Sterility and Hypermutability in the P-M System of Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

Inbred wild strains of Drosophila melanogaster derived from the central and eastern United States were used to make dysgenic hybrids in the P-M system. These strains possessed P elements and the P cytotype, the condition that represses P element transposition. Their hybrids were studied for the mutability of the P element insertion mutation, snw, and for the incidence of gonadal dysgenesis (GD) sterility. All the strains tested were able to induce hybrid dysgenesis by one or both of these assays; however, high levels of dysgenesis were rare. Sets of X chromosomes and autosomes from the inbred wild strains were more effective at inducing GD sterility than were sets of Y chromosomes and autosomes. In two separate analyses, GD sterility was positively correlated with snw mutability, suggesting a linear relationship. However, one strain appeared to induce too much GD sterility for its level of snw destabilization, indicating an uncoupling of these two manifestations of hybrid dysgenesis.     

Radiation and transposon-induced genetic damage in Drosophila melanogaster: X-ray dose-response and synergism with DNA-repair deficiency.

The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. The X-ray dose-response of 330-1320 rad was monitored for sterility, fecundity and partial X/Y chromosome loss among F2 progeny derived from the dysgenic cross of M strain females xP strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, Hw. This finding suggests that the lesions induced by both mutator systems which produce the synergistic effect are two-break events. The effect of increasing dose on the decline of fecundity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and in partial Y chromosome loss measured by the loss of the Bs marker alone or together with the y+ marker. Interaction was detected in the loss of the y+ marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P element mobility was assessed by measuring transmission distortion in dysgenic males derived from the II2 P strain. X-Irradiation of spermatids significantly increased the preferential elimination of the P-element-bearing second chromosome in mei-41, DNA-repair-deficient dysgenic males, but had no effect in their DNA-repair-proficient brothers. These findings indicate that the post-replication repair pathway is required for processing lesions induced by the combined effect of P element mobility and X-rays, and that the unrepaired lesions ultimately lead to chromosome loss.     

A high level of hybrid dysgenesis in Drosophila: High thermosensitivity, dependence on DNA repair, and incomplete cytotype regulation

Summary An unusually high level of P-M hybrid dysgenesis in Drosophila melanogaster is characteristic of hybrid offspring originating from both, A (M × ) and B (P × M) crosses of a subline of the Harwich P strain, termed H ^s . The novel properties induced by mobility of P elements carried by H ^s paternal chromosomes include: very high (over 95%) gonadal dysgenesis (GD) in both sexes at the low restrictive temperature of 21°C, and highly premature sterility when males are reared at 18°C and aged at 21°C. Although all three major chromosomes of the H ^s subline contributed to this atypical pattern of gonadal dysgenesis, chromosome 3 had the largest effect. Gonadal dysgenesis showed a temperature- and sex-dependent repression pattern by the defective P elements of Muller-5 Birmingham chromosomes; at 21°C there was virtually no repression of male sterility, but most effective repression of GD in females. At 29°C repression was effective in males, but declined in females. The high thermosensitive sterility, low fecundity, and premature aging of the male germ line were greatly exacerbated when males derived from either A or B crosses were deficient either in excision repair (mei-9 mutation) or in post-replication repair (mei-41 mutation). These findings demonstrate that both DNA repair pathways are essential for the repair of lesions induced by P element transposition and support the hypothesis that P element-induced chromosome breaks are responsible for the virtual abolition of the germ line. The relatively high premature sterility of cross B DNA repair-deficient males, reared at 18°C and aged at 21°C, indicates that there is incomplete cytotype regulation in H ^s subline hybrids.     

The interaction between X-rays and transposon mobility in Drosophila: hybrid sterility and chromosome loss

Genetic traits associated with P-M hybrid dysgenesis in Drosophila melanogaster were synergistically affected by X-rays. The interaction between damages induced by these two mutator systems was evident when sterility and X/Y chromosome loss were used as endpoints. No interaction was detected in partial chromosome loss, monitored by the loss of BS and y+ markers. The synergism in sterility, measured either as all-or-none or premature sterility (fecundity) was observed when male hybrids derived from different P strains fathers, namely Harwich or II2, were X-irradiated and the effects compared relative to similarly treated non-dysgenic hybrids. Brooding of sperm showed that the effects of ionizing radiation were ionizing radiation were dependent upon the stage of spermatogenesis during which cells were irradiated. The highly synergistic effect on sterility was found when either spermatids or spermatocytes, but not mature sperm, were irradiated with 550 rad of X-rays. These findings were consistent with the higher radiosensitivity of spermatocytes and spermatids to genetic damage and with the correlation between the incidence of sterility and aging of dysgenic hybrids. The latter observation was particularly evident in the case of Harwich P strain-derived male hybrids whose fertility was greatly reduced due to P element mobility. The synergistic effect of X-rays in these dysgenic hybrids resulted in the virtual abolition of the germ line, increasing the sterility from 50% of the untreated 9-10-day old males, to 85% of the treated males when spermatocytes were irradiated. The synergism observed between transposon mobility and ionizing radiation can best the attributed to an interaction between X-ray-induced and P element-induced chromosome breaks. This interpretation is consistent with the more than additive increase in X or Y chromosome loss in irradiated, Harwich P strain-derived hybrid sons. The induction of these events was 1.164% in dysgenic irradiated males as compared to 0.234% in X-irradiated nondysgenic hybrids and 0.40% in dysgenic untreated males. No synergism was observed in X/Y loss in hybrids derived from II2 P strain fathers where the frequency of the events due to P element mobility alone was only one tenth (0.037%) of that found in Harwich-derived hybrids.     

