Repression of P Element-Mediated Hybrid Dysgenesis in Drosophila Melanogaster

Inbred lines derived from a strain called Sexi were analyzed for their abilities to repress P element-mediated gonadal dysgenesis. One line had high repression ability, four had intermediate ability and two had very low ability. The four intermediate lines also exhibited considerable within-line variation for this trait; furthermore, in at least two cases, this variation could not be attributed to recurring P element movement. Repression of gonadal dysgenesis in the hybrid offspring of all seven lines was due primarily to a maternal effect; there was no evidence for repression arising de novo in the hybrids themselves. In one of the lines, repression ability was inherited maternally, indicating the involvement of cytoplasmic factors. In three other lines, repression ability appeared to be determined by partially dominant or additive chromosomal factors; however, there was also evidence for a maternal effect that reduced the expression of these factors in at least two of the lines. In another line, repression ability seemed to be due to recessive chromosomal factors. All seven lines possessed numerous copies of a particular P element, called KP, which has been hypothesized to produce a polypeptide repressor of gonadal dysgenesis. This hypothesis, however, does not explain why the inbred Sexi lines varied so much in their repression abilities. It is suggested that some of this variation may be due to differences in the chromosomal position of the KP elements, or that other nonautonomous P elements are involved in the repression of hybrid dysgenesis in these lines.     

Amplification of Kp Elements Associated with the Repression of Hybrid Dysgenesis in Drosophila Melanogaster

Mobile P elements in Drosophila melanogaster cause hybrid dysgenesis if their mobility is not repressed. One type of repression, termed P cytotype, is a complex interaction between chromosomes carrying P elements and cytoplasm and is transmitted through the cytoplasm only of females. Another type of repression is found in worldwide M' strains that contain approximately 30 copies per individual of one particular P element deletion-derivative termed the KP element. This repression is transmitted equally through both sexes. In the present study we show that biparentally transmitted repression increases in magnitude together with a rapid increase in KP copy-number in genotypes starting with one or a few KP elements and no other deletion-derivatives. Such correlated increases in repression and KP number per genome occur only in the presence of complete P elements, supporting the interpretation that they are probably a consequence of the selective advantage enjoyed by flies carrying the highest numbers of KP elements. Analysis of Q strains also reveals the presence of qualitative differences in the way the repression of dysgenesis is transmitted. In general, Q strains not containing KP elements have the P cytotype mode of repression, whereas Q strains with KP elements transmit repression through both sexes. This difference among Q strains further supports the existence of at least two types of repression of P-induced hybrid dysgenesis in natural populations of D. melanogaster.     

A Role for the Kp Leucine Zipper in Regulating P Element Transposition in Drosophila Melanogaster

The KP element can repress P element mobility in Drosophila melanogaster. Three mutant KP elements were made that had either two amino acid substitutions or a single amino acid deletion in the putative leucine zipper domain found in the KP polypeptide. Each KP element was expressed from the actin 5C proximal promoter. The wild-type control construct strongly repressed P element mobility, measured by the GD sterility and sn(w) mutability assays, in a position-independent manner. The single amino acid deletion mutant failed to repress P mobility regardless of its insertion site, while repression of P element mobility by the double amino acid substitution mutants was position dependent. The results show that the leucine zipper of the KP polypeptide is important for P element regulation. This supports the multimer-poisoning model of P element repression, because leucine zipper motifs are involved in protein-protein interactions.     

Regulation of P-element transposase activity in Drosophila melanogaster by hobo transgenes that contain KP elements

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(+), Delta2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry(+), Delta2-3)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.     

