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.     

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.     

Comparison of the regulation of P elements in M and M' strains of Drosophila melanogaster

M and M' strains of Drosophila melanogaster in the P-M system of hybrid dysgenesis were compared in two series of tests, with the following results. (1) The singed-weak hypermutability regulation test showed that M' strains had lower P excision rates than M strains, suggesting that P-elements repression must occur in M' strains although it is not detectable by gonadal dysgenesis assays. (2) The evolution of mixed P+M and mixed P+M' populations was compared, using a strong P strain. The P+M cultures invariably evolved in a few generations into strong P cultures, while the P+M' cultures evolved into P-type cultures with reduced P-factor potentials. However, after 30 generations of culture, both these types of mixed cultures had similar P copy numbers, suggesting that regulation of copy number had occurred in them.     

The P cytotype in Drosophila melanogaster: a maternally transmitted regulatory state of the germ line associated with telomeric P elements

The incomplete P elements TP5 and TP6 are inserted in the TAS repeats near the left telomere of the Drosophila melanogaster X chromosome. These telomeric P elements repress P-induced gonadal dysgenesis and germ-line hypermutability in both sexes. However, their capacity to repress hypermutability is lost when they are transmitted patroclinously in a cross. TP5 and TP6 do not repress P-element activity in somatic cells, nor do they alter the somatic or germ-line phenotypes of P-insertion alleles. In the germ line, these elements suppress the phenotype of a P-insertion allele of the singed gene that is evoked by other P elements, presumably because these other elements encode repressor polypeptides. This suppression is more effective when the telomeric P elements are inherited maternally. Regulation by telomeric P elements parallels that of the P cytotype, a state that represses P-element activity in some strains of Drosophila. This state exists only in the germ line and is maternally transmitted along with the P elements themselves. Regulation by known repressor P polypeptides is not restricted to the germ line and does not require maternal transmission of the relevant P elements. Regulation by telomeric P elements appears to be epistatic to regulation by repressor P polypeptides.     

Cytotype Regulation of P Transposable Elements in Drosophila melanogaster: Repressor Polypeptides or piRNAs?

The telomeric P elements TP5 and TP6 are associated with the P cytotype, a maternally inherited condition that represses P-element-induced hybrid dysgenesis in the Drosophila germ line. To see if cytotype repression by TP5 and TP6 might be mediated by the polypeptides they could encode, hobo transgenes carrying these elements were tested for expression of mRNA in the female germ line and for repression of hybrid dysgenesis. The TP5 and TP6 transgenes expressed more germ-line mRNA than the native telomeric P elements, but they were decidedly inferior to the native elements in their ability to repress hybrid dysgenesis. These paradoxical results are inconsistent with the repressor polypeptide model of cytotype. An alternative model based on the destruction of P transposase mRNA by Piwi-interacting (pi) RNAs was supported by finding reduced P mRNA levels in flies that carried the native telomeric P elements, which are inserted in a known major piRNA locus.     

Gonadal Dysgenesis Determinants in a Natural Population of DROSOPHILA MELANOGASTER

Three populations of Drosophila melanogaster from northern California were surveyed for the ability to produce and resist gonadal dysgenesis in the P-M system of hybrid dysgenesis. Males from all three populations produced low to moderate levels of gonadal dysgenesis in crosses to Oregon-R M females. Most females had the P cytotype, but the M cytotype occurred occasionally. The three populations could not be statistically differentiated from one another, but were easily distinguished from populations from Australia and Wisconsin on the basis of gonadal dysgenesis potential. The California populations had higher levels of M cytotype than did the Wisconsin population. Thirteen X chromosomes and 11 pairs of autosomes were extracted from one of the California populations, using a modification of the standard balancer chromosome technique to suppress hybrid dysgenesis during extraction. All lines produced strongly skewed sterility distributions in crosses to M-strain females, and mean levels of sterility were less than 50%. There was evidence of nonadditive interactions between the autosomes. Most extraction lines had the P cytotype, but M and intermediate cytotypes were observed. Some of the intermediate cytotypes were stable over time. Lines were tested at two different times after extraction. Some lines evolved higher sterility potential as they were kept in the laboratory, even in the presence of P cytotype. The results point out a number of deficiencies in current genetic and population genetic models of hybrid dysgenesis and imply that gonadal dysgenesis is unlikely to be an important evolutionary force in this population.     

