Regulation of P-Element Transposase Activity in Drosophila melanogaster by hobo Transgenes That Contain KP Elements

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

Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry⁺, 2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry⁺, 2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA.

THE P family of transposable elements in Drosophila melanogaster is regulated by a complex blend of mechanisms (ENGELS 1989 ; RIO 1990 ). The primary mechanism restricts expression of the enzyme that catalyzes P-element excision and transposition to the germline tissues of both sexes. In the somatic tissues, this P-encoded enzyme, the 87-kD transposase, is not produced because the last of the three introns in the P coding sequence remains in the RNA (LASKI et al. 1986 ). Translation of this incompletely spliced RNA results in a 66-kD polypeptide that represses P-element mobility (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; GLOOR et al. 1993 ).

In the germline, where transposase can be produced, P-element mobility is also regulated. Early genetic studies identified a maternally transmitted state called the P cytotype that represses P-element excision and transposition (ENGELS 1979A ). The P cytotype is characteristic of most, but not all, Drosophila strains that have P elements in their genomes (BINGHAM et al. 1982 ). A complementary state, the M cytotype, is characteristic of strains that lack P elements. When P elements are introduced into the M cytotype by crossing P males to M females, the P elements are mobilized in the germline tissues of the hybrid offspring. This mobilization causes an increased frequency of mutation and chromosome breakage and sometimes kills enough germ cells to make the animal sterile. These deleterious effects of P-element excision and transposition have been consolidated into a syndrome known as hybrid dysgenesis (KIDWELL et al. 1977 ).

Although the P cytotype is maternally transmitted, it depends absolutely on the P elements themselves. When chromosomes bearing P elements are removed from the genome by segregation in females that had P-cytotype mothers, the cytotype switches abruptly from P to M (SVED 1987 ). However, when P-containing chromosomes are introduced into the genome by crosses between P males and M females, with subsequent backcrosses of the daughters to P males, the cytotype switches gradually from M to P (ENGELS 1979A , ENGELS 1981 ). The mechanistic basis of these phenomena is not understood. However, it is thought that the P cytotype involves P-element products, most likely polypeptides that repress transposase activity.

Different types of P elements are found in the genomes of P strains. Complete P elements, 2.9 kb long, encode the transposase and the 66-kD polypeptide; although the latter is clearly made in the soma, there is evidence that it is produced in the germline as well (MISRA and RIO 1990 ; SIMMONS et al. 2002 ). Thus, the 66-kD product of complete elements could be a component of the P cytotype. Numerous incomplete P elements, most apparently derived from complete elements by internal deletions, are also present in the genomes of P strains (O'HARE et al. 1992 ). These are structurally heterogeneous and some have been shown to play a role in germline P-element regulation. The best studied of these incomplete P elements was originally identified in a strain derived from the Russian city of Krasnodar; it is called KP (BLACK et al. 1987 ; JACKSON et al. 1988 ).

The KP element is 1.15 kb long and encodes a polypeptide of 207 amino acids, 199 of which are identical with the amino-terminal portion of the transposase. Screens of natural populations of Drosophila have demonstrated that the KP element is geographically widespread (BLACK et al. 1987 ; W. R. ENGELS and C. R. PRESTON, personal communication), which suggests that natural selection has favored it. In vitro studies have demonstrated that the KP polypeptide forms homodimers and that it interacts with P-element DNA at three distinct sites (LEE et al. 1996 , LEE et al. 1998 ): the transposase-binding sites near the 5'- and 3'-ends of each P element, the 11-bp internal inverted repeats located near the transposase-binding sites, and, at high concentrations, the 31-bp terminal inverted repeats. Dimerization of KP polypeptides is mediated by at least two separate regions in the protein, one of which is a leucine zipper, and binding to DNA appears to depend on a CCHC motif located in the amino-terminal portion of the polypeptide (LEE et al. 1998 ). In vivo studies have shown that naturally occurring KP elements and transgenes designed to produce the KP polypeptide repress some of the traits of hybrid dysgenesis, although with varying abilities (RASMUSSON et al. 1993 ; ANDREWS and GLOOR 1995 ; SIMMONS et al. 1996 ), and one study has shown that KP elements repress transcription from a P-element promoter (LEMAITRE et al. 1993 ). The leucine zipper motif in the KP polypeptide appears to be necessary for the ability to repress hybrid dysgenesis (ANDREWS and GLOOR 1995 ).