Molecular Analysis of the P-M Gonadal Dysgenesis Cline in Eastern Australian Drosophila Melanogaster

The latitudinal cline in P-M gonadal dysgenesis potential in eastern Australia has been shown to comprise three regions which are, from north to south respectively, P, Q, and M, with the P-to-Q and Q-to-M transitions occurring over relatively short distances. The P element complements of 30 lines from different regions of the cline were determined by molecular techniques. The total amount of P element-hybridizing DNA was high in all lines, and it did not correlate in any obvious way with the P-M phenotypes of individual lines. The number of potentially full-sized P elements per genome was high in lines from the P regions, but variable or low among lines from the Q and M regions, and thus declined overall from north to south. A particular P element deletion-derivative, the KP element, occurred in all the tested lines. The number of KP elements was low in lines from the P region, much higher in lines from the Q region, and highest among lines from the M region, thus forming a cline reciprocal to that of the full-sized P elements. Another transposable element, hobo, which has been described as causing dysgenic traits similar to those of P-M hybrid dysgenesis, was shown to be present in all lines and to vary among them in number, but not in any latitudinal pattern. The P-M cline in gonadal dysgenesis potential can be inferred to be based on underlying clinal patterns of genomic P element complements. P activity of a line was positively correlated with the number of full-sized P elements in the line, and negatively correlated with the number of KP elements. Among Q and M lines, regulatory ability was not correlated with numbers of KP elements.     

Inheritance of P-element regulation in Drosophila melanogaster.

The ability to repress P-element-induced gonadal dysgenesis was studied in 14 wild-type strains of D. melanogaster derived from populations in the central and eastern United States. Females from each of these strains had a high ability to repress gonadal dysgenesis in their daughters. Reciprocal hybrids produced by crossing each of the wild-type strains with an M strain demonstrated that repression ability was determined by a complex mixture of chromosomal and cytoplasmic factors. Cytoplasmic transmission of repression ability was observed in all 14 strains and chromosomal transmission was observed in 12 of them. Genomic Southern blots indicated that four of the strains possessed a particular type of P element, called KP, which has been proposed to account for the chromosomal transmission of repression ability. However, in this study several of the strains that lacked KP elements exhibited as much chromosomal transmission of repression ability as the strains that had KP elements, suggesting that other kinds of P elements may be involved.     

Repression of Hybrid Dysgenesis in Drosophila Melanogaster by Individual Naturally Occurring P Elements

Individual P elements that were genetically isolated from wild-type strains were tested for their abilities to repress two aspects of hybrid dysgenesis: gonadal dysgenesis and mutability of a double-P element-insertion allele of the singed locus (sn(w)). These elements were also characterized by Southern blotting, polymerase chain reaction amplification and DNA sequencing. Three of the elements were 1.1-kb KP elements, one was a 1.2-kb element called D50, and one was a 0.5-kb element called SP. These three types of elements could encode polypeptides of 207, 204, and 14 amino acids, respectively. Gonadal dysgenesis was repressed by two of the KP elements (denoted KP(1) and KP(6)) and by SP, but not by the third KP element (KP(D)), nor by D50. Repression of gonadal dysgenesis was mediated by a maternal effect, or by a combination of zygotic and maternal effects generated by the P elements themselves. The mutability of sn(w) was repressed by the KP(1) and KP(6) elements, by D50 and by SP, but not by KP(D); however, the SP element repressed sn(w) mutability only when the transposase came from complete P elements and the D50 element repressed it only when the transposase came from the modified P element known as Δ2-3. In all cases, repression of sn(w) mutability appeared to be mediated by a zygotic effect of the isolated P element. Each of the isolated elements was also tested for its ability to suppress the phenotype of a P-insertion mutation of the vestigial locus (vg(21-3)). D50 was a moderate suppressor whereas SP and the three KP elements had little or no effect. These results indicate that each isolated P element had its own profile of repression and suppression abilities. It is suggested that these abilities may be mediated by P-encoded polypeptides or by antisense P RNAs initiated from external genomic promoters.     