Hybrid Dysgenesis in Drosophila Simulans Lines Transformed with Autonomous P Elements

The molecular and phenotypic analysis of several previously described P element-transformed lines of Drosophila simulans was extended in order to determine whether they had the potential to produce a syndrome of P-M hybrid dysgenesis analogous to the one in Drosophila melanogaster. The transformed line with the highest number of P elements at the beginning of the analysis, DsP pi-5C, developed strong P activity potential and P element regulation, properties characteristic of D. melanogaster P strains. The subsequent analysis of sublines derived from 34 single pair matings of DsP pi-5C revealed that they were heterogeneous with respect to both their P element complements and P activity potentials, but similar with respect to their regulatory capabilities. The subline with the highest P activity, DsP pi-5C-27, was subsequently used as a reference P strain in the genetic analysis of the D. simulans transformants. In these experiments, the reciprocal cross effect was observed with respect to both gonadal sterility and male recombination. As in D. melanogaster, the induction of gonadal sterility in D. simulans was shown to be temperature-dependent. Molecular analysis of DsP pi-5C-27 revealed that it has approximately 30 P elements per genome, at least some of which are defective. The number of potentially complete P elements in its genome is similar to the number in the D. melanogaster P strain, Harwich-77. Overall our analysis indicates that P-transformed lines of D. simulans are capable of expressing the major features of P-M hybrid dysgenesis previously demonstrated in D. melanogaster and that P elements appear to behave in a similar way in the two sibling species.     

The P-M and the 23.5 Mrf (Hobo) Systems of Hybrid Dysgenesis in Drosophila Melanogaster Are Independent of Each Other

Strains of Drosophila melanogaster bearing the male recombination factor 23.5 MRF induce hybrid dysgenesis in a way which is highly reminiscent of the P-M system, and, most probably, causally related to the activity of the transposable element hobo. We have investigated potential interactions between the two systems of hybrid dysgenesis by studying mixed lines derived from bidirectional crosses between 23.5 MRF and P strains, and analyzed their potentials to induce or suppress the occurrence of dysgenesis. All new lines possess the P induction abilities, as determined by two different procedures, and have also acquired a P cytotype. In contrast, some of them lost their ability to induce the non-P-M dysgenesis, as well as to suppress the action of 23.5 MRF. This loss of the 23.5 MRF induction abilities parallels the selective loss of full-length hobo elements from the genome of these lines, providing further substantiation to the notion that the 23.5 MRF activity is directly linked to this transposable element.     

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.     

Hybrid Dysgenesis-Induced Quantitative Variation on the X Chromosome of Drosophila Melanogaster

To determine the ability of the P-M hybrid dysgenesis system of Drosophila melanogaster to generate mutations affecting quantitative traits, X chromosome lines were constructed in which replicates of isogenic M and P strain X chromosomes were exposed to a dysgenic cross, a nondysgenic cross, or a control cross, and recovered in common autosomal backgrounds. Mutational heritabilities of abdominal and sternopleural bristle score were in general exceptionally high-of the same magnitude as heritabilities of these traits in natural populations. P strain chromosomes were eight times more mutable than M strain chromosomes, and dysgenic crosses three times more effective than nondysgenic crosses in inducing polygenic variation. However, mutational heritabilities of the bristle traits were appreciable for P strain chromosomes passed through one nondysgenic cross, and for M strain chromosomes backcrossed for seven generations to inbred P strain females, a result consistent with previous observations on mutations affecting quantitative traits arising from nondysgenic crosses. The new variation resulting from one generation of mutagenesis was caused by a few lines with large effects on bristle score, and all mutations reduced bristle number.     

Hybrid Dysgenesis in DROSOPHILA MELANOGASTER: Nature and Inheritance of P Element Regulation

The genetic determination of the control of resistance or susceptibility to germ line changes mediated by P elements was studied in two strains and in derivatives of crosses between them. One strain, characterized as true M, completely lacked P elements. The second strain, pseudo-M (M'), carried a number of P elements, but these did not have the potential to induce the gonadal sterility that is associated with P-M hybrid dysgenesis. Individuals from the true M strain were invariably unable to suppress P factor activity (i.e., all daughters of outcrosses of M females and P males were sterile). In contrast, individuals from the M' strain showed variable degrees of suppression that were manifested in a wide range of gonadal sterility frequencies in standard tests. This continuous distribution pattern was reproducible for more than 25 generations.--The results of the genetic analysis indicate that a strain with a variable degree of suppression of gonadal dysgenesis is not necessarily in a transient state between the extreme conditions of P and M cytotype. A large variance in the ability to suppress gonadal dysgenesis with a mean value intermediate between the extremes of P and M cytotype may be a relatively stable strain characteristic. No reciprocal cross effect was observed in the suppression of sterility of F1 females from M X M' matings. Thus, the existence of M' strains indicates a Mendelian component in P element regulation and suggests that cytotype, which has an extrachromosomal aspect, may be only one of perhaps several mechanisms involved in regulation. Analysis of the effects of individual chromosomes from the M' strain showed that each chromosome contributed to the reduction of gonadal dysgenesis in the progeny of test matings. The results are consistent with a one-component titration model for P element regulation.     

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.     

Telomeric P elements associated with cytotype regulation of the P transposon family in Drosophila melanogaster

P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry(+), delta2-3)99B. For H(hsp/CP)2 and P(ry(+), delta2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry(+), delta2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype.     