Efforts to study KP elements have been complicated by the phenomenon of genetic instability. In the presence of the P transposase, natural KP elements and P-element transgenes carrying the KP coding region are excised and transposed. To circumvent this problem, we have used another transposable element transformation system to introduce KP transgenes into the Drosophila genome. This transformation system is based on hobo transposable elements, which, like P elements, are found in some Drosophila strains (BLACKMAN et al. 1989 ; CALVI et al. 1991 ). Structurally complete hobo elements encode a transposase that is transposon specific. Thus, it cannot catalyze P-element movement and, likewise, the P transposase cannot catalyze hobo movement. With the creation of hobo transgenes designed to express either the P transposase or the KP polypeptide, we have been able to address questions about KP-mediated regulation of the P-element family: How effective is KP-mediated regulation? Does it involve a maternal effect? Is KP-mediated regulation enhanced by interactions with other P-encoded polypeptides?

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila husbandry and genetic techniques:
The chromosomes and genetic markers used in the experiments are described in LINDSLEY and ZIMM 1992 or in other references cited in the text. The culturing conditions and genetic procedures that were used to measure P-transposase activity are described in SIMMONS et al. 2002 . Statistical differences were evaluated by z-tests when the sample sizes were large and by nonparametric Mann-Whitney rank sum tests when they were small. The germline transformation of embryos with hobo vectors and the analyses of transformed stocks were accomplished by standard methods; hobo transposons were inserted into stocks that were free of natural sources of the hobo transposase. Primary insertions of these transposons were jumped to new locations in the genome by crossing transformed flies to flies that carried P(ry⁺, HBL1) (CALVI et al. 1991 ), a P transgene encoding the hobo transposase, which was inserted on the TM3, Sb balancer chromosome. Offspring from these crosses were mated appropriately to screen for new insertions of the hobo transposon. Recombination was used to create stocks with two different transgenes in the genome; the presence of each transgene was verified by PCR amplification with appropriate primers.

