P Element Insertions and Rearrangements at the Singed Locus of Drosophila Melanogaster

DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.     

Alteration of chromosome structure in ovarian nurse cells of Drosophila melanogaster by hybrid dysgenesis

The impact of hybrid dysgenesis on the chromosome structure of Drosophila melanogaster ovarian nurse cells was studied. In the examined lines and interlinear hybrids (including those yielded by dysgenic crosses in the P-M and I-R systems of hybrid dysgenesis), disturbed chromosome synapsis was revealed. The disturbance was somewhat similar to that observed in interspecific hybrids. Quantitative analysis showed that the mean frequency of nuclei with defective chromosome pairing ranged from 60.4 to 76%. FISH analysis of ovarian nurse chromosomes of Canton S x Berlin hybrids showed differences in the label localization in asynaptic homologs of arm 2L, which probably results in disrupted homolog pairing and reveal interlinear differences in localization of mobile genetic elements. Our results conform to Sved's model stating that hybrid dysgenesis is based on disorganization of the germline nuclear space.     

Visible mutations induced by P-M hybrid dysgenesis in Drosophila melanogaster result predominantly from P element insertions.

This study supplied no evidence that P-M hybrid dysgenesis is a general release mechanisms for transposon movement. Newly induced mutations (23 singed, three yellow, and one white) were generated by hybrid dysgenesis and were molecularly analyzed for the presence or absence of P element insertions. Only one dysgenically-induced insertion mutation out of 27 analyzed was the result of a non-P insert; this frequency is not statistically above the non-dysgenic control level. Thus, it appears that individual transposable elements families are independently regulated.     

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.     

The molecular basis of P-M hybrid dysgenesis: The role of the P element,a P-strain-specific transposon family

We have shown previously that four of five white mutant alleles arising in P-M dysgenic hybrids result from the insertion of strongly homologous DNA sequence elements. We have named these P elements. We report that P elements are present in 30–50 copies per haploid genome in all P strains examined and apparently are missing entirely from all M strains examined, with one exception. Furthermore, members of the P family apparently transpose frequently in P-M dysgenic hybrids; chromosomes descendant from P-M dysgenic hybrids frequently show newly acquired P elements. Finally, the strain-specific breakpoint hotspots for the rearrangement of the π₂ P X chromsome occurring in P-M dysgenic hybrids are apparently sites of residence of P elements. These observations strongly support the P factor hypothesis for the mechanistic basis of P-M hybrid dysgenesis.     

Dysgenesis-Induced Instability of Rosy Locus Transformation in DROSOPHILA MELANOGASTER: Analysis of Excision Events and the Selective Recovery of Control Element Deletions

Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome. The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus. We have labeled this allele, sdry+. This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes. Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA. The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA. In addition to loss of rosy locus function, such excisions affect the scalloped locus expression.--A second dysgenesis experiment was carried out involving an ry+ transposon inserted in polytene section 16D on the X chromosome. A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%.--A successful pilot experiment is described that utilizes dysgenic perturbation of the sdry+ allele to select for small deletions of the 5' noncoding region of the rosy locus.     

Requirement for Cell-Proliferation Control Genes in Drosophila Oogenesis

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat+ gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco (dcole88) prevents egg development when homozygous in the germ line, whereas the dco18 allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allowed the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surrounded normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybrid dysgenesis-induced response to selection in Drosophila melanogaster

In Drosophila melanogaster, the P-M and I-R systems of hybrid dysgenesis are associated with high rates of transposition of P and I elements, respectively, in the germlines of dysgenic hybrids formed by crossing females of strains without active elements to males of strains containing them. Transposition rates are not markedly accelerated in the reciprocal, nondysgenic hybrids. Previous attempts to evaluate the extent to which hybrid dysgenesis-mediated P transposition contributes to mutational variance for quantitative characters by comparing the responses to selection of P-M dysgenic and nondysgenic hybrids have given variable results. This experimental design has been extended to include an additional quantitative trait and the I-R hybrid dysgenesis system. The selection responses of lines founded from both dysgenic and nondysgenic crosses showed features that would be expected from the increase in frequency of initially rare genes with major effects on the selected traits. These results differ from those of previous experiments which showed additional selection response only in lines started from dysgenic crosses, and can be explained by the occasional occurrence of large effect transposable element-induced polygenic mutations in both dysgenic and nondysgenic selection lines. High rates of transposition in populations founded from nondysgenic crosses may account for the apparently contradictory results of the earlier selection experiments, and an explanation is proposed for its occurrence.     

