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  • Drosophila Stocks and Fly Genetics: Larvae and flies were raised on standard cornmeal-yeast-agar-molasses medium containing propanoic acid at 25°C. In all experiments the roX1 mutation is roX1ex6 and the roX2 allele is Df (1) roX252 [24]. The [w+4Δ4.3] transgene supplies essential adjacent genes lost in the roX2 deletion. The full genotypes of the flies in Figure 1 are as follows: A–B) y w; [GmroX1-75C]. C–D) y w; ocmL714X/+; [GmroX1-75C]. E–F) y w;msl1+/+ ocmL714X; [GmroX1-75C]. G–H) y w; [GmroX1-58D]. I–J) y w; ocmQ1673X/+; [GmroX1-58C]. K–L) y wm4. M–N) y wm4; ocmG1646E/+. O) The wildtype flies are y w. The ocm allele used for viability assay is indicated beside the graph. The mutagenesis was carried out as described in [14] except that all flies were homozygous for the [w+ GMroX1] reporter at all times because pilot experiments showed the modifiers produced a more conspicuous phenotype when the reporter was paired. The [GMroX1-75C] and [GmroX1-58D] transgenic stocks used were also as previously described [14]. CyO, y+ balanced males from each of the modifier mutants were mated to comparable females and the presence of nonbalanced progeny (yellow body and flat wings) was scored. The crosses used for 2A. y w; ocmG1646E/CyO, y+ virgins crossed to y w/Y; msl ocmL1658N/CyO, y+ males where msl is either msl1L60, msl21, or mle1. The interesting class of progeny had flat wings and yellow body. 2B. The control classes were produced by w; msl1hypo/CyO, Roi sib crosses to produce homozygotes or crosses to y w; msl1L60/CyO to produce hemizygotes. The double mutants were made by mating y w/Y; msl1hypo ocmL1658N/CyO, y+ males to y w; msl1L60 ocmL1658N/CyO, y+ virgins. 2C.y w roX1 roX2 [w+ cos4Δ]; [w+ GMroX1-75C] virgins crossed to y w/Y; msl1L60 ocm/CyO, y+ males where the different ocm alleles are showed for each column. Standard P element transformation was used for BAC rescue experiments [43]. Five 20 kb BAC (bacterial artificial chromosomes), 113N09, 12L17, 08I22, 99L08, 117N13 derived from a P[acman] library made available from the Bellen lab, were used for the rescue [36]. Irrelevant secondary mutations were removed from ocm chromosomes by back crossing to the starting chromosome until BAC 117N13 could rescue ocm homozygotes to Mendelian frequencies. The following primer pairs were used to amplify genomic DNA from adult flies followed by Sanger sequencing separately from both strands. Several background polymorphisms relative to the reference sequence were found during sequencing, but not shown. Likewise, a few nonsense mutations carried nearby missense mutations as well, but only the nonsense mutation is shown. Six ocm alleles (ocmS1590F, ocmG1646E ocmV1334D, ocmV1286D, ocmQ1297X and ocm166Δ11 bp)were first recombined to the [FRT42D] transgene as described [44] Mitotic clones were then induced in the eyes by crossing them to y w; [FRT42D] [y+]++ ocm/[FRT42D] [y+] [GMR-hid] [w+ cl]+; [ey-GAL][UAS-FLP] as described [43] to assay for cell viability. For immunostaining, y w; [FRT42D] ocmQ1297X was crossed to y w [ey-flp]; [FRT42D], [Ubi-GFP]/Cyo or y w [Ubx-flp]; [FRT42D], [Ubi-GFP]/Cyo flies to generate mitotic clones in the eyes and wings discs respectively. The flies stocks were gifts from the Bellen’s lab. Immunostaining on imaginal discs and embryos were performed as described [45]. The ovaries were mounted with Prolong gold (P-36931, Invitrogen) with no pre-treatment. A segment of the gene spanning codon 1287–1773 was subcloned into the pMAL-C2X vector (N8076, NEB). The fusion protein was expressed and run on a SDS page acrylamide gel. The protein was excised from the gel and injected into guinea pigs (Cocalico Biologicals, Reamstown, PA). The animals were sacrificed and the anti-serum collected after 91 days. Adult flies and embryos were first dounced in 50 mM Tris-HCl (pH 8.5), 300 mM NaCl and 1% NP-40 containing protease inhibitor (04693124001, Roche) as described in [15]. The samples were then boiled with SDS loading dye for 5 min and loaded onto an 8% SDS-polyacrylamide gel. Proteins were transferred in Tris-glycine buffer (pH 8.3) containing 25 mM Tris and 192 mM glycine and 20% methanol, for 1.75 h at 600 mA. Membranes were incubated with OCM antibodies overnight at 4°C. After three 15-min washes in PBS-Tween and 2 h incubation at room temperature with the appropriate secondary horseradish peroxidase conjugate, the proteins were detected with luminol system (Santa Cruz Biotechnology). The membrane was then stripped in 60 mM Tris-HCl pH 6.8, 0.7% β-mercaptoethanol and 2% SDS and reprobed in anti-ATP5A antibody (MS407, ABCAM).
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