Targeted Transposition at the Vestigial Locus of Drosophila Melanogaster

Targeted transposition is the replacement of one P element with another. We are exploiting this unique property of P elements to study the complex regulatory domain of the Dopa decarboxylase (Ddc) gene in Drosophila melanogaster. P element constructs targeted to the same site in the genome will be subjected to the same position effect. This allows the subtle effects typical of most mutations in the Ddc regulatory region to be measured in the absence of the variable influences of position effects which are associated with the current method of germline transformation. We have investigated some of the parameters affecting targeted transposition of a Ddc transposon, P[Ddc], into a P element allele at the vestigial locus. These events were detected by an increased mutant vg phenotype. The location of the donor transposon in cis or in trans to the target had little effect on the frequency of targeting. Likewise, the mobility of different donor elements, as measured by their rate of transposition to a different chromosome, varied nearly 20-fold, while the rate of targeted transposition was very similar between them. All targeted alleles were precise replacements of the target P element by P[Ddc], but in several cases the donor was inserted in the opposite orientation. The targeted alleles could be described as the result of a replicative, conversion-like event.     

Molecular Analysis of an Unstable P Element Insertion at the Singed Locus of Drosophila Melanogaster: Evidence for Intracistronic Transposition of a P Element

In a companion study, a number of P element insertions into the singed locus were characterized. Here is reported a detailed analysis of the structure and mutability of another P element insertion at sn, known as sncm. Under conditions which mobilize P elements, sncm mutates at high frequency to both wild-type (sn+) and to a much more extreme allele (snext). Wild-type revertants appear to represent precise or nearly precise excisions of the P element. Certainly two, and most likely all five, of the snext alleles studied result from the insertion of a duplicate copy of this P element into a nearby site in an inverted orientation. We propose a model in which both the sn+ and snext mutational events can be explained by excision of the P element from one chromatid followed by reintegration into the sister chromatid at a nearby site (intracistronic transposition). Finally, it is shown that the snext alleles are themselves unstable and the structure of a resulting chromosome aberration is examined.     

Cytotype Control of Drosophila Melanogaster P Element Transposition: Genomic Position Determines Maternal Repression

P element transposition in Drosophila is controlled by the cytotype regulatory state: in P cytotype, transposition is repressed, whereas in M cytotype, transposition can occur. P cytotype is determined by a combination of maternally inherited factors and chromosomal P elements in the zygote. Transformant strains containing single elements that encoded the 66-kD P element protein zygotically repressed transposition, but did not display the maternal repression characteristic of P cytotype. Upon mobilization to new genomic positions, some of these repressor elements showed significant maternal repression of transposition in genetic assays, involving a true maternal effect. Thus, the genomic position of repressor elements can determine the maternal vs. zygotic inheritance of P cytotype. Immunoblotting experiments indicate that this genomic position effect does not operate solely by controlling the expression level of the 66-kD repressor protein during oogenesis. Likewise, P element derivatives containing the hsp26 maternal regulator sequence expressed high levels of the 66-kD protein during oogenesis, but showed no detectable maternal repression. These data suggest that the location of a repressor element in the genome may determine maternal inheritance of P cytotype by a mechanism involving more than the overall level of expression of the 66-kD protein in the ovary.     

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.     

A Stable Genomic Source of P Element Transposase in Drosophila Melanogaster

A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable. Its transposase activity is higher than that of an entire P strain, but it rarely undergoes internal deletion, excision or transposition. This element was constructed by F. Laski, D. Rio and G. Rubin for other purposes, but we have found it to be useful for experiments involving P elements. We demonstrate that together with a chromosome bearing numerous nonautonomous elements it can be used for P element mutagenesis. It can also substitute efficiently for "helper" plasmids in P element mediated transformation, and can be used to move transformed elements around the genome.     

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.     

P Element Transposition Contributes Substantial New Variation for a Quantitative Trait in Drosophila Melanogaster

The P-M system of transposition in Drosophila melanogaster is a powerful mutator for many visible and lethal loci. Experiments using crosses between unrelated P and M stocks to assess the importance of transposition-mediated mutations affecting quantitative loci and response to selection have yielded unrepeatable or ambiguous results. In a different approach, we have used a P stock produced by microinjection of the ry506 M stock. Selection responses were compared between transposition lines that were initiated by crossing M strain females with males from the "co-isogenic" P strain, and ry506 M control lines. Unlike previous attempts to quantify the effects of P element transposition, there is no possibility of P transposition in the controls. During 10 generations of selection for the quantitative trait abdominal bristle number, none of the four control lines showed any response to selection, indicative of isogenicity for those loci affecting abdominal bristle number. In contrast, three of the four transposition lines showed substantial response, with regression of cumulative response on cumulative selection differential ranging from 15% to 25%. Transposition of P elements has produced new additive genetic variance at a rate which is more than 30 times greater than the rate expected from spontaneous mutation.     

