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.     

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.     

Temporal distribution of P elements in Drosophila melanogaster strains from natural populations in Japan

Genetic and molecular investigations were carried out with 10 Japanese Drosophila melanogaster strains on P-M system of hybrid dysgenesis. The strains used here were collected in the years from 1952 to 1984 from various natural populations, and have been maintained in our laboratory. The whole genomic Southern hybridization was performed by using the 2.9-kb P element and the internal fragments as probes. Five strains possessed no P element copy and the other 5 strains possessed mainly incomplete P elements which had internal deletions. The former 5 strains were M, 2 of the latter were Q, and the remaining 3 were M' strains. Hikone-R, collected in 1952, had no P element copy, while Hikone-H, collected in 1957, was the earliest observed to possess multicopies of an incomplete P element. This revealed that P elements in Drosophila melanogaster were present more than 30 years ago in Japan, as already shown to have been the case on the American continent.     

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.     

Evolution des potentialités dysgénésiques du système P-M dans des populations expérimentales mixtes P,Q, M et M′ de Drosophila melanogaster

Change of hybrid dysgenesis potentials in P-M system of Drosophila melanogaster — In the P-M system of hybrid dysgenesis, three types of Drosophila melanogaster strains have been described in relation to hybrid gonadal sterility: P, Q and M. When M strain females were mated with P strain males, the P factors resulted in variable level of sterility in their progeny. The Q strain had no significant potential for sterility in any hybrid strain combination. To observe the dynamics of chromosomal contamination, due to the P transposable elements in different genetic context, mixed populations of these three types of strains were set up and monitored for their gonadal sterility potential during at least 30 generations.A first set of 16 experimental populations was set up; each of these was initiated with a mixture of 50% of individuals from the Harwich strain (a strong P strain) and 50% of individuals from a M or Q strain collected in natural populations. The M activity levels of these strains corresponded to a range from 100% to 0%. For all of these populations, the M activity potential disappeared during the five first generations. However, the P activity potential reached an equilibrium level positively correlated with the M activity potential level introduced at the beginning. It is proposed that the force of invasion of the P type by chromosomal contamination through the transposition of the P elements is dependent on the copy number of P sequences present on the chromosome of the M strain in competition.A second set of 18 experimental populations was set up with a mixture of P, M or Q strains collected in France between 1965 and 1982 (this period probably corresponds to the invasion of the P elements in France). After 30 generations, all of these populations (except one) had lost all dysgenic sterility potentiality and seemed to be of the Q type. Taking into account the results obtained from the two sets of experimental populations, the temporal and geographical distribution of P elements in the world could be explained by a progressive diffusion of autonomous P elements, from America with an accompanying decrease of their ability to transpose.

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Ce travail a été réalisé dans le cadre de l'A.T.P. Biologie des populations et de l'UA 693 du C.N.R.S.     

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.     

Dynamic equilibrium between insertion and excision of P elements in highly inbred lines from an M′ strain of Drosophila melanogaster

Six highly inbred lines of Drosophila melanogaster extracted from an M strain (in the P/M system of hybrid dysgenesis) were studied for the evolution of the number and chromosomal location of complete and defective P elements through generations 52–200. These lines possessed full-sized P elements but differed in their cytotype (M or P). Three lines with P cytotype and full-sized P elements at site 1A had a constant P copy number over generations with low rates of insertion and excision. Three lines with M cytotype and at least one full-sized P element accumulated P copies over the generations and reached a plateau near generation 196, at which rates of transposition and excision were equal to 1.2 × 10^–3 to 3 × 10^–3 events per element per generation. At that time these three lines still presented an M cytotype, produced transposase, and were able to regulate P copy number. The similarity at equilibrium between insertion and excision rates was exactly what was expected from theoretical models for a self-regulated element. The large number of generations necessary to attain the equilibrium in copy number indicates, however, that caution may be de rigueur when testing theoretical models of copy-number containment based on transposition and excision-rate comparison.     

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.     

Evolutionary flux of P element regulation in a Drosophila melanogaster hybrid dysgenesis cline.

