted pH(hsp/CP), was verified by DNA sequencing. Two differences between the P sequence in pH(hsp/CP) and the canonical P sequence were noted: a deletion of 2864G, which lies outside the transposase coding region, and a synonymous substitution of C for 1104T in a valine codon.

Southern analysis of genomic DNA was accomplished by capillary transfer of the DNA from an agarose gel to Hybond membranes using an alkaline transfer solution. After air drying, the membranes were hybridized with a ³²P-labeled DNA probe generated by randomly priming DNA synthesis from a purified P-element PCR product. This product was made by using primers complementary to sequences in the inverted terminal repeats to amplify genomic DNA from a strain containing a single complete P element. The blots were washed and exposed to X-ray film to produce autoradiograms.

Genetic transformation with pH(hsp/CP):
A mixture of the plasmids pH(hsp/CP) at 300 ng/µl and pHBL1 (CALVI and GELBART 1993 ) at 100 ng/µl was injected into preblastoderm embryos from a hobo- and P-element-free y w^67c23 stock using standard procedures for the genetic transformation of Drosophila; pHBL1 is a "helper" plasmid that encodes the hobo transposase. Surviving embryos were reared to adulthood and mated individually to hobo- and P-element-free w; TM3, Sb/TM6, Tb flies; any offspring showing pigmented eyes were mated individually to w; TM3, Sb/TM6, Tb flies to establish stocks transformed with the H(hsp/CP) transposon. The chromosomal location of the transposon in these stocks was determined by analyzing segregation patterns in crosses with balancer chromosomes carrying dominant markers.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

H(hsp/CP), a hobo transgene that produces the P-element transposase in the germlines of male and female Drosophila:
The structure of the hobo transformation vector containing the gene for the P-element transposase fused to the promoter of the Drosophila hsp70 gene is shown in Fig 1. This vector, denoted pH(hsp/CP), is 13 kb long; in addition to the hsp70/P transposase gene fusion, it contains the mini-white gene as a phenotypic marker. Both the hsp70/P transposase gene fusion and the mini-white gene in the vector are situated between the ends of an incomplete hobo element. In the presence of the hobo transposase, the modified 10-kb-long hobo transposon in pH(hsp/CP) can be inserted into the Drosophila genome.

View larger version (14K): In this window In a new window Download PPT slide   Figure 1. Structure of pH(hsp/CP). The plasmid was constructed by inserting the hsp70/CP fusion gene into the unique NotI site of the marked hobo element in the pHawN transformation vector; pBS-KS is the Bluescript backbone of this transformation vector. Bent arrows indicate the direction of transcription. Enzyme symbols: B, BamHI; H, HindIII; K, KpnI; N, NotI; P, PstI; R, EcoRI; RV, EcoRV; S, SalI; Ss, SstI; Xb, XbaI; Xh, XhoI. The regions showing the restriction sites flanking the hsp70/CP fusion gene are not drawn to scale.

One insertion of this transposon was obtained by germline transformation. This insertion was mapped to chromosome 2 and a stock homozygous for it was obtained by inbreeding. Southern blotting experiments with BamHI- or SalI-digested genomic DNA indicated that the stock carried a single insertion of the H(hsp/CP) transgene, which, because it is situated on chromosome 2, is denoted H(hsp/CP)2.

The sn^w mutability assay was used to determine if the H(hsp/CP)2 transgene could produce the P transposase in the male and female germlines. Homozygous w sn^w females were crossed to homozygous y w; H(hsp/CP)2 males and their sons and daughters were crossed appropriately to detect P-transposase-catalyzed mutations of sn^w occurring in the germline. The test crosses were reared at 25° without the administration of heat shocks. The results (Table 1) show that the H(hsp/CP)2 insertion could induce sn^w mutability in both the male and female germlines. The mutation rate, which indicates the level of transposase activity, is >50% in the male germline and >10% in the female germline. However, these rates are not directly comparable because the data for the males include the pseudo-wild derivatives of sn^w whereas those for the females do not. If the sn⁺ flies are excluded from the male data, the mutation rate is 0.382, which is still more than three times the observed mutation rate in females. This difference could be due to greater expression of H(hsp/CP)2 in the male germline or to some aspect of the mutation process itself; for example, a greater number of cell divisions during the development of the male germline could provide more opportunities for mutations to occur. The males that were tested in these experiments were also examined for evidence of sn^w mutability in the soma, i.e., for sn^e or sn⁺ bristles on the body. No such evidence was found.