Analysis of Dysgenesis-Induced Lethal Mutations on the X Chromosome of a Q Strain of DROSOPHILA MELANOGASTER

The Q strain known as v6 was tested for its ability to induce X-linked lethal mutations in male and female hybrids from crosses with M strains in the P-M system of hybrid dysgenesis. All measurements of the mutation rate were made on the X chromosome derived from the v6 strain. The lethal rate for young hybrid males from the cross M female X v6 male was 1.11% per chromosome. For older males, it was only 0.44%, suggesting that there is less mutational or more repair activity in the germ cells of the older males or that mutant cells are selectively eliminated as the hybrid males age. The lethal rate for hybrid females from comparable crosses was approximately the same for both ages that were tested. However, it was substantially less than the rate for the hybrid males--only 0.26% per chromosome. Genetically identical hybrid females from reciprocal crosses also showed a low mutation rate, 0.13% per chromosome. Again, there was no difference between young and old flies. Mapping experiments established that most of the lethal mutations that were recovered from the male and female hybrids were located in two regions on the X chromosome, one between bands 14B13 and 15A9 , the other between bands 19A1 and 20A , which encompasses the maroonlike locus. More refined mapping of the lethals in the maroonlike region demonstrated that the vast majority of these affected a single gene located in band 19C4 . Cytological analysis of the lethal chromosomes revealed that several carried rearrangements, including inversions, duplications and deficiencies. Chromosome breakage occurred primarily in bands 14D1 -3 and 18F- 20A , and most of the breaks in the latter segment were located in 19C . However, rearrangements involving 19C and mutations of the gene in 19C4 were mutually exclusive events. In situ hybridization of a P element probe to the chromosomes of v6 demonstrated that P elements reside at a minimum of five sites on the X chromosome. These P element sites correspond to the mutational and breakage hot spots on that chromosome. The combined genetic and cytological data imply that most of the X-linked lethal mutations that occur in M X v6 hybrids are due to local P element action. Consideration of these and other data suggest that v6 is a weak P strain in the P-M system of hybrid dysgenesis and that other Q strains might also be regarded in this way.     

Radiation-induced and transposon-induced chromosome damage in Drosophila: translocations and transmission distortion

The possible interaction between X-ray- and transposon-induced chromosome damage was monitored in the P-M system of hybrid dysgenesis in Drosophila melanogaster. One- to two-day-old F1 dysgenic males originating from a cross between M strain females and P strain males were irradiated with 5.5 Gy (550 rad) or used as controls to monitor X-Y translocations and transmission ratio distortion. Two 3-day sperm broods were sampled for the former and two 4-day broods for the latter to detect damage induced in the most radiosensitive cells. F1 nondysgenic males derived from M female to M male crosses (controls) were treated identically. X-Y chromosome translocations induced by P element mobility alone declined sharply with a decrease in temperature (18 versus 21 degrees C) and they were significantly reduced with aging of hybrid males from brood 2, 4-8 days of age, to brood 3, 7-11 days of age. No significant increase in translocations was observed when X irradiation and P-M dysgenesis were combined, showing no interaction between damages induced by the two mutator systems. In contrast, interaction was observed in transmission ratio distortion which was significantly increased by X irradiation of hybrid males derived from both reciprocal M X P and P x M crosses. The preferential elimination of P element-bearing autosomes occurred when either spermatocytes or spermatids were irradiated. An aging effect was also observed, resulting in less distortion in 9- to 14-day-old dysgenic males compared to 5- to 10-day-old hybrids.     

Somatic Effects of P Element Activity in Drosophila melanogaster: Pupal Lethality

Nonautonomous P elements normally excise and transpose only when a source of transposase is supplied, and only in the germline. The germline specificity depends on one of the introns of the transposase gene which is not spliced in somatic cells. To study the effects of somatic P activity, a modified P element (delta 2-3) lacking this intron was used as a source of transposase. Nonautonomous P elements from a strain called Birmingham, when mobilized in somatic cells by delta 2-3, were found to cause lethality, although neither component was lethal by itself. The three major Birmingham chromosomes acted approximately independently in producing the lethal effect. This lethality showed a strong dependence on temperature. Although temperature sensitivity was limited to larval stages, the actual deaths occurred at the pupal stage. Survivors, which could be recovered by decreasing the temperature or by reducing the proportion of the Birmingham genome present, often showed multiple developmental anomalies and reduced longevity reminiscent of the effects of cell death from radiation damage. Although the genetic damage occurred in dividing imaginal disc cells, the phenotypic manifestations--death and abnormalities--are not observed until later. The survivors also showed gonadal dysgenic (GD) sterility, a well-known characteristic of P-M hybrid dysgenesis. To explain these findings, we suggest that pupal lethality and GD sterility are both caused by massive chromosome breakage in larval cells, resulting from excision and transposition of genomic P elements acting as substrate for the transposase.     

