Establishment and Maintenance of the P Cytotype Associated With Telomeric P Elements in Drosophila melanogaster

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

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

P elements inserted near the left telomere of the X chromosome are associated with the P cytotype, a maternally transmitted condition that strongly regulates the activity of the P transposon family in some strains of Drosophila. The regulatory abilities of two such elements, TP5 and TP6, are stable in homozygous stocks over many generations. However, these regulatory abilities are attenuated when the telomeric P elements are transmitted through heterozygous females, and they are utterly lost when the elements are transmitted through males. Paternally transmitted telomeric P elements reacquire regulatory ability when they pass through a female germ line. This reacquisition is enhanced if the females in which it occurs came from mothers who carried a telomeric P element. The enhancement has two components: (1) a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element ("presetting" or the "pre-P cytotype") and (2) a zygotic effect associated with inheritance of the mother's telomeric P element. One telomeric P element can enhance the reacquisition of another's regulatory ability. When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop strong regulatory abilities in a few generations. However, some of the stocks do not attain the regulatory ability of the original population.

THE P elements of Drosophila melanogaster have been extensively used as tools in genetic analysis (ENGELS 1989 ). These elements were discovered through their involvement in hybrid dysgenesis, a phenomenon observed in the offspring of crosses between different strains of Drosophila (KIDWELL et al. 1977 ). P elements are found in P strains but not in M strains. Crosses between P males and M females produce offspring with a syndrome of abnormalities in the germ line, including a high frequency of sterility and elevated mutation rates. These traits are usually not seen in the offspring of crosses between P females and M males. The difference between reciprocal crosses therefore indicates that the phenomenon of hybrid dysgenesis is regulated by a maternally transmitted condition characteristic of P strains. This condition is called the P cytotype (ENGELS 1979A ). M strains have a complementary condition called the M cytotype, which is permissive for hybrid dysgenesis. Genetic analyses have shown that the P cytotype depends on maternal transmission of the P elements themselves (ENGELS 1979A , ENGELS 1979B ; SVED 1987 ).

For many years it was thought that the repression of hybrid dysgenesis by the P cytotype involved polypeptides encoded by the P elements. Complete P elements, 2.9 kb long, encode an 87-kD polypeptide, the P transposase, which catalyzes P-element excision and insertion (KARESS and RUBIN 1984 ). Incomplete P elements, <2.9 kb because some DNA sequences have been deleted, do not encode the P transposase. However, they can be excised and transposed if a complete P element that makes the P transposase is present in the genome (ENGELS 1984 ). The excision of particular incomplete P elements has been used to monitor transposase activity in genetic experiments (ENGELS 1989 ). In addition to the transposase, complete P elements encode a 66-kD repressor polypeptide (LASKI et al. 1986 ; RIO 1990 ). This polypeptide is translated from an incompletely spliced P-element RNA. In the soma, only the 66-kD polypeptide is made. In the germ line, both the 66-kD repressor and the 87-kD transposase are produced.

These facts have led to the hypothesis that the P cytotype is a state in which the 66-kD polypeptide more or less completely represses the synthesis or activity of the P transposase (ROCHE et al. 1995 ). However, this hypothesis has been called into question by the discovery that incomplete P elements situated near the left telomere of the X chromosome are powerful regulators of the P-element family (MARIN et al. 2000 ; STUART et al. 2002 ). Because of their structure, these elements cannot produce the 66-kD polypeptide, although they may produce smaller repressor polypeptides like, for example, the KP element, which produces a polypeptide that binds to P elements and represses their transposition (LEE et al. 1998 ). However, unlike KP, particular telomeric P elements are not geographically widespread (STUART et al. 2002 )—a feature that would be expected if natural selection had favored them in Drosophila populations. Thus, repression by the telomeric P elements may not involve P-encoded polypeptides. Other mechanisms involving the organization of chromatin around the telomeric P elements have been proposed to explain their regulatory properties (ROCHE and RIO 1998 ; RONSSERAY et al. 1998 , RONSSERAY et al. 2001 ; STUART et al. 2002 ).

One key feature of the regulation mediated by the telomeric P elements is that it shows a reciprocal-cross effect. Telomeric P elements repress hybrid dysgenesis only when they are transmitted maternally. When transmitted paternally, a telomeric P's regulatory ability is lost (STUART et al. 2002 ; SIMMONS et al. 2004 , this issue). Regulation by the telomeric P elements therefore follows the same pattern as regulation by the P cytotype. This parallel is the primary reason for associating telomeric P elements with the P cytotype.