Captured Segment Exchange: A Strategy for Custom Engineering Large Genomic Regions in Drosophila melanogaster

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be “captured” as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.     

A P Element Containing Suppressor of Hairy-Wing Binding Regions Has Novel Properties for Mutagenesis in Drosophila Melanogaster

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.     

Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H^attP, reintroduced a wild type H genomic and a cDNA-construct (H^gwt, H^cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H^LD, H^iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N⁵⁴¹⁹) and Delta (Dl^B2) mutants were addressed. Overall, phenotypes were largely as expected: both H^LD and H^iD were similar to the H^attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H^gwt and H^cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H^cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H^gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.     

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.     

An Inverse Pcr Screen for the Detection of P Element Insertions in Cloned Genomic Intervals in Drosophila Melanogaster

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of ``local' P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of ~10(-4) are distributed every 40-50 kb.     

Nucleotide Variation at the Gpdh Locus in the Genus Drosophila

The Gpdh locus was sequenced in a broad range of Drosophila species. In contrast to the extreme evolutionary constraint seen at the amino acid level, the synonymous sites evolve at rates comparable to those of other genes. Gpdh nucleotide sequences were used to infer a phylogenetic tree, and the relationships among the species of the obscura group were examined in detail. A survey of nucleotide polymorphism within D. pseudoobscura revealed no amino acid variation in this species. Applying a modified McDonald-Kreitman test, the amino acid divergence between species in the obscura group does not appear to be excessive, implying that drift is adequate to explain the patterns of amino acid change at this locus. In addition, the level of polymorphism at the Gpdh locus in D. pseudoobscura is comparable to that found at other loci, as determined by a Hudson-Kreitman-Aguade test. Thus, the pattern of nucleotide variation within and between species at the Gpdh locus is consistent with a neutral model.     

P Element Transposition in Drosophila Melanogaster: An Analysis of Sister-Chromatid Pairs and the Formation of Intragenic Secondary Insertions during Meiosis

The gap-repair model proposes that P elements move via a conservative, ``cut-and-paste' mechanism followed by double-strand gap repair, using either the sister chromatid or homolog as the repair template. We have tested this model by examining meiotic purturbations of an X-linked ry(+) transposon during the meiotic cycle of males, employing the mei-S332 mutation, which induces high frequency equational nondisjunction. This system permits the capture of both sister-X chromatids in a single patroclinous daughter. In the presence of P-transposase, transpositions within the immediate proximity of the original site are quite frequent. These are readily detectible among the patroclinous daughters, thereby allowing the combined analysis of the transposed element, the donor site and the putative sister-strand template. Molecular analysis of 22 meiotic transposition events provide results that support the gap-repair model of P element transposition. Prior to this investigation, it was not known whether transposition events were exclusively or predominantly premeiotic. The results of our genetic analysis revealed that P elements mobilize at relatively high frequencies during meiosis. We estimated that approximately 4% of the dysgenic male gametes have transposon perturbations of meiotic origin; the proportion of gametes containing lesions of premeiotic origin was estimated at 32%.     

Change in State following Transposition of a Regulatory Element of the Enhancer System in Maize

From an original A2 allele (colored aleurone), a mutable allele, a2-m-4 1629, that changes from a2 to A2 is described. Mutability is expressed as a very distinct pattern limited to the last cell division.—The mutability of a2-m-4 1629 is autonomously controlled by an En at the a2 locus. This En, inactive on standard a testers for En, is partially active on a2-m-1, an a2 tester for En, and expresses varied levels of activity from limited to nearly full suppression of the a2-m-1 color phenotype.—When the En of the a2-m-4 1629 allele transposes from the a2 locus, it behaves, at the new position, like a standard En in triggering a2-m-1, a-m-1 and a-m(r), which express colored spots on a colorless background. The activity of En is therefore different following the change in chromosome location. This finding supports the "position" hypothesis that has been proposed to explain diverse patterns observed among controlling elements. In this case mutation is related to the terminal cell state and not to tissue differences as shown with some phase-variation regulatory elements.     

A Non-Coding Genomic Duplication at the HMX1 Locus Is Associated with Crop Ears in Highland Cattle

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.