Construction of hobo transformation vectors and molecular techniques:
The pH(hsp/KP) and pH(hsp/SP) transformation vectors were created in the same manner as the pH(hsp/CP) transformation vector described in SIMMONS et al. 2002 . The pH(hsp/BP1) transformation vector was created by PCR amplifying the BP1 element (SIMMONS et al. 1996 ) from base pair 39 to base pair 2871 using a cloned BP1 element as template DNA and primers with restriction enzyme recognition sites for BamHI at their termini. The PCR product was digested with BamHI and ligated into the BamHI site of pMartini/hsp, a plasmid derived from pBS-SK that contains the hsp70 promoter inserted between the EcoRI and BamHI restriction sites in the polycloning region. The BP1 sequence was inserted in the correct orientation downstream of the hsp70 promoter. The resulting plasmid was digested with NotI, which cleaves on either side of the polycloning region, and the liberated fragment containing the hsp/BP1 fusion was cloned into the unique NotI site of the pHawN transformation vector (B. CALVI, personal communication) to produce pH(hsp/BP1). The P elements in each of the three hobo transformation vectors were sequenced to verify their structures. In the KP element, nucleotide 2866A (in the complete P sequence described in O'HARE and RUBIN 1983 ), which lies beyond the coding region, was deleted. Standard procedures were used to extract and manipulate DNA. PCR amplifications used Taq DNA polymerase, 0.2 mM each dNTP, 1x buffer with an additional 1.5 mM MgCl₂, and appropriate primers (1.5 ng each) in a volume of 25 ml; template DNA was typically a crude extract from single adult flies, although in some reactions cloned DNA was used. PCR amplifications typically involved 30 cycles, with each cycle consisting of 1-min denaturation at 92°, 2-min annealing at 55°, and 3-min polymerization at 72°.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Three hobo transgenes containing incomplete P elements:
Fig 1 shows the structures of hobo transformation vectors containing different P elements fused to the Drosophila hsp70 promoter. CP is a terminally truncated but otherwise complete P element. The three incomplete P elements are KP, a 1.15-kb element known to encode a repressor polypeptide of 207 amino acids; SP, a 0.5-kb element capable of encoding a polypeptide of 14 amino acids; and BP1, a 2.8-kb element capable of encoding a polypeptide of 717 amino acids in the germline and one of 541 amino acids in the somatic cells (SIMMONS et al. 1996 ). Terminally truncated copies of each of these incomplete P elements were inserted into the pHawN transformation vector downstream of a hsp70 promoter; all three P elements also contained an intact P-element promoter. The resulting plasmids were injected into y w^67c23 embryos along with pHBL1 (CALVI and GELBART 1993 ), a helper plasmid that encodes the hobo transposase, to obtain genetically transformed flies, which were recognized by the expression of a visible marker (the mini-white gene) present in the hobo transposon. Each insertion of a hobo transposon was localized to a chromosome by segregation analysis, and homozygous stocks were established by inbreeding. Additional insertions of these transposons were obtained by using a P transgene that encodes the hobo transposase to jump the primary insertions to new locations. Southern blotting experiments identified which transformed stocks carried a single hobo transposon, and only these stocks were used in subsequent analyses. Because these stocks lacked natural sources of the hobo transposase, they were genetically stable.

View larger version (25K): In this window In a new window Download PPT slide   Figure 1. Structures of hobo transformation vectors containing the CP, KP, SP, and BP1 elements. The vectors were constructed by inserting the hsp70/P 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 endpoints of the deletions in the incomplete P elements are given with reference to the coordinates in the canonical P-element sequence (O'HARE and RUBIN 1983 ). The regions showing the restriction sites flanking the hsp/P fusion genes are not drawn to scale.

Effects of maternally transmitted hobo transgenes on P-transposase activity:
The various hobo transgenes were tested for repression of P-transposase activity encoded by two different transposase sources, H(hsp/CP)2, a hobo transgene inserted on chromosome 2 that produces the P transposase in the germline (SIMMONS et al. 2002 ), and P(ry⁺, 2-3)99B, a stable P transgene inserted on chromosome 3 that produces the P transposase in the soma as well as in the germline (ROBERTSON et al. 1988 ). Although the transposase gene in H(hsp/CP)2 is fused to a heat-shock-inducible promoter, this gene produces the P transposase even in the absence of heat shock (SIMMONS et al. 2002 ). The P(ry⁺, 2-3)99B transgene is immobile because of abnormalities in its termini (ROBERTSON 1996 ). These two transgenes were used to destabilize sn^w, a double-P-element-insertion mutation of the X-linked singed (sn) bristle gene (ENGELS 1979B ; ROIHA et al. 1988 ), by crossing males homozygous for one or the other of them to females homozygous for sn^w. The sons and/or daughters from these crosses were then individually tested for germline sn^w mutability. When the P(ry⁺, 2-3)99B transgene provided the P transposase, the F₁ flies were also examined for somatic sn^w mutability.