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 in Drosophila melanogaster: a new type of gonadal dysgenic sterility expressed at the pre-pupal stage

Two sublines, B-202 and B-207, which were derived from crosses between Or-R (M) females and Cy/Pm (P) males were found to cause a new type of gonadal dysgenic sterility, designated as the GD-3. GD-3 sterility showed a typical reciprocal cross effect under the P-M system. It was caused at the frequency close to 100% in dysgenic offsprings reared above 25 degrees C, of which gonads were morphologically clearly different from those of usual GD sterility, whereas there was no indication of GD-3 sterility at temperatures below 24 degrees C. Temperature sensitive period of GD-3 sterility was estimated to the prepupal stage by shift-down experiment. In the B-202 subline, the 2nd chromosomes marked with Pm alone carried GD-3 elements. Those of the B-207 subline, however, were estimated to locate both on the 2nd and 3rd chromosomes, acting synergistically with each other.     

P transposable element in Drosophila melanogaster: horizontal transfer]

The P transposable element family in Drosophila melanogaster is responsible for the syndrome of hybrid dysgenesis which includes chromosomal rearrangements, male recombination, high mutability and temperature sensitive agametic sterility (called gonadal dysgenesis sterility). P element activity is controlled by a complex regulation system, encoded by the elements themselves, which keeps their transposition rate low within the strain bearing P elements and limits copy number by genome. A second regulatory mechanism, which acts on the level of RNA processing, prevents P mobility to somatic cells. The oldest available strains, representing most major geographical regions of the world, exhibited no detectable hybridization to the P-element. In contrast, all recently collected natural populations that were tested carried P-element sequences. The available evidence is consistent with the hypothesis of a worldwide P-element invasion of D. melanogaster during the past 30 years. Timing and direction of the invasion are discussed. The lack of P-element in older strains of Drosophila melanogaster as well as in the species must closely related to Drosophila melanogaster, suggests that P entered the Drosophila melanogaster genome recently, probably by horizontal transfer from an other species. The analysis of P-element elsewhere in the genus Drosophila reveals that several more distantly related species carried transposable elements with sequences quite similar to P. The species with the best-matching P-element is D. willistoni. A P-element from this species was found to match all but one of the 2907 nucleotides of the Drosophila melanogaster P-element. The phylogenic distributions and the likely horizontal transfers of the two other Drosophila transposable elements are discussed.     

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.     

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.     

Chromosomal Effects on Mutability in the P-M System of Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

Two manifestations of hybrid dysgenesis were studied in flies with chromosomes derived from two different P strains. In one set of experiments, the occurrence of recessive X-linked lethal mutations in the germ cells of dysgenic males was monitored. In the other, the behavior of an X-linked P-element insertion mutation, snw, was studied in dysgenic males and also in dysgenic females. The chromosomes of one P strain were more proficient at causing dysgenesis in both sets of experiments. However, there was variation among the chromosomes of each strain in regard to the ability to induce lethals or to destabilize snw. The X chromosome, especially when it came from the stronger P strain, had a pronounced effect on both measures of dysgenesis, but in combination with the major autosomes, these effects were reduced. For the stronger P strain, the autosomes by themselves contributed significantly to the production of X-linked lethals and also had large effects on the behavior of snw, but they did not act additively on these two characters. For this strain, the effects of the autosomes on the X-linked lethal mutation rate suggest that only 1/100 P element transpositions causes a recessive lethal mutation. For the weaker P strain, the autosomes had only slight effects on the behavior of snw and appeared to have negligible effects on the X-linked lethal mutation rate. Combinations of chromosomes from either the strong or the weak P strain affected both aspects of dysgenesis in a nonadditive fashion, suggesting that the P elements on these chromosomes competed with each other for transposase, the P-encoded function that triggers P element activity. Age and sex also influenced the ability of chromosomes and combinations of chromosomes to cause dysgenesis.     