In this article we consider questions about how the P cytotype is established and maintained. Is the P cytotype stably maintained when a telomeric P element is transmitted maternally? Is it reestablished when a paternally transmitted telomeric P element is returned to the female germ line? What factors influence the process of reestablishment? We address these questions by studying the regulatory abilities of two incomplete P elements, TP5 and TP6, inserted at the same site in the TAS repeats near the left telomere of the X chromosome. TP5 is 1.8 kb long and TP6 is 1.9 kb long. Neither of these elements encodes a known repressor polypeptide; however, they both have strong abilities to repress transposase activity in the germ line (STUART et al. 2002 ; SIMMONS et al. 2004 , this issue).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and mutability assay for P transposase activity:
Genetic symbols for the Drosophila stocks are explained in LINDSLEY and ZIMM 1992 or in other references cited in the text. Experimental cultures were maintained on a standard cornmeal-molasses-dried yeast medium at 25° unless stated otherwise. The X-linked telomeric P elements TP5 and TP6 were derived from natural populations by recombination with pure M strains (STUART et al. 2002 ). Subsequently, these elements were combined on the same X chromosome with the double P-insertion mutation singed weak (sn^w), a hypomorphic allele of the singed bristle locus. In the presence of the P transposase, sn^w becomes hypermutable due to the excision of one or the other of the two incomplete P elements inserted in the 5' region of the singed gene (ROIHA et al. 1988 ). When one P element is excised, the sn^w allele changes to sn^e, an allele with an extreme mutant phenotype. When the other P element is excised, sn^w changes to sn⁺, an allele that is phenotypically indistinguishable from wild type. To detect these changes, males in which they were occurring were mated individually to C(1)DX, y f females with attached-X chromosomes and their sons were scored for bristle phenotype. Sons were counted on day 13 or 14 after the test cultures were established and again on day 17. The combined frequency of sn^e and sn⁺ flies among those scored within a culture was used to estimate the sn^w mutation rate.

Polymerase chain reactions:
Crude DNA solutions were obtained from single flies by squashing the flies in 100 µl buffer with a sterile toothpick (GLOOR and ENGELS 1992 ). Samples (2 µl) from these solutions were used to seed amplification reactions catalyzed by Taq DNA polymerase. Each reaction contained the four deoxyribonucleotides, polymerase, polymerase buffer, MgCl₂, and appropriate primers. The reactions were carried through 30 cycles of amplification, with each cycle consisting of 1 min at 92°, 2 min at 60°, and 3 min at 72°. During the first cycle, the time at 72° was extended by 4 min. Reaction products were analyzed on 0.7% agarose gels by electrophoresis. Two types of reactions were carried out: (1) amplification with a primer complementary to a segment of the P element's terminal inverted repeat and (2) amplification with an element-specific primer (either TP5 or TP6 specific) and a primer complementary to a segment near the 3' end of the P element. The sequences and positions of these primers within the complete P element are given in STUART et al. 2002 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Stocks with telomeric P elements repress transposase activity consistently over time:
Previous analyses have demonstrated that stocks carrying either the TP5 or the TP6 telomeric P elements repress P-element excisions in the germ line. To investigate the consistency of this repression over time, we tested homozygous TP5 and TP6 stocks at 6-month intervals for repression of transposase-catalyzed excisions from sn^w, a double P-insertion allele of the X-linked singed bristle locus. In males, the sn^w allele causes a weak malformation of the bristles. When one or the other of the P elements inserted in the sn^w allele is excised by transposase action, alleles with different phenotypes—either extremely malformed bristles (sn^e) or essentially wild-type bristles (sn⁺)—are created. The frequency of these phenotypes estimates the sn^w mutation rate, which can be used as an index of transposase-catalyzed P-excision activity in the father's germ line. Repression of this activity is indicated by a reduction in the sn^w mutation rate.