The first set of experiments (Table 1) evaluated the effect of two naturally occurring KP elements (RASMUSSON et al. 1993 ) and four H(hsp/KP) transgenes on H(hsp/CP)2-induced sn^w mutability in the male germline. Two H(hsp/SP) transgenes and one H(hsp/BP1) transgene were also tested. Females homozygous for sn^w and the KP element or hobo transgene were mated to homozygous H(hsp/CP)2 males and their sons were crossed to C(1)DX, y f females for the sn^w mutability test. Heat-shock treatments were not administered to the flies in these experiments. The sn^w mutation rate for the control group (0.579) is consistent with previous estimates obtained under comparable conditions (SIMMONS et al. 2002 ). Similar rates were obtained with flies heterozygous for the H(hsp/SP) or H(hsp/BP1) transgenes. Thus, neither the hsp70 promoter nor the SP or BP1 sequences in these transgenes had any effect on the transposase activity encoded by H(hsp/CP)2. However, one of the natural KP elements (KP1) and all four H(hsp/KP) transgenes reduced sn^w mutability by 15–20% of the control value. By z-tests, these reductions are statistically significant. Individual KP elements, either naturally occurring or as a part of a transgene construct, can therefore function as weak repressors of P-transposase activity, and the KP transgenes can do so without induction by heat shock.

The second set of experiments (Table 2) evaluated the ability of two of the H(hsp/KP) transgenes to repress sn^w mutability in the germlines of males and females that had been heat-shocked daily throughout their lives. The heat-shock treatments might be expected to enhance expression of both the KP polypeptide and the H(hsp/CP)2-encoded transposase. The tested flies were obtained by crossing females homozygous for sn^w and a KP, SP, or BP1 hobo transgene with H(hsp/CP)2 males. The sn^w mutation rate for control males was not significantly greater than it was in the absence of heat shocks, which is consistent with previous experimental results (SIMMONS et al. 2002 ). However, the rates for the males carrying the H(hsp/SP) transgenes were somewhat elevated (cf. Table 1); in one case, the increase was 15% of the non-heat-shock value. The rates for the males that were heterozygous for the H(hsp/KP) transgenes were also somewhat greater than they were without heat shocks; however, by z-tests they were still significantly less than the corresponding control value. Thus, under heat-shock conditions the H(hsp/KP) transgenes still function as weak repressors of H(hsp/CP)2-encoded transposase activity in the male germline.

The data in Table 2 also demonstrate that these transgenes repress transposase activity in the female germline. Individual females heterozygous for sn^w;H(hsp/CP)2 and a KP, SP, or BP1 transgene were crossed appropriately to allow their germline sn^w mutation rates to be estimated. However, these rates are not directly comparable to those estimated for males because not all the mutational events that occur in the female germline can be detected by the genetic scheme; furthermore, in the female germline the expression of the H(hsp/CP)2 transgene is significantly increased by heat shock (SIMMONS et al. 2002 ). The mutation rate for the control group in this experiment (0.193) therefore includes a heat-shock-inducible component. Both of the H(hsp/KP) transgenes that were tested reduced this value by 50%. This statistically significant effect indicates moderate repression of transposase activity.

The third set of experiments tested the KP, SP, and BP1 transgenes for repression of somatic transposase activity. Because P(ry⁺, 2-3)99B lacks the intron that regulates the tissue-specific production of the P transposase, this transgene produces the transposase in somatic cells. Flies carrying the sn^w mutation and the P(ry⁺, 2-3)99B transgene therefore may have as many as three different bristle phenotypes on their bodies due to the transposase-catalyzed instability of sn^w during somatic development. To determine if any of the hobo transgenes could repress this instability, females homozygous for sn^w and a particular transgene were crossed to homozygous P(ry⁺, 2-3)99B males and their sons were examined for bristle mosaicism. As Table 3 shows, none of the transgenes was able to repress somatic transposase activity effectively, either with or without heat-shock treatments.