Additional experiments were performed to compare the sn^w mutability induced by H(hsp/CP)2 in the male germline to that induced by different P strains. These strains included Harwich-w (KIDWELL et al. 1977 ) and ₂ (ENGELS 1979A , ENGELS 1979B ), both known to be strong inducers of sn^w mutability and gonadal dysgenesis; Nem12 and Still2 (listed as N12 and S2 in KOCUR et al. 1986 ), both known to be moderate inducers of sn^w mutability and gonadal dysgenesis; and ₆ (ENGELS and PRESTON 1981 ), a strain known to induce some sn^w mutability but little gonadal dysgenesis. Males from each strain were crossed to w sn^w females at 21° (to prevent gonadal dysgenesis in the offspring) and their sons were tested at 25° for sn^w mutability. The results in Table 2 show that in the male germline H(hsp/CP)2 is a more powerful inducer of sn^w mutability than any of the P strains tested. The observed mutation rate for the w sn^w; H(hsp/CP)2/+ flies, 0.326, is, however, less than the value of 0.526 obtained in the preliminary experiments described above. Thus, the culturing temperature of the initial cross affects the level of P-transposase activity encoded by the H(hsp/CP)2 transgene.

Transposase activity of two insertions of H(hsp/CP):
A second insertion of the H(hsp/CP) transgene was obtained on chromosome 3. Preliminary experiments demonstrated that this insertion, denoted H(hsp/CP)3, was homozygous viable and fertile and that it was capable of destabilizing the sn^w mutation in both the male and female germlines, but not in the male soma. Crosses between the homozygous H(hsp/CP)2 and H(hsp/CP)3 strains, followed by inbreeding, produced a strain that was homozygous for both of these insertions. Several sets of experiments were then conducted to compare the combined transposase activities of these two insertions with their separate transposase activities. In each experiment, homozygous w sn^w females were crossed with males homozygous for one or two of the insertions, and the offspring were tested for germline sn^w mutability.

Transposase activity in the male germline:

Four sets of experiments were carried out to investigate the transposase activity of the H(hsp/CP) transgenes in the male germline (Table 3). In sets I and II, transposase activity was measured both with and without a heat shock administered to the tested flies. In sets III and IV, no heat shock was given; in addition, in set III the tested flies were reared at 21° instead of 25°.

View this table: In this window In a new window   Table 3. Destabilization of snw by H(hsp/CP)2 and P(ry+, 2-3) transgenes under different conditions in the male germline

When H(hsp/CP)2 was tested by itself in the absence of any heat shocks, the estimated mutation rate for flies reared at 21° was 0.318, which is consistent with the previous estimate (0.326). The three estimates for flies reared at 25° were 0.498, 0.563, and 0.593, which are reasonably consistent with each other and with the preliminary estimate (0.526); the lowest of these values came from an experiment in which the flies were scored only on day 14. When H(hsp/CP)3 was tested by itself in the absence of any heat shocks, the estimated mutation rate for the flies reared at 21° was 0.328. For the flies reared at 25°, the estimated mutation rates were 0.434, 0.497, and 0.484. These results indicate that at 25°, H(hsp/CP)3 is slightly less effective than H(hsp/CP)2 at inducing sn^w mutability. [By z-tests, two of the three comparisons between H(hsp/CP)2 and H(hsp/CP)3 were statistically significant at the 5% level.] At 21°, however, the two insertions appeared to be equally effective at causing sn^w mutability.

When heat shocks were administered to the tested flies, all the mutation rates were increased by small amounts. For H(hsp/CP)2, these rates were 0.575 (by a z-test significantly greater than the corresponding non-heat-shock value) and 0.605 (not significantly greater), and for H(hsp/CP)3, they were 0.528 (significantly greater) and 0.536 (not significantly greater). Heat-shock treatment therefore appears to enhance H(hsp/CP)-encoded transposase activity in the male germline, albeit slightly.