Females were sampled from homozygous TP5 w sn^w (w, white eyes), TP6 w sn^w, and control w sn^w stocks on three occasions separated by 6-month intervals. The TP5 w sn^w and TP6 w sn^w stocks were created 3 years prior to sampling by making a single X chromosome homozygous in each case (STUART et al. 2002 ). During and before the sampling period, these stocks were maintained at 21° by mass transfers of adult flies to new cultures every generation. The only P elements present in these stocks were the two incomplete P elements inserted in the singed locus, an incomplete P element tightly linked to singed (ROIHA et al. 1988 ), and a telomeric P element, which was also incomplete. Because no complete P elements were present, the stocks were not selected for repression of transposase activity during this time. The collected (TP) sn^w females were crossed en masse to males homozygous for H(hsp/CP)2, a hobo transgene on chromosome 2 that encodes the P transposase (SIMMONS et al. 2002A ), and their (TP) sn^w; H(hsp/CP)2/+ sons were assayed for sn^w mutability. Each son was crossed to 3 or 4 C(1)DX, y f (y, yellow body; f, forked bristles) females carrying attached-X chromosomes, and the male progeny were scored for the weak singed, extreme singed, and wild bristle phenotypes.

The results of these experiments are shown in the top of Table 1. The control sn^w mutation rates, which ranged from 0.500 to 0.533, are consistent with previous estimates for flies carrying the H(hsp/CP)2 transposase source (SIMMONS et al. 2002A ; STUART et al. 2002 ). Both the TP5 and the TP6 stocks strongly repressed sn^w mutability. At all three sampling times, the mutation rates for the flies with either of the telomeric P elements were low compared to the controls. Thus, during the year-long sampling period, the stocks with the telomeric P elements maintained a strong ability to repress transposase activity similar to that observed when they were first tested [2.5 years earlier; see STUART et al. 2002 (Table 4)].

The repression ability associated with a telomeric P element is lost by passage through the male germ line:
Previous analyses have indicated that telomeric P elements lose their ability to repress transposase activity when they are transmitted through the male germ line (STUART et al. 2002 ). To verify this result, males collected from the TP5 w sn^w, TP6 w sn^w, and control w sn^w stocks at the second time point in the study described above were crossed to C(1)DX, y f ; H(hsp/CP)2 females and their sn^w; H(hsp/CP)2/+ sons were assayed for sn^w mutability. The bottom of Table 1 presents the results of these experiments. The control mutation rate, 0.571, is somewhat higher than the corresponding rate from the experiment in which the sn^w allele was maternally derived. This greater value might be due to maternal transmission of the transposase activity encoded by the H(hsp/CP)2 transgene (SIMMONS et al. 2002B ). The mutation rates of the TP5 and TP6 stocks are indistinguishable from this higher control rate. Thus, the ability of a telomeric P element to repress sn^w mutability is lost when that element is transmitted from father to son.

Paternally transmitted telomeric P elements regain their ability to repress transposase activity when they are reestablished in homozygous stocks:
To determine if paternally transmitted telomeric P elements can regain their regulatory abilities when they are returned to the female germ line, we "extracted" TP w sn^w (TP is TP5 or TP6) X chromosomes from homozygous stocks by crossing individual males to C(1)DX, y f females from a pure M strain and then made the extracted X chromosomes homozygous using an FM7 balancer X chromosome marked with the semidominant mutation Bar (B) eyes. This work was initiated at the same time as the analysis of sample 1 in Table 1. Sons from the cross involving the C(1)DX, y f females were mated to FM7/sc⁷ l (sc, scute bristles; l, recessive lethal) females from a pure M stock, and their TP w sn^w/FM7 daughters were backcrossed to TP w sn^w males carrying the extracted chromosome to establish a homozygous TP w sn^w stock. Each of these stocks was maintained in small, mass-mated cultures without selection for repression of hybrid dysgenesis. At generations 2, 7, and 11, females from these stocks were mated to H(hsp/CP)2 males to obtain TP w sn^w; H(hsp/CP)2/+ sons, which were tested for germ-line sn^w mutability by crossing them individually to C(1)DX, y f females.

Table 2 summarizes the results of these tests. The M strain controls show that the H(hsp/CP)2 transgene induced a high rate of sn^w mutability (0.532–0.577). All of the lines extracted from the TP5 and TP6 stocks were able to repress this mutability significantly. In the first test after they were made homozygous, most of the lines repressed sn^w mutability to a level below 0.10. However, a few lines (e.g., TP5.5, TP6.1, TP6.3, TP6.8, and TP6.9) were less effective as repressors, with mutabilities ranging from 0.141 to 0.405. PCR with TP5- and TP6-specific primers was used to determine if the telomeric P elements were present in 10–20 test males from three of these lines (TP5.5, TP6.1, and TP6.3); all the males in these samples proved to carry the appropriate telomeric P element.