Repression of Hybrid Dysgenesis in Drosophila Melanogaster by Heat-Shock-Inducible Sense and Antisense P-Element Constructs

Sets of sense and antisense P-element constructs controlled by a heat-shock-inducible promoter were tested for their ability to repress manifestations of P-element activity in vivo. As a group, the antisense constructs repressed pupal lethality, a somatic manifestation of P activity, and this repression was significantly enhanced by heat shock. Three of the 11 antisense constructs also repressed gonadal dysgenesis, a manifestation of P activity in the female germ line; however, none had any effect on P-element-mediated mutability in the male germ line. Among the 13 different heat-shock-inducible sense constructs that were tested, those containing the KP and DP elements were strong repressors of pupal lethality, gonadal dysgenesis and P-element-mediated mutability; however, individual lines carrying these constructs varied in their ability to repress each of these traits, presumably because of genomic position effects. With the exception of the sense construct that contained a complete P element, none of the sense or antisense constructs repressed a lacZ reporter gene driven by the P-element promoter. Overall, the experimental results suggest that in nature, P-element activity could be regulated by P-encoded polypeptides and by antisense P RNAs.     

Genetic and molecular analysis of repression in the P-M system of hybrid dysgenesis in Drosophila melanogaster.

Twelve inbred lines derived from an M' strain of Drosophila melanogaster were used to study the repression of P-element-mediated hybrid dysgenesis. Initial assessments indicated that the lines differed in the ability to repress gonadal dysgenesis, and that this ability was highly correlated with the ability to repress snw hypermutability. Later assessments indicated that most of the lines with low or intermediate repression potential evolved to a state of higher repression potential; however, Southern analyses failed to reveal significant changes in the array of genomic P elements that could account for this evolution. In addition, none of the lines possessed the incomplete P element known as KP, which has been proposed to explain repression in some D. melanogaster strains. One of the lines maintained intermediate repression potential throughout the period of study (52 generations), indicating that the intermediate condition was not intrinsically unstable. Genetic analyses demonstrated that in some of the lines, repression potential was influenced by factors that were inherited maternally through at least two generations; however, these factors were not as influential as those in a classic P cytotype strain. Additional tests with a dysgenesis-inducing X chromosome called T-5 indicated that repression itself was mediated by a combination of maternal effects and paternally inherited factors that were expressed after fertilization. These tests also suggested that in some circumstances, the P transposase, or its message, might be transmitted through the maternal cytoplasm.     

Genomic P elements content of a wild M' strain of Drosophila melanogaster: KP elements do not always function as type II repressor elements

The P element is one of the best-studied DNA transposons as a model system to study evolution of mobile DNAs. The P element is a causative factor for P-M hybrid dysgenesis in Drosophila melanogaster and the P-M phenotype (P, Q, or M) has been thought to reflect genomic P elements content. Recent survey of natural populations showed that full-size P (FP) and KP elements are predominant in almost all current populations, irrespective of their phenotype variation. It was also suggested that some P elements are functionally inactive and their inactivation plays an important role in determining P-M phenotype. In order to know how the genomic P elements are inactivated, we characterized molecular features and insertion sites of them in an M' strain. We isolated 20 P elements, one FP, 15 KP, and four other internally deleted defective elements, all of which appeared thoroughly inactive. These FP and KP elements had canonical sequences in each case, but no mutations abolishing their function. In addition, they were mostly located in or within the vicinity of presumably active genes. Our results suggest that inactivation of P elements is associated with neither mutations nor constitutional suppression by heterochromatinization in M' strains and that only a few elements inserted in some special chromosomal regions are likely to be involved in determination of the phenotype of individual flies. Existence of many copies of canonical, but inactive, KP elements in the M' strain is inconsistent with the assumption that type II repression of the KP element is the main reason for its increase in the wild populations of D. melanogaster.     