When the H(hsp/CP)2 and H(hsp/CP)3 insertions were combined in the same genotype, sn^w mutability was greater than when the insertions were tested by themselves in four of six comparisons, but it was significantly greater in only one of these comparisons. Furthermore, heat shocks did not increase sn^w mutability in the double-insertion flies. These results suggested that the transposase activity of the H(hsp/CP) transgenes is limited in the male germline.

In experimental sets II and III, the stable P(ry⁺, 2-3)99B transgene was also tested for induction of sn^w mutability in the male germline. This transgene contains a modified P element that expresses the transposase in the soma as well as in the germline (ROBERTSON et al. 1988 ). Homozygous w sn^w females were crossed to homozygous P(ry⁺, 2-3) males and their w sn^w; P(ry⁺, 2-3)/+ sons, all mosaic for bristle phenotype because of the somatic activity of the 2-3-encoded transposase, were tested for germline sn^w mutability. For the flies that had been reared at 25°, the observed mutation rate was 0.765 without heat shock and 0.748 with heat shock; for the flies that had been reared at 21°, the observed mutation rate was 0.616. By z-tests, these rates are significantly greater than any of those seen with the H(hsp/CP) transgenes, even when the H(hsp/CP) transgenes were combined, under comparable experimental conditions. Thus, the transposase activity encoded by the P(ry⁺, 2-3)99B insertion is not subject to the same limitation as that encoded by the H(hsp/CP) insertions.

Transposase activity in the female germline:

The H(hsp/CP) insertions were also tested for their abilities to destabilize sn^w in the female germline (Table 4). In two sets of experiments, both heat-shocked and non-heat-shocked flies were tested; in a third set of experiments, no heat shocks were given. The results of the first two sets of experiments indicate that the heat-shock treatment increased the mutation rate by a factor of 2–3. These increases were seen in each of the tested groups within each set of experiments. By the nonparametric sign test, this pattern of results is statistically significant (P = 0.0156). Thus in the female germline, the transposase activity of the H(hsp/CP) transgenes is enhanced by heat shocks.

With or without heat shocks, the observed mutation rates of females carrying two H(hsp/CP) transgenes were not significantly greater than those of females carrying only one. Thus, the transposase activity of the H(hsp/CP) transgenes appears to be limited in the female germline as well as in the male germline.

Repression of the somatic transposase activity of P(ry⁺, 2-3)99B by H(hsp/CP) insertions:
The P(ry⁺, 2-3)99B insertion produces the P transposase in somatic tissues because the last intron in the P-transposase gene, which is not removed in these tissues by splicing, has been deleted by construction. A male carrying this insertion and the sn^w allele has a mosaic of weak singed, extreme singed, and wild-type bristles on its cuticle because the transposase produced by P(ry⁺, 2-3) destabilizes sn^w in the bristle precursor cells during development (LASKI et al. 1986 ). The H(hsp/CP) insertions, by contrast, cannot produce the P transposase in the somatic tissues because they possess the last intron of the P-transposase gene. Instead, these insertions would be expected to produce a truncated polypeptide that acts as a repressor of transposase activity (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ). To determine if the H(hsp/CP) insertions could produce this repressor, w sn^w females were crossed to w; H(hsp/CP) males, and then their w sn^w; H(hsp/CP)/+ sons were crossed to C(1)DX, y f; P(ry⁺, 2-3)99B females and their w sn^w/w +; H(hsp/CP)/+ daughters were crossed to w; P(ry⁺, 2-3)99B males. The male offspring from these "reciprocal" crosses were classifed by eye color [pigmented, i.e., carrying H(hsp/CP), or not pigmented, i.e., not carrying H(hsp/CP)] and by bristle phenotype, and those with either a weak singed or a mosaic bristle phenotype were counted. In one set of experiments daily heat shocks were administered to all the crosses and in another set, they were not.

The results of these experiments (Table 5) show that H(hsp/CP)2 repressed somatic transposase activity, although only partially, whereas H(hsp/CP)3 did not. Even with heat shocks, H(hsp/CP)3 had little ability to repress somatic transposase activity. Repression by H(hsp/CP)2 was stronger when the transgene was transmitted maternally; however, administration of heat shocks to the H(hsp/CP)2 test cultures did not seem to affect its repression ability.