The extracted lines were retested for repression of sn^w mutability in generations 7 and 11. All the lines except TP6.1 and TP6.3 repressed sn^w mutability to a level below 0.10, and most of them repressed it to a level below 0.05. The observed mutabilities for TP6.1 and TP6.3 ranged from 0.121 to 0.284. In generation 13, males from these two lines were examined by PCR for the presence of the TP6 element. All the tested males (15 from TP6.1 and 14 from TP6.3) proved to carry this element.

These experiments demonstrate that telomeric P elements that have been passed through the male germ line can regain repression ability after being made homozygous. However, sometimes the original level of repression ability associated with a telomeric P element is not achieved.

Telomeric P elements maintain repression ability when they are transmitted maternally:
We conducted a series of genetic experiments to investigate the role of the female germ line in the establishment and maintenance of repression by TP5 and TP6. Each experiment was performed at three different times to assess the reproducibility of the results. The first and second of these replications coincided with the tests of samples 2 and 3 in Table 1.

The first type of experiment was designed to determine if the repression ability of the telomeric P element could persist through three generations of maternal transmission. During two of these generations, the element was heterozygous. Homozygous sn^w females were collected from the TP5, TP6, and control stocks and mated to y sn^w or FM6, y dm B males; the y locus is tightly linked to the left telomere of the X chromosome and FM6 is a balancer X chromosome. The (TP) w sn^w/y sn^w or (TP) w sn^w/FM6 heterozygous daughters from these crosses were mated to y sn^w males, and their (TP) w sn^w/y sn^w daughters were mated to homozygous H(hsp/CP)2 males. The y sn^w (yellow body) and (TP) w sn^w (non-yellow body) sons from these last matings—both heterozygous for the H(hsp/CP)2 transposase source—were then individually tested for sn^w mutability by crossing them to C(1)DX, y f females.

Table 3 summarizes the results of the three replications of this experiment. In the first two replications, only the y⁺ flies from the last mating were tested for sn^w mutability. In the third replicate, both the y⁺ and y flies from this mating were tested. The y flies, which did not carry a telomeric P element, provided an opportunity to see if repression ability could be transmitted through the egg cytoplasm independently of the telomeric P element itself.

The mutation rates for the control flies ranged from 0.471 to 0.554 and were similar to previous estimates of sn^w mutability obtained using the H(hsp/CP)2 transposase source. The mutation rates for the TP5 and TP6 flies were significantly less than the control rates. When a maternally transmitted TP5 was present in the tested flies, the rates ranged from 0.033 to 0.177, and when a maternally transmitted TP6 was present, they ranged from 0.116 to 0.301. Thus, sn^w mutability was repressed by telomeric P elements that had been transmitted through females for three generations; in two of these generations, the elements were heterozygous. Furthermore, TP5 appeared to be a stronger repressor than TP6.

The mutation rates from the y flies in replicate III were similar to the control mutation rates—if anything, slightly higher—even though in two cases, a telomeric P element was present in the mothers of the tested males. Thus, as STUART et al. 2002 showed with a slightly different type of experiment, repression ability is not transmitted through the egg cytoplasm independently of the telomeric P elements themselves.

Paternally transmitted telomeric P elements reacquire repression ability during one generation in a female:
Having shown that a telomeric P element retains repression ability during transmission through heterozygous females, we next analyzed whether a paternally inherited telomeric P element could acquire repression ability during one generation in a female (Fig 1). In this experiment homozygous w sn^w females were crossed to y sn^w or FM6, y dm B males and their w sn^w/y sn^w or w sn^w/FM6 daughters were crossed to TP w sn^w males. The two types of females that were produced by these F₁ crosses, (a) TP w sn^w/w sn^w females, which had white eyes, and (b) TP w sn^w/y sn^w or TP w sn^w/FM6 females, which had red eyes, were mated to homozygous H(hsp/CP)2 males, and the resulting F₃ males were tested for sn^w mutability. From the type a females, the males that lacked the telomeric P element were used as controls. These males were distinguished from their TP sn^w brothers by performing PCR with an element-specific primer after they had mated. From the type b females, only the sons that carried the telomeric P element were tested for sn^w mutability. These males could be distinguished from their non-TP brothers by body color (y⁺ rather than y).