View this table: In this window In a new window   Table 3. Effect of H(hsp/P) transgenes on P(ry+, 2-3)99B-induced snw mutability in somatic cells

The fourth set of experiments evaluated naturally occurring KP elements and H(hsp/KP) transgenes for repression of sn^w mutability induced in the male germline by P(ry⁺, 2-3)99B. H(hsp/SP) and H(hsp/BP1) transgenes were also tested. In these experiments, females homozygous for sn^w and a KP element or hobo transgene were crossed to males homozygous for P(ry⁺, 2-3)99B and their sons were crossed to C(1)DX, y f females from a P strain for the sn^w mutability test. Because somatic transposase activity is repressed in the offspring of this cross, the sons could be scored unambiguously for bristle phenotype. The results are given in Table 4. As the control data show, the P(ry⁺, 2-3)99B transgene was a more effective inducer of sn^w mutability than the H(hsp/CP)2 transgene; the mutation rate was 0.75. In the presence of a H(hsp/SP) or H(hsp/BP1) transgene, the mutation rate was increased slightly. However, in the presence of KP1 or any of the four H(hsp/KP) transgenes that were tested, the mutation rate was significantly lower (by z-tests), in one case 50% lower. Thus, although P(ry⁺, 2-3)99B is a stronger inducer of sn^w mutability than H(hsp/CP)2, it is more effectively repressed by the KP1 element and KP transgenes. One of the naturally occurring KP elements, KP6, did not repress P(ry⁺, 2-3)99B-induced sn^w mutability. However, it also did not repress H(hsp/CP)2-induced sn^w mutability (see Table 1).

View this table: In this window In a new window   Table 4. Effect of maternally transmitted KP elements and H(hsp/P) transgenes on P(ry+, 2-3)99B-induced snw mutability in the male germline

Identifying the maternal and zygotic effects of KP elements:
In all the previous experiments the KP elements and H(hsp/KP) transgenes were transmitted maternally to the progeny that were tested for sn^w mutability. To determine whether the observed reduction in mutation rates was due to maternal or zygotic effects of the element or transgene, three different types of experiments were performed.

In the first experiment, sn^w males carrying either the KP1 element or the H(hsp/KP)7 transgene were crossed to attached-X females homozygous for either the H(hsp/CP)2 or P(ry⁺, 2-3)99B transgenes, and their sons were tested for sn^w mutability. Because the KP elements in these crosses were paternally derived, any repression of sn^w mutability must be due to a zygotic effect of the element. For comparison, reciprocal crosses were also carried out; sn^w females homozygous for the KP element or transgene were mated to males homozygous for one of the transposase transgenes and their sons were tested for sn^w mutability. In these reciprocal crosses, repression of sn^w mutability could be due to a combination of maternal and zygotic effects. Table 5 summarizes the results of this experiment.

View this table: In this window In a new window   Table 5. Effects of maternally or paternally transmitted KP element or H(hsp/KP) transgene on H(hsp/CP)2- or P(ry+, 2-3)99B-induced snw mutability in the male germline

The control data from the two types of crosses show that with H(hsp/CP)2, but not with P(ry⁺, 2-3)99B, there is more sn^w mutability when the transposase source is maternally derived. This effect is presumably due to maternal transmission of the transposase activity encoded by the H(hsp/CP)2 transgene. The P(ry⁺, 2-3)99B transgene does not transmit transposase activity through the egg cytoplasm (M. J. SIMMONS, K. J. HALEY and S. J. THOMPSON, unpublished data). When the results from the crosses involving the KP element or transgene are compared with their respective controls, it appears that both the element and the transgene repressed sn^w mutability, no matter if they were paternally or maternally derived. For the P(ry⁺, 2-3)99B transposase source, the repression was statistically significant by z-tests, but for the H(hsp/CP)2 transposase source it was not. Thus, as seen in previous experiments, the stronger transposase source, P(ry⁺, 2-3)99B, was the one more effectively repressed by the KP element and transgene. Furthermore, the element and the transgene repressed P(ry⁺, 2-3)99B-encoded transposase activity with equal effectiveness and without any obvious maternal effect.