Repression of the germline transposase activity of P(ry⁺, 2-3)99B by H(hsp/CP)2:
Tests with two insertions of the H(hsp/CP) transgene indicated that the germline transposase activity of these transgenes is significantly less than that of the P(ry⁺, 2-3)99B transgene. To determine if a H(hsp/CP) transgene could affect the germline transposase activity of the P(ry⁺, 2-3)99B transgene, two replicate stocks homozygous for both H(hsp/CP)2 and P(ry⁺, 2-3)99B were constructed. Males from these stocks were then crossed to w sn^w females and their w sn^w; H(hsp/CP)2/+; P(ry⁺, 2-3)99B/+ sons were tested for sn^w mutability; w sn^w; H(hsp/CP)2/+ and w sn^w; P(ry⁺, 2-3)99B/+ males were also tested for comparison. All the test cultures were incubated at 25° and no heat shocks were applied. From the results (Table 6), it is clear that the H(hsp/CP) transgene adversely affects the transposase activity of the P(ry⁺, 2-3)99B transgene. With the P(ry⁺, 2-3)99B transgene alone, the sn^w mutation rate was 0.780, which is consistent with previous results obtained under similar conditions (cf. Table 3); when the H(hsp/CP)2 transgene was combined with P(ry⁺, 2-3)99B, the sn^w mutation rate decreased significantly. Thus, the H(hsp/CP)2 transgene actually represses the transposase activity of the P(ry⁺, 2-3)99B transgene in the male germline.

View this table: In this window In a new window   Table 6. Regulation of P(ry+, 2-3)99B-induced germline snw mutability by H(hsp/CP)

Repression of H(hsp/CP)-encoded transposase activity by the P cytotype:
The P cytotype is jointly determined by the P elements on the chromosomes and by maternally inherited cytoplasmic factors (ENGELS 1979A ). Two types of analyses were performed to determine if the transposase activity encoded by the H(hsp/CP) transgenes could be regulated by the P cytotype. First, H(hsp/CP) transgenes were tested for their ability to induce gonadal dysgenesis (GD) in the daughters of P-cytotype females and, second, H(hsp/CP) transgenes were tested for their ability to destabilize sn^w in the grandsons of P-cytotype females.

Effect of the P cytotype on H(hsp/CP)-induced GD:

Five P-cytotype strains were studied in the gonadal dysgenesis experiments. Southern analysis indicated that all five possessed numerous P elements in their genomes. Two M-cytotype strains were also included in these studies: y w, a true M strain that is devoid of P elements and Sexi.4, a pseudo-M (or M') strain that has numerous incomplete P elements in its genome (RASMUSSON et al. 1990 ). Table 7 presents data showing the basic characteristics of these seven strains. First, four of the five P strains were able to induce significant frequencies of gonadal dysgenesis when males from them were crossed to females from the true M strain. The only exception was ₆, which, because of its weak ability to induce gonadal dysgenesis, has previously been classified as a special type of P strain called Q (ENGELS and PRESTON 1981 ). Neither the M or M' strains induced significant gonadal dysgenesis in these crosses. Second, all five P strains (including ₆) were effective repressors of the gonadal dysgenesis induced when Harwich-w males were crossed to M-cytotype females, either with or without heat shocks. Thus, all five strains clearly possess the P cytotype.

Table 8 shows the results of reciprocal crosses between the seven test strains and a strain homozygous for two insertions of the H(hsp/CP) transgene. When the test strains provided the male parent for the cross, the frequency of gonadal dysgenesis among the daughters was about the same as that seen in comparable crosses with the control M strain (cf. Table 7). However, there were two exceptions. When males from either the ₆ or Sexi.4 strains were used in the crosses, the frequency of gonadal dysgenesis among the daughters was increased (30.2% for ₆ and 14.1% for Sexi.4) over that seen in the control crosses (8.2% for ₆ and 1.0% for Sexi.4); by the Mann-Whitney rank sum test, both increases are significant. Thus, the doubly homozygous transgene strain cannot repress the induction of gonadal dysgenesis by different P strains—i.e., it has the M cytotype—and this strain actually enhances the dysgenesis-inducing potential of Q and M' males, presumably by activating the P elements they transmit to the offspring.

