Molecular isolation and analysis of the erect wing locus in Drosophila melanogaster.

Abstract

The molecular study of the erect wing (ewg) locus was initiated by isolating DNA in the lA8-lB1 interval of the X chromosome. Previous developmental genetic analyses of the mutant alleles at the ewg locus have demonstrated that the wild-type ewg product is essential during embryogenesis and is required postembryonically at least for the development of indirect ffight muscle. To define the ewg-encoding DNA, chromosomal breakpoints that genetically flank the ewg locus were used. P-element-mediated transformation followed by subsequent rescue of the ewg-lethal alleles has defined a 11.5-kilobase genomic fragment as encoding the ewg locus. Northern blot analysis of transcription from this DNA has revealed a complex pattern of transcription with respect to both size and developmental profile. Tissue distribution of putative ewg transcription was examined by in situ hybridization to 6-to 14-h-old embryonic sections. These sections revealed that the expression of putative ewg messages is limited to the central nervous system-derived structures and not observed within the mesoderm during this developmental stage.In the fruit fly, Drosophila melanogaster, a large array of mutations have been discovered through phenotypic wing position abnormalities or flight abnormalities (13). Several mutants with impaired flight display defects in muscle structure or organization (8,18,23,26,27). A major fraction of these mutations are expected to reside in genes that specify muscle-specific proteins. This expectation is corroborated in the cases of a few genes whose wild-type products have been characterized (1,16,17,23,26,27). A second set of mutations may represent genes expressed in the muscle which also have expression in other tissues. Finally, a third class of mutations may identify an interesting group of genes whose expression lies outside of the mesodermal tissue but is essential to the proper development or function of muscle. The involvement of a functional nervous system in the proper development and maintenance of muscle has been recognized in vertebrate and invertebrate systems (3, 12, 14, 19, 25, 30). The recent work of Lawrence and Johnston (20) has revealed neural influence during the differentiation and development of insect musculature.We have been studying the erect wing (ewg) locus in Drosophila melanogaster because of the interesting phenotypes associated with mutations at this locus (11; R. J. Fleming, Ph.D. thesis, Brandeis University, Waltham, Mass., 1987 blastoderm, the area from which the mesoderm and the nervous system are derived (11). Thus, both the phenotypic defects associated with mutant ewg alleles and the genetic mosaic analyses are consistent with the suggestion that the ewg gene product may be required in the musculature or the nervous system.To directly address the question of tissue specificity of expression and function of the ewg gene, we have initiated a molecular characterization of the ewg-encoding DNA. The proximal to distal order for known genes in the 1A8-1B1-2 interval of the X chrom...

Loss of Flight and Associated Neuronal Rhythmicity in Inositol 1,4,5-Trisphosphate Receptor Mutants ofDrosophila

“…In all cases, the responses obtained were indistinguishable from wild type (data not shown). The observed changes in wing posture and flight thus arise from specific defect(s), which could be an alteration in indirect flight muscle function in turn caused by developmental and/or physiological changes (Fleming et al, 1989;DeSimone and White, 1993). The physiological changes could lie in the muscle or in any part of a neural circuit for flight.…”

Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila

“…pBSscXR3.7 contains a 3.7 kb XbaI-EcoRI fragment that adjoins the sc 8 breakpoint (subcloned from λsc101, Campuzano et al, 1985). The −1.9 kb probe was from pBSscBP0.8, a 0.8 kb BgIII-PstI fragment from λsc101; the −54 kb probe corresponds to pBSTG3R3.5, which contains a 3.5 kb EcoRI fragment subcloned from λTG-3 (Fleming et al, 1989); and the −103 kb probe was derived from pBS6.1HR1.75, whose 1.75 kb HindIII-EcoRI insert was obtained from a cosmid walk generated in our laboratory (cos 6.1).…”

“…Only the restriction sites flanking the sc 8 breakpoint (coordinate 0) are shown for the following enzymes: X (XhoI), H (HindIII), E (EagI), B (BamHI), R (EcoRI), V (EcoRV), NC (NcoI), G (BgIII), and Sa (SaII). The locations of the erect wing (ewg, coordinates −92 to −100 kb), yellow (y, −20 to −25 kb), and achaete (ac, −10 to −13 kb) genes are indicated (Campuzano et al, 1985;Fleming et al, 1989). (B) DNA inserts were prepared from imaginal discs and brains of sibling third instar larvae (X/O males) either lacking (Dp−) or containing (Dp+) one copy of Dp1187.…”

The Neuron-Enriched Splicing Pattern of Drosophila erect wing Is Dependent on the Presence of ELAV Protein

“…Canton-S was used as the wild-type strain. The alleles and transgenes used in this study are as follows (abbreviated names are given, followed by full descriptions): ewg l1 , an ewg lethal allele which does not allow expression of the 116-kDa EWG protein (27); ewgR19-1 and ewgR19-2, two independent inserts of ewg genomic transgene P{ry ϩ ϭewg ϩ } that provide full rescue of ewg l1 (18); elav-EWG, a transgene, P{w ϩ ϭelav-EWG} (in which ewg cDNA [SC3 ORF] is fused to the elav promoter [53]), which provides full rescue of ewg l1 and is referred to in reference 13 as EWG NS ; elav-ewg, a transgene, P{w ϩ ϭelav-ewg}28 (in which ewg genomic transcribed sequences are fused to the neuron-specific elav promoter [53]), which provides full rescue of ewg l1 (25); elav edr , a specific insert of the transgene P{w ϩ ϭelav ϩ } which has a wild-type elav genomic rescue fragment (ELAV expression of P{w ϩ ϭelav ϩ }edr insert is specifically reduced in photoreceptor neurons, but expression in brain neurons is less affected due to the transgene insertion site [25,26] virgin females were crossed to w/Y; c309 males; in the control male larvae, there is no wing disc transcription from the ewg-ELAV neuron-specific minitransgene. Somatic clones were generated using the method described in reference 27 by crossing virgin elav e5 ; P{w ϩ ϭelav)DmORF2/TM3 virgin females to yw/Y; Kip P (⌬2-3 ry ϩ ) males, a source of transposase (43).…”

“…ewgR19 is a genomic transgene starting 1,852 nt 5Ј to exon B, including exons B to J and introns 2 to 6 (Fig. 1A) (18). elav-ewg is a genomic transgene encompassing exons B to J and introns 2 to 6 (EcoRI-PstI fragment from the ewg gene fused to the elav promoter [ Fig.…”