Clines of P-induced hybrid dysgenesis provide a means for monitoring the evolution of transposition repression over space and time. We have studied the molecular and phenotypic profiles of flies taken from a 2900 km cline along the eastern coast of Australia, which had previously been characterized over 10 years ago as having P populations in the north, Q populations at central sites and M' populations in the south. We have found that Q and M' populations of flies have increased their range within the cline at the expense of P lines. Q populations were found to be in the north of the cline and M' populations in the south. Some of the northern Q lines transmit repression through both sexes and type I deletion elements have been isolated from them. We suggest that these elements are responsible for Q type repression. The results support our model that populations made up of Q individuals with strong biparentally transmitted repression form an evolutionarily stable strategy for the repression of hybrid dysgenesis in Drosophila 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.     

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.     

Components of Hybrid Dysgenesis in a Wild Population of DROSOPHILA MELANOGASTER

Hybrid dysgenesis is a condition found in certain interstrain hybrids of Drosophila melanogaster caused by the interaction of chromosomal and cytoplasmic factors. Germ-line abnormalities, including sterility, high mutability and male recombination, appear in the affected individuals. There are at least two distinct systems of hybrid dysgenesis. We examined a Wisconsin wild population in two consecutive years to determine the distribution of the chromosomal P factor and the extrachromosomal M cytotype that together cause one kind of hybrid dysgenic sterility. The P factor was found to be very common in the population, with all three major chromosomes being polymorphic for it. This polymorphism was strongly correlated with variability for male recombination elements, suggesting that these two traits are part of the same system of hybrid dysgenesis. There was a slight tendency for the P factor to be lost in lines taken from this population and inbred in the laboratory for many generations. A large-scale search for the M cytotype, which causes susceptibility to the P factor, showed that it is present in the population at only very low frequencies. Further evidence that the population is mostly immune to the action of the P factor was our finding of a general lack of dysgenic sterility in the wild flies themselves. However, we were able to isolate several wild strains that consistently showed the M cytotype. In some cases, the frequency of the M cytotype could be maintained in these lines, but it could not usually be increased by artificial selection. Some possible consequences of hybrid dysgenesis for the evolutionary biology of Drosophila are suggested.     

Hybrid dysgenesis in Drosophila melanogaster: predominance of Q factor in Japanese populations and its change in the laboratory

The P-M system of hybrid dysgenesis was investigated in two Japanese regions, Katsunuma and Ohzu. Most strains were Q in that they did not produce appreciable gonadal dysgenesis with P males or with M females. A few strains produced ambiguous results. Some of these were unstable and on later testing were P, Q or M. Eleven laboratory strains which had been derived from various sites in Japan and had been maintained for 8 to 27 years were also examined. Eight (including all four that were 27 years old) were M, two were Q, and one which had been kept as a large cage population showed some P activity.     

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.     

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.     

Hybrid dysgenesis in natural populations ofDrosophila melanogaster in Japan

The P-M system of hybrid dysgenesis inDrosophila melanogaster was investigated on the basis of gonadal dysgenesis, using 1,590 strains from 28 natural populations in Japan, and 20 populations from Southeast Asia, the Pacific area and Africa. Strong P strains were found sporadically in several populations in Japan. Few strong M strains were observed. Q strains were present at a high frequency in most populations. Thus, most populations in these areas were regarded as Q populations. The distribution of the P element and the evolution of P, Q and M populations are also discussed.     

Artificial and epigenetic regulation of the I factor, a nonviral retrotransposon of Drosophila melanogaster

The I factor (IF) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.     

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.     

DYNAMICS OF P-M HYBRID DYSGENESIS IN P-TRANSFORMED LINES OF DROSOPHILA SIMULANS

An autonomous P element from Drosophila melanogaster was introduced by microinjection into the germ line of its sibling species, Drosophila simulans. The invasion kinetics of P elements was studied in seven independent lines over 60 generations, using gel blotting, in situ hybridization, and dysgenic crosses. Some of the main phenotypic and molecular characteristics of P-M hybrid dysgenesis were observed, i.e., gonadal dysgenesis (GD sterility), chromosome rearrangements, and the occurrence of degenerate P elements. At least four lines reached a steady state with complete or nearly complete P-element regulation but with a moderate number of P elements (10–24 per haploid genome) and P activity (10–35% GD sterility). This failure to obtain strong P strains in D. simulans could be due to experimental conditions, a host-dependent component of P transposition, or more severe selection against the deleterious effects of this transposon.     

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.