An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function

Abstract

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.

Figure 1.—

Crossing scheme for genetic screen. A total of 75,000 chromosomes were mutagenized and screened for the adult glossy-eye phenotype. A total of 90 glossy-eye mutants were isolated, and larval eye-disc clones were induced to study their BrdU incorporation and ELAV expression. Twenty-three failed to incorporate BrdU but expressed ELAV normally and were studied further. m* represents the EMS-induced mutation. The cl-R3 mutation is cell lethal and is used to eliminate cells that are homozygous for the chromosome that does not contain m*. Similarly, for larval clones, cells homozygous for the RpS3 (Minute) mutation are eliminated. Cells heterozygous for the cl-R3 and RpS3 mutations grow more slowly, allowing m*/m* cells to form large clones.

Figure 2.—

Map positions and molecular lesions of identified mutations. Deficiency stock names and their approximate endpoints, as annotated by FlyBase, are indicated. Solid black lines, alleles identified from the screen; red stars, point mutations; brackets, deletions; red bars, deficiencies that fail to complement the mutant; green bars, deficiencies that complement the mutant.

Figure 3.—

Representative adult and larval phenotypes of mutants from mapped complementation groups. (A–N) Eyes of adult flies examined by scanning electron microscopy (A–G) and bright-field microscopy (H–N). An eye containing mock clones is mosaic in color (H), but the entire eye is faceted as in wild-type tissue (A). In eyes mutant for the indicated mitochondrial genes, cells heterozygous for the mutation are faceted (B–G) and red (I–N) as in mock clones, while homozygous clones are glossy and white. In all images, the representative allele is indicated in parentheses and posterior is to the left. (O–U) 30-min BrdU incorporation (red) in third instar larval eye discs with mock (O) and mutant (P–U) clones. Armadillo (blue) marks the morphogenetic furrow. In an eye disc with mock clones (O), both green and nongreen tissue are wild type. BrdU is randomly incorporated anterior to the furrow (“a”); posterior to the furrow, a single band of BrdU incorporation (arrowhead) marks the SMW. In mutant eye discs, BrdU incorporation is lost in homozygous mutant tissue, marked by the lack of GFP, both anterior and posterior to the furrow. Incorporation remains normal in clones heterozygous for the mutation (green). For comments on the apparent local nonautonomy of this phenotype (arrow), see Mandal et al. (2005). (V–B′) ELAV antibody stainings of eye discs with mock (V) and mutant (W–B′) clones. Expression of ELAV is largely normal in all mutant clones.

Figure 4.—

GatA and ATL localize to the mitochondrion. Co-immunostaining of GatA–GFP and ATL–GFP fusion proteins (A and D, green), respectively, with MitoTracker Orange CM-H2TMRos (Molecular Probes, Eugene, OR) (B and E) shows mitochondrial localization of the fusion proteins (C and F, yellow). Full-length cDNAs were cloned using the Gateway cloning system (Invitrogen, San Diego) into the pAWG vector containing the actin promoter and sequence coding for a C-terminal GFP protein. Blue, DAPI staining (C) and TOPRO3 (F) staining.

Figure 5.—

Schematic of complexes involved in oxidative phosphorylation in the mitochondrion. Proteins highlighted in pink are encoded by mutant genes identified from this screen or described in Mandal et al. (2005).