View larger version (19K): In this window In a new window Download PPT slide   Figure 1. Reacquisition of repression ability by a paternally inherited telomeric P element (TP).

The results of three replica experiments are summarized in Table 4. The mutation rates for the control flies, which did not carry a telomeric P element, ranged from 0.522 to 0.636. The rates for the flies that carried telomeric P elements, derived from either type a or type b females, were consistently lower. For the flies carrying TP5, the rates ranged from 0.291 to 0.462, and the median value was 0.338. For the flies carrying TP6, the rates ranged from 0.394 to 0.458, and the median was 0.435. In a comparable experiment, STUART et al. 2002 observed similar mutation rates—0.288 for TP5 and 0.400 for TP6. These results therefore indicate that a telomeric P element that came from a male and passed through a female for one generation reacquires some ability to repress sn^w mutability. On average TP5 seems to reacquire more repression ability than TP6. In six of seven comparisons based on the data in Table 4 and the data from STUART et al. 2002 , flies carrying TP5 had lower mutation rates than flies carrying TP6 (P = 0.054 by the sign test).

Reacquisition of repression ability is enhanced by the maternal effect of a telomeric P element:
To determine if the reacquisition of repression ability by a paternally derived telomeric P element is influenced by maternally transmitted factors associated with a telomeric P element, we crossed TP_i w sn^w males to TP_j w sn^w/y sn^w or TP_j w sn^w/FM6 F₁ females that were obtained by mating homozygous TP_j w sn^w females with y sn^w or FM6 males (Fig 2). The F₂ TP_i w sn^w/y sn^w or TP_i w sn^w/FM6 daughters from these crosses were then mated to males homozygous for the H(hsp/CP)2 transgene, and their TP_i w sn^w; H(hsp/CP)2/+ sons, identified by having y⁺ body color, were tested for sn^w mutability. The objective of these experiments was to see if TP_j, the telomeric P element in the F₁ females, enhanced the reacquisition of repression ability by the paternally derived element TP_i through a strictly maternal effect, i.e., one transmitted to the F₂ females independently of TP_j itself. RONSSERAY et al. 1993 called this effect the "pre-P cytotype." We call it "presetting" and refer to TP_j as the "presetting" telomeric P element.

View larger version (23K): In this window In a new window Download PPT slide   Figure 2. Maternal effect of a telomeric P element (TPj) on reacquisition of repression ability by a paternally inherited telomeric P element (TPi).

The results of three replica experiments are presented in Table 5. To evaluate them, we must refer to the data showing the reacquisition of repression ability by a paternally derived telomeric P element when no presetting telomeric P element was present; these data are in the bottom third of Table 4. A comparison of the data in Table 4 and Table 5 indicates that the reacquisition of repression ability by a paternally derived TP5 or TP6 was significantly enhanced by a strictly maternal effect of either element. In the absence of a presetting telomeric P element, the mutation rates for TP5 ranged from 0.291 to 0.374 (bottom third of Table 4), whereas with TP5 as the presetting element, they ranged from 0.069 to 0.094 (Table 5, top left), and with TP6 as the presetting element, they ranged from 0.126 to 0.249 (Table 5, bottom left). In the absence of a presetting telomeric P element, the mutation rates for TP6 ranged from 0.399 to 0.458 (bottom third of Table 4), whereas with TP5 as the presetting element, they ranged from 0.154 to 0.300 (Table 5, top right), and with TP6 as the presetting element, they ranged from 0.213 to 0.341 (Table 5, bottom right). For both of the paternally derived telomeric P elements, z-tests established that the mutation rates when presetting elements were present were significantly less than the corresponding rates when these elements were absent.

The data in Table 5 indicate that a strictly maternal effect of either TP5 or TP6 can enhance reacquisition of repression ability by either of these telomeric P elements—that is, the reacquisition of repression ability in a female is preset if her mother carried a telomeric P element even though that P element is not present in the female herself. In these experiments TP5 reacquired greater repression ability than TP6, and TP5 was the more effective presetting element. It is possible that presetting is due to the inheritance of one of the sn^w alleles that had coexisted with the telomeric P element in the F₁ (presetting) females. However, this hypothesis is not supported by the data from replicate III, in which the FM6 balancer chromosome was used to preclude transmission of sn^w from the F₁ females to the F₂ females. Presetting therefore seems to involve a feature of the maternal cytoplasm that is transmitted to the F₂ females independently of either the telomeric P element or the sn^w allele.