In the second experiment, sn^w females were crossed to males homozygous for a transposase transgene and a hobo transgene carrying a KP, SP, or BP1 element, and their sons were tested for sn^w mutability. Thus, unlike the first experiment, this one evaluated the repression ability of paternally derived hobo transgenes when the transposase transgenes were also paternally derived. Five of the six H(hsp/KP) transgenes that were tested repressed H(hsp/CP)2-induced sn^w mutability significantly (Table 6). However, the H(hsp/SP) and H(hsp/BP1) transgenes did not. The strongest repressor among the KP transgenes, H(hsp/KP)7, and the H(hsp/SP)B transgene were also tested in combination with P(ry⁺, 2-3)99B. Both of the H(hsp/KP)7; P(ry⁺, 2-3)99B stocks that were synthesized for this purpose proved to be effective repressors of transposase activity, whereas the H(hsp/SP)B; P(ry⁺, 2-3)99B stock was not. Thus, H(hsp/KP) transgenes repress transposase activity when they are paternally derived along with either type of transposase source. Repression by these transgenes therefore clearly involves a zygotic effect.

In the third experiment, the H(hsp/KP)7 transgene was tested for repression through a strictly maternal effect, i.e., one transmitted independently of the transgene itself. Reciprocal matings between w sn^w and w sn^w; H(hsp/KP)7 flies produced w sn^w; H(hsp/KP)7/+ daughters that were crossed to w; P(ry⁺, 2-3)99B males. The progeny of these crosses included pigmented sons (class A), which carried the H(hsp/KP)7 transgene, and nonpigmented sons (class B), which did not. Because these flies all carried sn^w and the paternally derived transposase transgene, they could be tested for sn^w mutability. If the H(hsp/KP)7 transgene repressed transposase activity through a strictly maternal effect, the males in class B would show a reduced mutation rate.

The results of the experiment (Table 7) indicate that the males of class A, but not those of class B, repressed sn^w mutability. The mutation rates of class B were actually greater than the negative control rate obtained from tests with w sn^w; P(ry⁺, 2-3)99B/+ males derived from crosses between w sn^w females and P(ry⁺, 2-3)99B males. In contrast, the mutation rates of class A were significantly less than the negative control rate but not significantly greater than the positive control rate obtained from tests with w sn^w; H(hsp/KP)7/+; P(ry⁺, 2-3)99B/+ males derived from crosses between w sn^w; H(hsp/KP)7 females and P(ry⁺, 2-3)99B males. In this experiment, it did not matter which grandparent in the crossing scheme carried the H(hsp/KP)7 transgene (cross I, grandfather; cross II, grandmother). In either case, H(hsp/KP)7 did not repress transposase activity through a strictly maternal effect.

View this table: In this window In a new window   Table 7. Partitioning H(hsp/KP)7-mediated repression of P(ry+, 2-3)99B-induced snw mutability in the male germline into grandmaternal, maternal, and zygotic effects

Testing for synergistic repression of transposase activity by maternally transmitted H(hsp/KP) and H(hsp/CP) transgenes:
The KP polypeptide might interact with the transposase or the 66-kD polypeptide encoded by complete P elements to create a stronger repressor protein. Two types of experiments were performed to evaluate this hypothesis.

The first experiments tested for synergistic repression of gonadal dysgenesis (GD) by maternally transmitted KP and CP transgenes. Individual females homozygous for a H(hsp/KP) transgene or for a H(hsp/KP) transgene and the H(hsp/CP)2 transgene were mated to males from the Nem12 P strain (listed as N12 in KOCUR et al. 1986 ) and their daughters were examined for gonadal dysgenesis. In control crosses with y w true M females, Nem12 induced 50% GD (Table 8). By Mann-Whitney rank sum tests, significantly less GD was induced in crosses with KP1 or KP6 females and with H(hsp/KP)3, H(hsp/KP)7, H(hsp/KP)13, or H(hsp/KP)14 females. However, approximately control levels of GD were induced in crosses with H(hsp/KP)10, H(hsp/KP)11, and H(hsp/SP)B females. Thus, the two naturally occurring KP elements and four of the six KP transgenes that were tested repressed Nem12-induced GD. In the presence of the H(hsp/CP)2 transgene, Nem12 induced more GD (76.8%). All six of the H(hsp/KP) transgenes repressed GD under these conditions, but only H(hsp/KP)7 was a stronger repressor than it was in the absence of H(hsp/CP)2. Thus, there is no compelling evidence for synergistic repression of GD by the H(hsp/KP)-H(hsp/CP) combination.