The Influence of Nonautonomous P Elements on Hybrid Dysgenesis in Drosophila melanogaster

An inbred line of the M' strain Muller-5 Birmingham was studied for its abilities to affect P-M hybrid dysgenesis. This strain possesses 57 P elements, all of which are apparently defective in the production of the P transposase. In combination with transposase-producing elements, these nonautonomous elements can enhance or diminish the incidence of hybrid dysgenesis, depending on the trait that is studied. Dysgenic flies that have one or more paternally-derived chromosomes with these elements partially repress the instability of the P element insertion mutation, snw; however, such flies have elevated frequencies of another dysgenic trait, GD sterility, and also show distorted segregation ratios. An explanation is presented in which all of these phenomena are unified as manifestations of the kinetics of P element activation in the germ line. The progeny of Muller-5 Birmingham females exhibit partial repression of both snw instability and GD sterility. This repression appears to involve a factor that can be transmitted maternally through at least two generations. This mode of repression therefore conforms to the pattern of inheritance of the P cytotype, the condition that brings about nearly total repression of P element activity in some strains. Models in which this repression could arise from the nonautonomous P elements of Muller-5 Birmingham are discussed.     

Phenotypic stability of the P-M system in wild populations of Drosophila melanogaster

The P element appears to be one of the most recently invaded transposons of D. melanogaster. To study the dynamics and long-term fate of P elements in natural populations of D. melanogaster, 472 isofemale lines newly collected from 27 localities of Japan were examined for the P element-associated characteristics (abilities to induce and repress of P element transposition) and genomic P element composition (size classes and their numbers). There was variation in the P element-related phenotypes among local populations, but genomic P composition did not correlate strongly with the phenotype of each line: full-size P and KP elements predominated in their genomes (FP+ KP predominance). Comparison with previous results suggests a stability in the P-M system in local populations over about 15 years. In some populations, phenotypic stability for particularly long times was found: for 30 years or more Q strains predominated in Hikone and Tanushimaru, P or Q strains around Inakadate, and M' or Q strains around Tozukawa. There was no clear evidence of structural destruction underlying functional variation of P elements during this period. These results suggest that the current evolutionary status of P elements in the gene pool of D. melanogaster is not intermediary stage predicted by the original recent invasion hypothesis, and that several other factors such as the position effect play important roles.     

Localization of P elements, copy number regulation, and cytotype determination in Drosophila melanogaster

Seventeen highly-inbred lines of Drosophila melanogaster extracted from an M' strain (in the P/M system of hybrid dysgenesis) were studied for their cytotype and the number and chromosomal location of complete and defective P elements. While most lines were of M cytotype, three presented a P cytotype (the condition that represses P-element activity) and one was intermediate between M and P. All lines were found to possess KP elements and only eight to bear full-sized P elements. Only the lines with full-sized P elements showed detectable changes in their P-insertion pattern over generations; their rates of gain and of loss of P-element sites were equal to 0.12 and 0.09 per genome, per generation, respectively. There was no correlation between these two rates within lines, suggesting independent transpositions and excisions in the inbred genomes. The results of both Southern blot analysis and in situ hybridization of probes made from left and right sides of the P element strongly suggested the presence of a putative complete P element in region 1A of the X chromosome in the three lines with a P cytotype; the absence of P copy in this 1A region in lines with an M cytotype, favours the hypothesis that the P element inserted in 1A could play a major role in the P-cytotype determination. Insertion of a defective 2 kb P element was also observed in region 93F in 9 of the 13 M lines. The regulation of the P-element copy number in our lines appeared not to be associated with the ratio of full-length and defective P elements.     

Genetic Transformation of Drosophila melanogaster with an Autonomous P Element: Phenotypic and Molecular Analyses of Long-Established Transformed Lines

Following transformation of a Drosophila melanogaster true M strain with an autonomous P element, six lines were established and monitored for their molecular and phenotypic properties during a 4-yr period. The number of P elements increased with time in all the lines but the rate of increase differed among lines. Furthermore, degenerate elements arose in each of the lines during propagation. By the end of the 4th yr, the total number of elements in every line was similar to that of a very strong P strain.--At the phenotypic level, all of the transformed lines evolved high P activity, but only three developed complete or nearly complete regulatory ability. The other three lines attained only intermediate levels of regulation over the 4-yr period. One of these lines was particularly noteworthy. Although it contained as many as 55 P elements per genome (20 of which were potentially complete) and had extremely high P activity potential, it continued to exhibit limited regulatory ability. In addition, when females of this line were maintained at high temperatures, the ability to suppress P activity was even further diminished. A strain with this combination of molecular and phenotypic properties, in an apparently stable configuration, has not been previously described.--The results are discussed in the context of the possible role of degenerate elements in regulating P element expression.     