On the Identification of the ROSY Locus DNA in DROSOPHILA MELANOGASTER: Intragenic Recombination Mapping of Mutations Associated with Insertions and Deletions

DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered.     

Somatic mutation in the wings of Drosophila melanogaster females dysgenic due to P elements when reared at 29 degrees C.

The possibility of somatic mobilisation of P elements in Drosophila melanogaster was investigated. Flies, trans-heterozygous for the genetic markers mwh and flr3, were obtained by crossing males containing transposition-competent P elements with females having M cytotype. The hybrid dysgenic flies were reared at 29 degrees C and their wings examined for mutant clones. The frequency of mutant spots found on the wings of the female flies was significantly higher than that of female control flies. We postulate that this increase in frequency may be due to P element mobilisation at high temperature in the wing cells of dysgenic hybrids. This is in direct contrast to the large body of research which indicates that P-transposition-mediated mutation is restricted to the germline.     

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.     

Dominant manifestation of pluripotency in embryonic stem cell hybrids with various numbers of somatic chromosomes

Developmental potential was assessed in 8 intra-specific and 20 inter-specific hybrid clones obtained by fusion of embryonic stem (ES) cells with either splenocytes or fetal fibroblasts. Number of chromosomes derived from ES cells in these hybrid clones was stable while contribution of somatic partner varied from single chromosomes to complete complement. This allowed us to compare pluripotency of the hybrid cells with various numbers of somatic chromosomes. Three criteria were used for the assessment: (i) expression of Oct-4 and Nanog genes; (ii) analyses of teratomas generated by subcutaneous injections of the tested cells into immunodeficient mice; (iii) contribution of the hybrid cells in chimeras generated by injection of the tested cells into C57BL blastocysts. All tested hybrid clones showed expression of Oct-4 and Nanog at level comparable to ES cells. Histological and immunofluorescent analyses demonstrated that most teratomas formed from the hybrid cells with different number of somatic chromosomes contained derivatives of three embryonic layers. Tested hybrid clones make similar contribution in various tissues of chimeras in spite of significant differences in the number of somatic chromosomes they contained. The data indicate that pluripotency is manifested as a dominant trait in the ES hybrid cells and does not depend substantially on the number of somatic chromosomes. The latter suggests that the developmental potential derived from ES cells is maintained in ES-somatic cell hybrids by cis-manner and is rather resistant to trans-acting factors emitted from the somatic one.     

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.     

Analysis of the Behavior of Mitochondria in the Ovaries of the Earthworm Dendrobaena veneta Rosa 1839

We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher “volume density” of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS) revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy organelles to the next generation.     

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.     

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.     

An improved procedure for protoplast microelectrofusion and culture of Nicotiana tabacum intraspecific somatic hybrids: plant regeneration and initial proofs on organelle segregation

Somatic hybrids were produced between haploid Nicotiana tabacum L. cv. Petite Havana (wild type) and haploid streptomycin resistant (SR1) mutant by an improved version of microelectrofusion of preselected pairs of protoplasts and the culture of fusion products in a nurse culture. Resistance of diploid plants regenerated from 20 somatic hybrid clones was tested at low concentration of streptomycin in the light as well as at high concentrations of streptomycin in the dark. In two independent hybrid lines, plants resistant in the light but sensitive in the dark were found. The existence of this plant type indicates a segregation of chloroplasts and mitocondria in somatic hybrid clones. It is suggested that microelectrofusion of preselected pairs of protoplasts combined with a reliable nurse culture might be a good technique for controlled somatic hybridization, cell reconstitution and partial gene transfer to different plant species. It might also be used to follow and analyse organelle segregation in somatic hybrid clones. The possibility that mitochondria might be resistant to streptomycin in the SR1 mutant is also discussed.