In the second experiments to investigate synergistic repression by KP and CP transgenes, the two transgenes were variously transmitted in crosses, separately or together and maternally or paternally, to obtain females that were tested for sn^w mutability in their germlines. The results of experiments with two KP transgenes are given in Table 9. In the absence of any KP transgene, the CP transgene induced 11% sn^w mutability when it was paternally derived and 16% when it was maternally derived. The difference between these groups may be due to maternal transmission of transposase activity. As expected, the KP transgenes reduced the sn^w mutation rates significantly, no matter which parent they came from; however, there was no evidence that maternal transmission of both the KP and CP transgenes enhanced the repression effect.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Because hobo transgenes are stable in the presence of the P transposase, we have been able to combine transgenes that express the KP polypeptide with transgenes that express the P transposase to study KP-mediated regulation of the P-element family. The two principal assays for regulation, repression of gonadal dysgenesis and repression of sn^w mutability, demonstrated that H(hsp/KP) transgenes were approximately as effective as naturally occurring KP elements in regulating the P-element family. However, in either assay, the observed repression of transposase activity was much less than that seen in flies with the P cytotype. Previous studies with naturally occurring KP elements or P(hsp/KP) transgenes also reported incomplete repression of gonadal dysgenesis and sn^w mutability, even when the transgenes were subjected to heat shocks (RASMUSSON et al. 1993 ; SIMMONS et al. 1996 ). More complete repression was seen in a study with P transgenes in which the KP coding sequence was fused to the actin5C promoter (ANDREWS and GLOOR 1995 ). Vigorous transcription from this promoter may have led to abundant synthesis of the KP polypeptide in the germline tissues. (Another explanation is discussed below.) In nature it is possible that the strong repression characteristic of the P cytotype is due to the contributions of many KP elements scattered throughout the genome. However, an accumulation of KP elements is not a guarantee that the P cytotype will develop because some strains that have many KP elements in their genomes are incapable of repressing hybrid dysgenesis (BLACK et al. 1987 ; SIMMONS et al. 1990 ). The expression of each KP element is probably influenced by its genomic position. An element inserted near an enhancer that stimulates transcription in the germline could behave like the P(actin5C/KP) transgenes and repress P activity almost completely.

In this study, KP elements and transgenes repressed sn^w mutability no matter if they were maternally or paternally derived. The fact that they repressed when they were paternally derived demonstrates that repression can involve a purely zygotic effect. Maternally derived KP elements or transgenes did not seem to repress more strongly than paternally derived elements or transgenes. Furthermore, experiments with the strongest repressor transgene failed to produce any evidence for repression through a strictly maternal effect. Thus, these results suggest that KP acts zygotically rather than maternally to regulate transposase activity. These findings are consistent with those of LEMAITRE et al. 1993 who demonstrated that naturally occurring KP elements partially repress the expression of P-lacZ transgenes through a zygotic effect.

The absence of a true maternal effect is consistent with analyses of strains carrying transposase-producing transgenes. These analyses have shown that transposase activity is transmitted maternally in the egg cytoplasm of H(hsp/CP)/+ females, but not in that of P(ry⁺, 2-3)99B females (M. J. SIMMONS, K. J. HALEY and S. J. THOMPSON, unpublished results). One possible explanation for this difference is that the H(hsp/CP) transgene contains the last P-element intron, whereas the P(ry⁺, 2-3) transgene does not. KP elements also do not possess this intron. Thus, if the last intron is essential for maternal transmission of P-element RNA, we would not expect KP-mediated repression of hybrid dysgenesis to involve a strictly maternal effect. However, some experiments have indicated that naturally occurring KP elements repress gonadal dysgenesis through a maternal effect (RASMUSSON et al. 1993 ). The reason for this discrepancy between the results of the sn^w and GD assays for KP-mediated repression is not clear.