Long-term patterns of genomic P element content and P-M characteristics of Drosophila melanogaster in eastern Australia

A latitudinal cline in characteristics associated with the P DNA transposable element is well known in eastern Australian populations of Drosophila melanogaster. In order to survey the long-term patterns of P-M system characteristics and genomic P element content, we established 292 isofemale lines from 54 localities in 1996-1997 and evaluated them for gonadal dysgenesis (GD) sterility and the ratio of KP to full-size P elements (KP/FP ratio). The results were compared to those from collections made in 1983-1986 and 1991-1994. Over 10-14 years, 1) the cross A GD scores of the northern-middle populations declined dramatically; 2) the clinal pattern of the cross A* GD scores did not change; 3) the latitudinal pattern of the KP/FP ratio did not change. The results suggest that only a few P elements determine P-M characteristics and that there has been selection for genomes with fewer active P elements, but not for a great change in proportions of size classes.     

Genomic P elements and P-M characteristics of eastern Australian populations of Drosophila melanogaster

As part of our effort to monitor changes in the clinal pattern of P element-associated traits in eastern Australian Drosophila melanogaster, we investigated the genomic P elements of 293 isofemale lines collected in the period 1991–1994 from 45 localities. P elements were present in many copies in all genomes examined, with full-size P and KP element size classes accounting for the large majority. SR elements were not present in at least 92% of the lines tested. South of about 26° south Latitude (°SLat), the ratio of KP to full-size P elements (KP/P ratio) increased, correlating weakly with the P-M phenotypes of the populations, from moderately P populations (26–29°SLat) to M populations (37–38°SLat) North of 26°SLat, in weak P populations, the KP/P ratio was higher than between 26 and 29°Slat. The KP/P ratio appears to be higher in the northern populations than it was when previous studies were done. Overall, a high KP/P ratio among lines correlated roughly with a lack of P activity, but it also correlated with reduced repressor function. In a sample of 30 lines, a maternal effect of repressor function did not show a pattern with latitude, nor with KP/P ratio, nor with presence or absence of P activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

KP elements repress P-induced hybrid dysgenesis in Drosophila melanogaster.

Molecular and genetic analysis has revealed a specific P factor deletion derivative (the KP element) which is able to repress P-induced hybrid dysgenesis. All naturally occurring strains lacking the P cytotype (M') that were examined, throughout the world contain up to 30 copies of KP per haploid genome together with complete P factors. The KP element is derived from the P factor by an internal deletion of 1753 bp removing nucleotides 808-2560 and is transcribed to yield an abundant 0.8-kb poly(A)+ RNA with the coding capacity for an in-frame 207 amino acid polypeptide. Genetic crosses show that KP elements preferentially accumulate in the presence of P factors and suppress hybrid dysgenesis. Suppression is transmitted through both sexes and is thus distinct from the maternally transmitted P cytotype mode of suppression. The spread of KP elements is probably due to the continual selection of individuals with the highest numbers of KP elements in which P-induced hybrid dysgenesis is suppressed.     

Full-size P and KP elements predominate in wild Drosophila melanogaster

We analyzed the genomic P elements of 57 wild-derived Drosophila melanogaster isofemale lines from Africa, Australia and Asia. All carried many P sequences per genome, and the full-size P and the internally deleted KP elements were very common or predominant in the populations. The genomic content of full-size P and KP elements does not correlate well with the P transposition-inducing and -repressing abilities of a line. Our results show that a large majority of type I repressor elements are full-size P elements, and almost all type II repressor elements are KP elements in the natural populations of D. melanogaster from these parts of the world.     

The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.