When males from the doubly homozygous transgene stock were crossed to P, Q, M, and M' females, either with or without heat shock, little gonadal dysgenesis was observed among the daughters except when the cross involved M' females, in which case the GD frequency was 35.9% without heat shock and 63.9% with heat shock. These highly significant results indicate that paternally derived H(hsp/CP) transgenes induce gonadal dysgenesis in the offspring of M' females and that this induction is enhanced by heat shocks. However, these transgenes do not induce gonadal dysgenesis in the offspring of true M females, which do not have P elements to attack, nor do they induce it in the offspring of P females, which inherit the repressive P cytotype.

Effect of the P cytotype on H(hsp/CP)-induced sn^w mutability:

P-cytotype control of H(hsp/CP)-encoded transposase activity was also investigated by using the sn^w mutability assay. Harwich-w P-cytotype females were crossed to w sn^w males and their w sn^w/Harwich-w daughters were crossed to y w males or to males homozygous for one or two H(hsp/CP) transgenes. The w sn^w sons of these crosses, which carried zero, one, or two H(hsp/CP) insertions in their chromosomes, were then tested for germline sn^w mutability. In one set of experiments daily heat shocks were given to the flies in the last two generations; in another set they were not.

In the absence of H(hsp/CP) transgenes, germline sn^w mutability was 0.208 with heat shock and 0.222 without heat shock. Transposase-producing P elements derived from the Harwich-w genome evidently were able to destabilize sn^w in these test groups. In the presence of H(hsp/CP) transgenes, higher mutation rates were observed, but the increases were not statistically significant (Table 9). Moreover, these rates were not nearly so high as those seen in flies derived from crosses lacking the Harwich-w grandmaternal contribution, i.e., in the absence of the P cytotype (cf. Table 3). Thus, the induction of sn^w mutability by the H(hsp/CP) transgenes is repressed by the P cytotype.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The H(hsp/CP) transgenes are effective producers of the P transposase in the germlines of male and female Drosophila. The transposase is produced constitutively from these transgenes, either because transcription is initiated at the hsp70 promoter without heat-shock induction or because it is initiated at the P-element promoter, which is structurally intact and located downstream of the hsp70 promoter. Constitutive expression of P(hsp70/CP) transgenes has been reported previously (STELLER and PIRROTTA 1986 ; SIMMONS et al. 1996 ). STELLER and PIRROTTA 1986 argued that the transcription of these transgenes is initiated at the hsp70 promoter because a transgene in which the gene for neomycin resistance (neo) had been inserted between the hsp70 promoter and the transposase gene generated much lower levels of transposase activity than a transgene without this insertion. STELLER and PIRROTTA 1986 hypothesized that the neo insertion interfered with the proper translation of RNAs that had been initiated at the hsp70 promoter. These investigators also found that heat-shock treatments significantly increased the level of transposase activity encoded by the P(hsp70/neo/CP) transgene, but not that encoded by the P(hsp70/CP) transgene. In their experiments, transposase activity was assessed by measuring sn^w mutability in the male germline; mutability in the female germline was not studied. In our experiments, heat-shock treatments significantly increased the transposase activity encoded by the H(hsp/CP) transgenes in the female germline; in the male germline the heat-shock treatments had little effect.

The H(hsp/CP) transgenes are genetically stable as long as hobo elements encoding the hobo transposase are not present. In contrast, P(hsp/CP) transgenes are inherently unstable because they produce the transposase that catalyzes their own movement. For most genetic applications, H(hsp/CP) transgenes should therefore be superior to P(hsp/CP) transgenes. Only one genetically stable transposase-encoding P transgene, P(ry⁺, 2-3)99B, is currently available. When this transgene inserted into the genome, it was altered so that it cannot be acted on effectively by the transposase (ROBERTSON 1996 ). Thus, it does not excise or transpose at noticeable frequencies. The Jumpstarter element, another P transgene that encodes the P transposase (COOLEY et al. 1988 ), is genetically unstable.