Reacquisition of repression ability is enhanced by a maternally inherited telomeric P element:
The reacquisition of repression ability by a paternally derived telomeric P element is enhanced by the strictly maternal effect of a presetting P element. What happens if this strictly maternal effect is combined with the zygotic effect of the presetting P element? To answer this question, we crossed TP_i w sn^w males with TP_j w sn^w/y sn^w or TP_j w sn^w/FM6 F₁ females to obtain TP_i w sn^w/TP_j w sn^w F₂ females, which were mated to homozygous H(hsp/CP)2 males (Fig 3). The two types of sons, TP_i w sn^w; H(hsp/CP)2/+ and TP_j w sn^w; H(hsp/CP)2/+, were then tested for sn^w mutability. Males carrying different telomeric P elements were distinguished by PCR with element-specific primers after the males had mated.

View larger version (22K): In this window In a new window Download PPT slide   Figure 3. Maternal and zygotic effects of a maternally inherited telomeric P element (TPj) on reacquisition of repression ability by a paternally inherited telomeric P element (TPi).

The results of three replica experiments are shown in Table 6. First, we consider the cases in which TP_i and TP_j were identical in the F₂ females (top half of Table 6). These cases are similar to the situation in the TP5 and TP6 stocks because the F₂ females that were crossed to H(hsp/CP)2 males were homozygous for one or the other of the telomeric P elements. In the case where TP_i = TP_j = TP5, the observed sn^w mutation rates from the tested males were low, similar to values obtained by sampling a homozygous TP5 stock (Table 1). Likewise, in the case where TP_i = TP_j = TP6, the mutation rates were similar to those obtained by sampling a homozygous TP6 stock. Thus, under these conditions, repression ability is much like that in the original TP5 and TP6 stocks.

Next, we consider the cases in which TP_i and TP_j were different in the F₂ females (bottom half of Table 6). In either case (TP5 paternally derived, TP6 maternally derived, or vice versa), the paternally derived telomeric P element invariably showed more repression ability, i.e., a lower sn^w mutation rate, than it did in the simple test for reacquisition of repression ability (cf. Table 4; P = 0.016 by the sign test) or in the test for reacquisition of repression ability with a presetting effect (cf. Table 5; P = 0.016 by the sign test). Furthermore, in five of six comparisons, the maternally derived telomeric P element showed more repression ability than it did in the test for simple maternal transmission of repression ability (cf. Table 3; P = 0.094 by the sign test). Thus, these results suggest that, in the F₂ females, the maternally and paternally derived telomeric P elements mutually facilitate the establishment of repression ability and that the maternally derived P element has both a presetting and a zygotic effect on the acquisition of repression ability by the paternally derived P element.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Stocks homozygous for the telomeric P elements TP5 or TP6 have maintained a strong ability to repress hybrid dysgenesis from the time they were first tested to the time of the experiments reported here—a period of 3.5 years. The regulatory abilities of these elements are therefore stable in stocks over many generations. However, genetic analysis demonstrates that both TP5 and TP6 lose their regulatory abilities when they are transmitted through the male germ line. If this loss were irreversible, the repression abilities of the TP5 and TP6 stocks would be expected to dissipate over time since an increasing fraction of the X chromosomes in these stocks would have passed through males. The fact that homozygous TP5 and TP6 stocks maintain strong repression ability over many generations indicates that paternally inherited telomeric P elements are restored to full repression ability when they pass through the female germ line. In every generation the regulatory abilities of these elements must therefore be reestablished.

Before attempting to dissect the reestablishment process, we monitored the repression abilities of TP5 and TP6 transmitted maternally through three generations—from homozygous females to heterozygous females to heterozygous females and then to hemizygous males in which repression ability was measured. Under these conditions, both TP5 and TP6 maintained repression ability, although this ability was less than that seen in males derived directly from homozygous TP females. Transmission through heterozygous females therefore attenuates the repression ability of a telomeric P element; furthermore, the attenuation is greater for TP6, which is the weaker of the two repressing elements.

Paternally transmitted telomeric P elements reacquire repression ability by passing through the female germ line. If the female does not carry another telomeric P element and if her mother did not carry such an element, the extent of this reacquisition is rather limited—and it is more limited for TP6 than for TP5. Nevertheless, the repression ability of telomeric P elements in males derived from females in which reacquisition takes place is significant.

