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We have determined that two P elements, P[21-3] and P[21r36], residing in the 5'-UTR of the vestigial wing gene, encode functional repressors in eye tissue. However, neither element fits a previous categorization of repressor- making elements as Type I or II. Both elements encode polypeptides that are shorter than the canonical elements they most closely resemble. DNA sequencing reveals that P[21r36] encodes an intact THAP domain that is missing in the P[21] element, which does not encode a functional repressor. Recovery of P[21-3] at sites other than vestigial (where it causes the wing mutant, [vg.sup.21-3]) reveals that the element can make repressor in wing tissue of sufficient activity to repress the mutant phenotype of [vg.sup.21-3]. Why the P[21-3] element fails to produce repressor when located at vestigial may be explained by our observation that three different mutants in the RNA interference pathway cause a partial reversion of [vg.sup.21-3]. We speculate that the vg and P-initiated transcripts that arise at the vg locus in the [vg.sup.21-3] mutant trigger an RNA interference response that results in the mutual degradation of both transcripts. Key words: P element, repressor, THAP domain, RNA interference, Drosophila. Les auteurs ont determine que deux elements P, P[21-3] et P[21r36], situes dans la region 5'-UTR du gene vestigial, codent pour des represseurs fonctionnels dans les tissus oculaires. Cependant, aucun des deux elements ne rencontre les criteres permettant de le categoriser comme un element represseur de Type I ou II. Les deux elements codent pour des polypeptides qui sont plus courts que les elements canoniques auxquels ils ressemblent le plus. Le sequencage a permis de montrer que P[21r36] code pour un domaine THAP intact qui est absent chez l'element P[21], lequel ne code pas pour un represseur fonctionnel. L'observation de l'element P[21-3] a des sites autres que vestigial (oU il cause un phenotype mutant de l'aile, [vg.sup.21-3]) revele que cet element produit un represseur suffisamment actif dans les tissus de l'aile pour reprimer le phenotype mutant [vg.sup.21-3]. Pourquoi l'element P[21-3] est incapable de produire suffisamment de represseur lorsqu'il est situe dans le gene vestigial pourrait s'expliquer par l'observation que trois mutants differents dans le sentier de l'interference a l'ARN causent une reversion partielle de [vg.sup.21-3]. Les auteurs speculent que les transcrits vg et P inities au locus vg chez le mutant [vg.sup.21-3] declencheraient une interference a l'ARN qui se traduirait par une degradation mutuelle des deux transcrits. Mots-cles : element P, represseur, domaine THAP, interference a l'ARN, Drosophila. [Traduit par la Redaction], Introduction In an earlier paper (Anderson et al. 2006), we have described a P element replacement experiment in which we attempted to replace the P element responsible for the mutant [...]

Abstract: The DOPA decarboxylase gene (Ddc) belongs to the 'early-late' class of ecdysone-inducible genes in Drosophila melanogaster. Its expression is up-regulated in epidermal tissues by the ecdysone receptor acting through [...]

Scalloped (SD), a TEA/ATTS-domain-containing protein, is required for the proper development of Drosophila melanogaster. Despite being expressed in a variety of tissues, most of the work on SD has been restricted to understanding its role and function in patterning the adult wing. To gain a better understanding of its role in development, we generated [sd.sup.47M] flip-in mitotic clones. The mitotic clones had developmental defects in the leg and eye. Further, by removing the VG domains involved in activation, we created a reagent (VG[DELTA]ACT) that disrupts the ability of SD to form a functional transcription factor complex and produced similar phenotypes to the flip-in mitotic clones. The VG2[DELTA]ACT construct also disrupted adult CNS development. Expression of the VG[DELTA]ACT construct in the wing alters the cellular localization of VG and produces a mutant phenotype, indicating that the construct is able to antagonize the normal function of the SD/VG complex. Expression of the protein:protein interaction portion of SD is also able to elicit similar phenotypes, suggesting that SD interacts with other cofactors in the leg, eye, and adult CNS. Furthermore, antagonizing SD in larval tissues results in cell death, indicating that SD may also have a role in cell survival.

Histochemical staining and DNA manipulations help target an enhancer trap of the p element alleles vg(lacZ1) to the S-1 region of the vestigial gene (vg+) that is essential for the formation of wings. The allele has high mutant phenotypic characteristics and contains high level haltere expression of beta-galactosidase in the dorsal wing surface. This targeting process of the p elements is applicable to genes that have low primary p element insertion rates.

We report here the isolation of a new P-element-induced allele of the vestigial locus [vg.sup.2a33], the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was [vg.sup.21], which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing [vg.sup.2a33] in a dysgenic cross, we isolated the [vg.sup.2a33] allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3'-proximal copy of the 11-bp internal repeat from the P element of [vg.sup.21]. To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5'-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from [vg.sup.21]. Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.