In this study we observed that KP elements and transgenes were more effective repressors of the transposase activity of P(ry⁺, 2-3)99B than that of H(hsp/CP)2, even though P(ry⁺, 2-3)99B was the stronger transposase source. The reduced susceptibility of H(hsp/CP)2 to KP-mediated repression might be due to some feature of its structure or genomic position; for example, the hsp70 promoter might impair the ability of the KP-repressor polypeptide to inhibit transcription of H(hsp/CP)2. Alternately, H(hsp/CP)2 might be less sensitive to KP-mediated repression because the 66-kD polypeptide it can produce interferes with the function of the KP repressor. The 66-kD polypeptide can repress transcription from the P-element promoter (LEMAITRE and COEN 1991 ). It might, therefore, reduce the synthesis of RNA from KP elements and transgenes and, thereby, limit the production of the KP polypeptide.

We had hypothesized that the KP polypeptide might interact with the 66-kD polypeptide or with the transposase, to produce a more effective repressor of P-element activity. A KP/66-kD or KP/transposase multimer might, for example, bind tightly to P-element DNA and either block the authentic transposase from attacking the DNA or silence transcription from the P promoter. Either of these mechanisms could effectively repress P-element activity. However, genetic tests of this hypothesis failed to provide any evidence to support it. Combinations of KP and CP transgenes, even when maternally transmitted, were no more effective in repressing sn^w mutability or gonadal dysgenesis than were KP transgenes by themselves; in some cases, the KP-CP transgene combinations were actually less effective repressors than the individual KP transgenes. Thus, although KP elements can contribute to P-element regulation, there is no evidence that they interact synergistically with complete P elements to do so.

This study did not specifically address the mechanism of KP-mediated repression. The KP polypeptide might interact with the transposase and impair its function, or it might interact with the P-element promoter and inhibit transcription. ANDREWS and GLOOR 1995 favored the first hypothesis because KP-mediated repression is compromised when a leucine zipper motif in the KP polypeptide is mutated. Leucine zippers have been implicated in various protein-protein interactions. However, in vitro analyses have indicated that the KP polypeptide binds to P-element DNA in the vicinity of the P promoter (LEE et al. 1996 , LEE et al. 1998 ) and that this binding is essential for repression of P transposition. Furthermore, truncated KP polypeptides lacking the leucine zipper bind to P DNA and repress transposition in vitro. Thus, it is plausible that in vivo the KP polypeptide inhibits P-element transcription by binding to the P promoter. The demonstration by LEMAITRE et al. 1993 that KP elements partially repress the expression of P-lacZ transgenes is consistent with this hypothesis.

A role for the KP polypeptide as a transcriptional repressor might explain why KP-mediated repression is a weak-to-moderate component of P-element regulation. As a transcriptional repressor, this polypeptide would feed back negatively to repress its own synthesis, thereby limiting its availability to repress transcription from transposase-encoding P elements. Strong regulation by P(actin5C/KP) transgenes has been observed (ANDREWS and GLOOR 1995 ). However, these transgenes lacked the P-element promoter and would therefore not be subject to the hypothesized negative feedback mechanism. Natural KP elements and KP transgenes in which the P promoter has been retained exhibit weak-to-moderate repression ability. The strong regulation that is characteristic of the P cytotype most likely involves some other factor.

	ACKNOWLEDGMENTS

Technical assistance was provided by Carina Belinco, Paul Kocian, Bradley Morrison, Dan Owens, Sarah Thompson, and Jeremy Stuart. The hobo transformation system was provided by 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.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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