Maternal Repression of the P Element Promoter in the Germline of Drosophila Melanogaster: A Model for the P Cytotype

The transposition of P elements in Drosophila melanogaster is regulated by products encoded by the P elements themselves. The P cytotype, which represses transposition and associated phenomena, exhibits both a maternal effect and maternal inheritance. The genetic and molecular mechanisms of this regulation are complex and not yet fully understood. In a previous study, using P-lacZ fusion genes, we have shown that P element regulatory products were able to inhibit the activity of the P promoter in somatic tissues. However, the repression observed did not exhibit the maternal effect characteristic of the P cytotype. With a similar approach, we have assayed in vivo the effect of P element regulatory products in the germline. We show that the P cytotype is able to repress the P promoter in the germline as well as in the soma. Furthermore, this repression exhibits a maternal effect restricted to the germline. On the basis of these new observations, we propose a model for the mechanism of P cytotype repression and its maternal inheritance.     

Spread of the autonomous transposable element hobo in the genome of Drosophila melanogaster

The transposable element hobo has been introduced into the previously empty Drosophila melanogaster strain Hikone so that its dynamics can be followed and it can be compared with the P element. Five transformed lines were followed over 58 generations. The results were highly dependent on the culture temperature, the spread of hobo element being more efficient at 25 degrees C. The multiplication of hobo sequences resulted in a change in the features of these lines in the hobo system of hybrid dysgenesis. The number of hobo elements remained low (two to seven copies) and the insertions always corresponded to complete sequences. Our findings suggest that, despite their genetic similarities, P and hobo elements differ in many aspects, such as mobility and regulation mechanisms.      

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.     

Cytotype regulation by telomeric P elements in Drosophila melanogaster: interactions with P elements from M' strains

P strains of Drosophila are distinguished from M strains by having P elements in their genomes and also by having the P cytotype, a maternally inherited condition that strongly represses P-element-induced hybrid dysgenesis. The P cytotype is associated with P elements inserted near the left telomere of the X chromosome. Repression by the telomeric P elements TP5 and TP6 is significantly enhanced when these elements are crossed into M′ strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M′ P elements must coexist in females for this enhanced repression ability to develop. However, once established, it is transmitted maternally to the immediate offspring independently of the telomeric P elements themselves. Females that carry a telomeric P element but that do not carry M′ P elements may also transmit an ability to repress dysgenesis to their offspring independently of the telomeric P element. Cytotype regulation therefore involves a maternally transmissible product of telomeric P elements that can interact synergistically with products from paternally inherited M′ P elements. This synergism between TP and M′ P elements also appears to persist for at least one generation after the TP has been removed from the genotype.     

Rapid spread of transposable p elements in experimental populations of Drosophila melanogaster

The invasion of P elements in natural populations of Drosophila melanogaster was modeled by establishing laboratory populations with 1%, 5% and 10% P genomes and monitoring the populations for 20 generations. In one experiment, the ability of flies to either induce or suppress gonadal sterility in different generations was correlated with the amount of P element DNA. In a second experiment, the percentage of genomes that contained P elements, and the distribution of P elements among individual flies was monitored. The ability to induce gonadal dysgenesis increased rapidly each generation. However, the increase in P cytotype lagged behind by five to ten generations. The total amount of P element DNA and the frequency of flies containing P elements increased each generation. The number of P elements within individual genomes decreased initially, but then increased. Finally, the distribution of P elements within the genomes of individuals from later generations varied considerably, and this pattern differed from the parental P strain. These results suggest that the interaction between the assortment and recombination of chromosomal segments, and multiplicative transposition could result in the rapid spread of P elements in natural populations.     

Evolution of Hybrid Dysgenesis Potential following P Element Contamination in Drosophila Melanogaster

P elements were introduced into M strain genomes by chromosomal contamination (transposition) from P strain chromosomes under conditions of P-M hybrid dysgenesis. A number of independently maintained contaminated lines were subsequently monitored for their ability to induce gonadal (GD) sterility in the progeny of reference crosses, over a period of 60 generations, in two experiments. The efficiency of chromosomal contamination was high; all tested lines acquired P elements following the association of M and P chromosomes in the same genome for a single generation. All the contaminated lines also sustained an initial unstable phase, marked by high frequencies of transposition and sterility within lines, in the absence of P element regulation. Subsequently, each of the lines rapidly evolved to one of three relatively stable strain types whose phenotypic and molecular properties correspond rather closely to those of the P, Q and M' strains that have previously been characterized. The numbers and structures of P elements and the presence or absence of P element regulation during the early generations appeared to be critical factors determining the subsequent course of evolution. On the basis of GD sterility frequencies, both the mean level of P activity, and the average capacity for P element regulation, were reduced in lines raised at 25 degrees, relative to those raised at 20 degrees, during the early generations. This latter result is consistent with the expectation that natural selection will tend to modify the manifestation of dysgenic traits, such as high temperature sterility, which cause a reduction of fitness. However, overall, stochastic factors appeared to predominate in determining the course of evolution of individual lines.