Reacquisition of repression ability is enhanced if the females in which the reacquisition occurs came from mothers who carried a telomeric P element. This enhancement has two components: (1) a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element and (2) a zygotic effect associated with inheritance of the mother's telomeric P element.

RONSSERAY et al. 1993 referred to the first component as the pre-P cytotype; we call it presetting. This component suggests that either a product of the mother's telomeric P element or an effect of this element on some aspect of chromatic organization is transmitted through the egg cytoplasm to the females in which the paternally derived telomeric P element is partially restored to regulatory function. However, previous analyses with TP5 and TP6 have shown that repression ability itself is not transmitted through the egg cytoplasm independently of the telomeric P element (STUART et al. 2002 ). Furthermore, the presetting effect does not seem to be mediated by an sn^w allele transmitted from the females that carry the presetting telomeric P element, and the presetting effect is not element specific. TP5 can preset the reacquisition of repression ability by a paternally derived TP6 or vice versa. However, TP5 is more receptive to the presetting effect—i.e., it reacquires more repression ability than TP6—and TP5 is also the better presetting element—i.e., it more strongly enhances the reacquisition of repression ability by a paternally derived TP5 or TP6. RONSSERAY et al. 1993 also obtained evidence that the pre-P cytotype is not element specific.

The strictly maternal effect of a presetting telomeric P element is further enhanced by a zygotic effect of that element. Maximal restoration of regulatory function is seen when a paternally inherited telomeric P element passes through a female who also inherited a telomeric P element from her mother. Under these circumstances, the repression ability of the paternally derived element is almost as strong as that seen in the homozygous TP5 and TP6 stocks. The repression ability of the maternally derived element is also boosted. Thus, maternally and paternally derived elements mutually facilitate the establishment of the P cytotype in the female germ line.

When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop a strong ability to repress hybrid dysgenesis. In a sample of 10 TP5 and 9 TP6 stocks, only two TP6 lines, TP6.1 and TP6.3, failed to reach the repression ability characteristic of the original TP6 stock. PCR experiments indicated that the TP6 element was present in each of these lines; thus, their diminished repression ability cannot be attributed to loss of the telomeric P element. It might, however, be due to a genetic factor—possibly a feature of the telomere—that impairs repression by the P element. This factor might have been segregating in the TP6 stock, and when lines were extracted from it, the factor might have been retained in some of them. A few test cultures in samples from the original TP6 stock yielded sn^w mutation rates as high as or higher than the average rates seen in the TP6.1 and TP6.3 lines. Thus, the moderate repression ability of these lines could reflect variation that was already present within the TP6 stock. Alternately, it could reflect a change that occurred when the lines were created.

The P cytotype is the paramount system for regulating P elements in the germ line. Genetic analysis has shown that this system is associated with P elements inserted near the left telomere of the X chromosome. It is not clear to what extent P elements inserted at other genomic locations may contribute to the P cytotype; however, some evidence suggests that they do (RONSSERAY et al. 1998 , RONSSERAY et al. 2001 ). Regulation by the P cytotype is established by the combined maternal and zygotic effects of telomeric P elements and, once established, is maintained stably over time. The mechanistic basis of this regulation is not known. One possibility is that it involves repression by some aspect of the chromatin organization of telomeres—a type of telomere position effect. However, the pre-P cytotype, or presetting effect, raises the possibility that a transmissible product of telomeric P elements, either a polypeptide or an RNA, is involved. It is not known if either TP5 or TP6 encodes a repressor polypeptide such as the one produced by the KP element. However, unlike KP, neither TP5 nor TP6 is widespread in natural populations of Drosophila (STUART et al. 2002 ). A broad geographic distribution of these elements would be expected if they had been spread by natural selection based on the production of repressor polypeptides. The repression abilities of TP5 and TP6 might therefore not be related to their polypeptide-coding capacities. It is also not known if either TP5 or TP6 produces an RNA that could function as a regulatory factor, perhaps by interfering with the expression of transposase-encoding RNAs. Further work will be needed to determine if the P cytotype involves a product of telomeric P elements.

	ACKNOWLEDGMENTS

Mark Liszewski provided technical help. Financial support was provided by National Institutes of Health grant GM-40263 and the Minnesota Medical Foundation.

Manuscript received June 24, 2003; Accepted for publication September 28, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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