When two H(hsp/CP) transgenes are present in the genome, their combined ability to destabilize the sn^w allele is not significantly greater than their separate abilities, but it is much less than the destabilizing ability of the P(ry⁺, 2-3)99B transgene. These observations imply that the transposase activity of the two H(hsp/CP) transgenes is limited. This limitation can be explained in three ways. First, the two transgenes might compete for transcription factors to initiate RNA synthesis, from either the hsp70 or the P-element promoters controlling the transposase gene. Second, the transcripts of the transgenes might compete for splicing factors that are required for correct processing of the transposase pre-mRNA, in particular, for removal of the last intron. Third, a transgene product might feed back negatively to limit transposase expression or function.

The key observation that discriminates among these hypotheses is that a H(hsp/CP) transgene reduces the transposase activity encoded by the P(ry⁺, 2-3)99B transgene, in both the soma and the germline. This effect is difficult to explain by competition for transcription or splicing factors because (1) the P(ry⁺, 2-3)99B transgene naturally produces a high level of transposase activity, (2) this activity does not depend on an hsp70 promoter for expression, and (3) the RNA transcribed from the P(ry⁺, 2-3)99B transgene is partially preprocessed. Rather, the repression of P(ry⁺, 2-3)99B is better explained by a product of H(hsp/CP) that acts as a negative regulator of transposase expression or function. This product is unlikely to be the transposase itself. A better candidate is the 66-kD polypeptide encoded by P-element RNAs that retain the last intron. Such RNAs are produced naturally in somatic tissues because of a factor that inhibits this intron's splicing, and they also appear to be produced in the germline (MISRA and RIO 1990 ; ROCHE et al. 1995 ).

Transgenes and plasmids designed to produce the 66-kD polypeptide repress P-element mobility in vivo (MISRA and RIO 1990 ; GLOOR et al. 1993 ; HANDLER et al. 1993 ); furthermore, the 66-kD polypeptide has been found in the oocytes of a P strain (MISRA and RIO 1990 ). It has therefore been proposed that this polypeptide naturally plays an important role in P-element regulation in the germline and that it might be the basis for the P cytotype. According to one model (O'HARE et al. 1992 ; ROCHE et al. 1995 ), P elements are weakly transcribed in the P cytotype because the 66-kD polypeptide acts as a transcriptional repressor. When little P-element RNA is produced, the splicing machinery tends to leave the last intron in P-element transcripts, and this leads to the synthesis of more 66-kD polypeptide instead of the 87-kD transposase. An autoregulatory loop in which the 66-kD polypeptide feeds back to minimize transcription from the P-element promoter is thereby established.

The 66-kD polypeptide can repress transcription from the P-element promoter in the soma (LEMAITRE and COEN 1991 ; LEMAITRE et al. 1993 ). However, it is not clear if it does so in the germline. The fact that the transposase activity encoded by the H(hsp/CP) transgenes is limited suggests that the 66-kD polypeptide has a genuine autoregulatory role in the germline. It is not clear, however, if the 66-kD polypeptide is the basis of the P cytotype.

This chromosomal/cytoplasmic state represses the transposase activity encoded by the H(hsp/CP) transgenes. When males carrying these transgenes are crossed to M' females, their daughters are dysgenic, especially when the cultures are treated with heat shocks. The M' females used in these crosses came from an inbred strain that has 43 incomplete P elements in its genome (RASMUSSON et al. 1990 ); however, none of these P elements has regulatory ability. The gonadal dysgenesis observed among the daughters of these crosses therefore indicates that the H(hsp/CP) transgenes can induce gonadal dysgenesis when they are combined with P-element targets for the transposase to attack. The fact that they do not do so in the daughters of P females demonstrates that the P cytotype can repress the transposase activity encoded by the H(hsp/CP) transgenes. Other experiments have demonstrated that the P cytoype represses sn^w mutability induced by the H(hsp/CP) transgenes. Thus, these transgenes could become useful genetic tools in efforts to elucidate the mechanistic nature of the P cytotype.

	ACKNOWLEDGMENTS

Technical assistance was provided by Mylee Bishop, Gretchen Cutler, Joseph Fong, Paul Kocian, Bradley Morrison, Dan Owens, and Sarah Thompson. The hobo transformation system was obtained from Brian Calvi and William Gelbart. Financial support was provided by National Institutes of Health grant R01-GM40263.

Manuscript received November 15, 2001; Accepted for publication February 11, 2002.

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