Quantitative Effects of P Elements on Hybrid Dysgenesis in Drosophila Melanogaster

Genetic analyses involving chromosomes from seven inbred lines derived from a single M' strain were used to study the quantitative relationships between the incidence and severity of P-M hybrid dysgenesis and the number of genomic P elements. In four separate analyses, the mutability of snw, a P element-insertion mutation of the X-linked singed locus, was found to be inversely related to the number of autosomal P elements. Since snw mutability is caused by the action of the P transposase, this finding supports the hypothesis that genomic P elements titrate the transposase present within a cell. Other analyses demonstrated that autosomal transmission ratios were distorted by P element action. In these analyses, the amount of distortion against an autosome increased more or less linearly with the number of P elements carried by the autosome. Additional analyses showed that the magnitude of this distortion was reduced when a second P element-containing autosome was present in the genome. This reduction could adequately be explained by transposase titration; there was no evidence that it was due to repressor molecules binding to P elements and inhibiting their movement. The influence of genomic P elements on the incidence of gonadal dysgenesis was also investigated. Although no simple relationship between the number of P elements and the incidence of the trait could be discerned, it was clear that even a small number of elements could increase the incidence markedly. The failure to find a quantitative relationship between P element number and the incidence of gonadal dysgenesis probably reflects the complex etiology of this trait.     

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.     

Gonadal Dysgenesis Reveals Sexual Dimorphism in the Embryonic Germline of Drosophila

In hybrid dysgenesis, sterility can occur in both males and females. At 27.5 degrees, however, we found that P element-induced germline death was restricted to females. This sex-specific gonadal dysgenesis (GD) is complete by the first larval instar stage. As such, GD at 27.5 degrees reveals the sexually dimorphic character of the embryonic germline. The only other known dimorphic trait of the embryonic germline is the requirement for ovo. ovo is required for germline development in females only and has been implicated in germline sex determination. Dominant mutations of ovo partially suppressed female GD. Although embryonic germ cells are undifferentiated and morphologically indistinguishable between males and females, the functional dimorphism seen in ovo requirement and GD at 27.5 degrees indicates that sexual identity in Drosophila germ cells is established in embryogenesis.     

Germline autonomous sterility of P-M dysgenic hybrids and their application to germline transfers in Drosophila melanogaster

The gonadal (GD) sterility of the P-M hybrid dysgenesis of Drosophila melanogaster was analyzed with reciprocal pole cell transfers. GD sterility was found to result from autonomous degeneration of germline cells; the death of individual germline cells could not be prevented by the surrounding tissues of nondysgenic flies. Germline cells of the M strains developed predominantly in the hybrid-dysgenic flies even at a low rate of GD sterility. The autonomous ability of germ plasm to induce functional germ cells was confirmed using hybrid-dysgenic hosts for transferring ectopically formed pole cells. The advantages of germline transfers using the hybrid-dysgenic hosts are discussed.     

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.     

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.     

Induction of unstable mutations in Drosophila melanogaster by microinjection of DNA from oncogenic viruses into the embryonic polar plasma. III. Genetic instability in the absence of an intact P-element

DNA of simian adenovirus Sa7 injected into polar plasma of early Drosophila melanogaster y1snw*; bw; st stock embryos induced one to three unstable visible X-linked mutations in the absence of intact P-element. Numerous mutational events (reversions, new mutations) occur only in four precisely destabilized by the Sa7 DNA loci of X-chromosome and take place during 4-5 generations; in the next generations the level of instability decreased. At the same time, Sa7 DNA induced reversions and new allelic mutations in the snw mutant locus, without exogenous intact P-element.     

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 MELANOGASTER: Sterility Resulting from Gonadal Dysgenesis in the P-M System

Crosses between two types of strains, called P and M, characteristically give high frequencies of F(1) sterility and other aberrant traits. Previous studies indicated that, in addition to the direction of the parental cross, many factors influence the manifestation of this phenomenon known as "hybrid dysgenesis."-The present study is concerned with the characteristics of GD (gonadal dysgenesis) sterility associated with the P-M system and its temperature dependence. Female sterility is accompanied by a complete absence of egg-laying, and this is not attributable to an inability to mate. Thus, it seems likely that sterility results from a defect in gametogenesis of hybrid individuals. This conclusion is supported by the morphological and cytological observations presented in an accompanying paper (Schaefer, Kidwell and Fausto-Sterling 1979).-A narrow, critical, developmental temperature range was found in which both female and male sterility rose sharply from a low level to a high maximum. The critical range was 27 to 29 degrees for males, slightly higher than the range of 24 to 26 degrees for females. Two other dysgenic traits, male recombination and transmission ratio distortion, were affected by developmental temperature, but temperature response curves were quite different from those for sterility. The temperature-sensitive stage for female sterility occurs during embryonic and early larval development.-GD sterility is compared and contrasted with SF sterility, another specific type of non-Mendelian sterility resulting from a different interstrain dysgenic interaction.     

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.