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.     

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.     

Characteristics of inserted mutations obtained in the P-M hybrid dysgenesis system in the 9F12-10A7 region of the Drosophila melanogaster X-chromosome

19 new mutations in the 9F12-10A7 region of Drosophila melanogaster X chromosome was obtained in the system of P-M hybrid dysgenesis. They appeared to be lethals, as judged from viability of homo- or hemizygous females. In situ hybridization of P DNA with polytene chromosomes revealed P-element insertion in the 10A1-2 band in the majority of the mutants. As a result of complementation analysis, all these mutations were localized at previously known loci: l(1)BP1, l(1)BP5, l(1)BP8, l(1)BP7. No insertion mutations were found at the vermilion locus. This can imply for non-random distribution of insertion mutations in the region studied. Further comparison of these mutations with previously EMS-induced ones revealed that insertion mutations are predominantly hypomorph lethals which do not influence the viability, morphology and fertility of homozygous males and females, but drastically reduce viability of hemizygous females.     

The Unusual Spectrum of Mutations Induced by Hybrid Dysgenesis at the Triplo-Lethal Locus of Drosophila Melanogaster

The Triplo-lethal locus (Tpl) is unique in its dosage sensitivity; no other locus in Drosophila has been identified that is lethal when present in three doses. Tpl is also haplo-lethal, and its function is still a mystery. Previous workers have found it nearly impossible to mutationally inactive Tpl other than by completely deleting the chromosomal region in which Tpl resides (83DE). We have utilized P-M hybrid dysgenesis in an effort to obtain new mutations of Tpl. We recovered 19 new duplications of Tpl, 15 hypomorphic mutations of Tpl (a previously rare class of mutation), and no null mutations. Surprisingly, 14 of the 15 hypomorphic alleles have no detectable P element sequences at the locus. The difficulty in recovering null mutations in Tpl suggests that it may be a complex locus, perhaps consisting of several genes with redundant functions. The relative ease with which we recovered hypomorphic alleles is in sharp contrast to previous attempts by others to mutagenize Tpl. A higher mutation rate with hybrid dysgenesis than with radiation or chemicals also suggests a peculiar genetic organization for the locus.     

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

The molecular basis of I-R hybrid dysgenesis in Drosophila melanogaster: identification, cloning, and properties of the I factor

We have analyzed two mutations of the white-eye gene, which arose in flies subject to I-R hybrid dysgenesis. These mutations are associated with insertions of apparently identical 5.4 kb sequences, which we have cloned. We believe that these insertions are copies of the I factor controlling I-R hybrid dysgenesis. The I factor is not a member of the copia-like or fold-back classes of transposable elements and has no sequence homology with the P factor that controls P-M dysgenesis. All strains of D. melanogaster contain I-factor sequences. Those present in reactive strains must represent inactive I elements. I elements have a remarkably similar sequence organization in all reactive strains and are located in peri-centromeric regions. Inducer strains appear to contain both I elements, located in peri-centromeric regions, and 10-15 copies of the complete I factor at sites on the chromosome arms.     

Characterization of the Mus308 Gene in Drosophila Melanogaster

Among the available mutagen-sensitive mutations in Drosophila, those at the mus308 locus are unique in conferring hypersensitivity to DNA cross-linking agents but not to monofunctional agents. Those mutations are also associated with an elevated frequency of chromosomal aberrations, altered DNA metabolism and the modification of a deoxyribonuclease. This spectrum of phenotypes is shared with selected mammalian mutations including Fanconi anemia in humans. In anticipation of the molecular characterization of the mus308 gene, it has been localized cytogenetically to 87C9-87D1,2 on the right arm of chromosome three. Nine new mutant alleles of the gene have been generated by X-ray mutagenesis and one was recovered following hybrid dysgenesis. Characterization of these new alleles has uncovered additional phenotypes of mutations at this locus. Homozygous mus308 flies that have survived moderate mutagen treatment exhibit an altered wing position that is correlated with reduced flight ability and an altered mitochondrial morphology. In addition, observations of elevated embryo mortality are potentially explained by an aberrant distribution of nuclear material in early embryos which is similar to that seen in the mutant giant nuclei.     

The Absence of Somatic Effects of P-M Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

The wings and abdomens of dysgenic and nondysgenic control flies were scored for the presence of clones of cells mutant for first and third chromosome markers. These exceptional clones can arise from mitotic recombination, de novo mutation or deletion, and P-M hybrid dysgenesis has been shown to increase the frequency of parallel processes occurring in germ-line cells. Particular attention was given to careful genetic and molecular characterization of all stocks and to providing adequate and appropriate controls so that even very small increases in somatic clone frequency due to P-M hybrid dysgenesis would be detected. No difference was found in the frequency, size distribution or anatomical distribution of mutant somatic clones correlated to hybrid dysgenesis, confirming previous indications. The potential adaptive significance of a germ-line restriction of P-M hybrid dysgenesis is discussed.     

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.     

Detection of new genes participating in the trans-regulation of locus yellow of MDG-4 in Drosophila melanogaster

The aim of the present work was to obtain mutations in the genes involved in regulation of the yellow locus and mdg4. For this purpose, we searched for mutations changing phenotypic expression of the y(2) mutation induced by mdg4 insertion into the regulatory region of the yellow locus. Mutations have been obtained in the earlier described system of prolonged genome instability, sometimes combined with P-M hybrid dysgenesis. The mutation mod(mdg4) in a novel gene, modifier of mdg4, was detected which either enhanced or suppressed a phenotypic expression of several mutations induced by mdg4 insertion. We suggest that mod(mdg4) controls the expression of mdg4. In addition, the mutations in five loci located on the X chromosome have been found which enhanced the mutation phenotype of several y alleles induced by insertions of different mobile elements in the regulatory region of the latter. Possibly, the protein products of these genes designated as enhancers of yellow-1, 2, 3, 4 and 5 are directly or indirectly involved in control of the yellow locus expression.     

Movement of Drosophila melanogaster transposable elements other than P elements in a P-M hybrid dysgenic cross

Summary The Drosophila melanogaster mobile DNA sequences P factors and P elements transpose at elevated rates when P strain males are mated to M strain females in a hybrid dysgenic cross (Engels 1983). Isofemale lines derived from such a cross were analysed by in situ hybridisation using cloned copies of the transposable elements copia, 412 and F. It was found that lines derived from dysgenic crosses showed a statistically significant number of new sites for these elements when compared to a non-dysgenic control cross. This result suggests a functional coupling of copia, 412 and F transposition and some component present in the P-M dysgenic system.     

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.     

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.     

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.     

Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster

We present results demonstrating that the hobo family of transposable elements can promote high rates of chromosomal instability. Using strains with a hobo element inserted within the decapentaplegic gene complex (DPP-C), we have recovered numerous DPP-C mutations involving chromosomal rearrangements and deletions with one endpoint in the vicinity of the pre-existing hobo element. This hypermutability occurred in the germ lines of hybrid progeny from crosses involving strains containing hobo elements to strains lacking them. In some crosses, the offspring had rudimentary gonads, reminiscent of GD sterility. The germline hypermutability and infertility are similar to those produced by P-element-mediated hybrid dysgenesis. Given the many genetic and molecular similarities of the P and hobo systems, we propose that a system analogous to P-M hybrid dysgenesis has been activated in the hobo+ X hobo- crosses.