Generating Drosophila β-Spectrin Mutants

A lethal deficiency in the β spectrin region of the X chromosome, Df(1)SD10, was produced by γ-ray mutagenesis of flies carrying the ry⁺ P-element insertion S6.9-11 (Wakimoto et al. 1986). In brief, adult males were exposed to 3,300 rads from a Cs¹³⁷ source, crossed to ry⁵⁰⁶/ry⁵⁰⁶ females, then scored for ry⁻ female progeny. A total of 32 ry⁻ revertant females were recovered from 145,000 females screened, and three hemizygous lethal lines were obtained. Df(1)SD10 was the only cytologically visible deficiency (16A-16DE) that lacked the β spectrin gene by polytene hybridization. Duplications of the β spectrin region on chromosome 3 were produced by γ-ray mutagenesis of the stock Tp(1;3)B^S3i, which is marked with the Bar-super dominant visible eye mutation. Tp(1;3)B^S3i males were irradiated with 4,000 rad, then crossed to Oregon R females. Bar-eye male progeny retaining the duplication were mated with ywf/ywf females, and crosses were scored for reversion of the male-sterile phenotype. Screening of 36,000 progeny yielded three male-fertile Bar-eye lines: Dp(1;3)B^S3iD1, Dp(1;3)B^S3iD2, and Dp(1;3)B^S3iD3. The extent of the duplications was determined by in situ hybridization and conventional cytology and balanced stocks of the recessive-lethal duplications were maintained over the TM6b chromosome.
A germ-line tranformant expressing an myc epitope-tagged wild-type β spectrin transgene on chromosome 2 was produced using a previously described strategy (Dubreuil et al. 1996). A BamHI-NotI fragment of the full-length β spectrin cDNA (Byers et al. 1992) was subcloned into the vector pWUMB. The vector was assembled from the w⁺ transformation vector pW8 (Klemenz et al. 1987), a 2-kb fragment of the Drosophila ubiquitin promoter (Lee et al. 1988), and a linker sequence encoding the myc epitope tag at the translation start site. The resulting construct encodes full-length wild-type β spectrin, except that the first 10 amino acids of β spectrin are replaced with the 10–amino acid myc epitope tag, which reacts with the mouse mAb 9E10 (Evan et al. 1985). The pWUMB-βspec construct was introduced into germ-line DNA by standard embyro microinjection. A single autosomal transformant P[βspec^T3I] was recovered on chromosome 2.
Mutations in the Drosophila β spectrin gene were produced by chemical mutagenesis. The screen was based on recovery of X-linked lethal mutations by complementation with Dp(1;3)B^S3iD3, a duplication of the 16A-F region of the X chromosome on chromosome 3. Males from an isogenized Oregon R stock were fed 24.5 mM ethyl methane sulfonate using standard methods (Grigliatti 1986), then mated to C(1)DX/y; Dp(1;3)B^S3i/+ females. F₁ male progenies were selected for presence of the duplication by their Bar-eye phenotype and crossed in single pair matings with C(1)DX/y females. Recessive X-linked lethal mutations in the 16A-F region were identified as crosses that yielded exclusively Bar-eye male progeny. 20 new mutations were recovered from a total of 10,744 chromosomes screened. The mutants were ordered into three intervals (I–III) by complementation tests with two additional duplications: Dp(1;3)B^S3iD1 and Dp(1;3)B^S3iD2. The mutants were assigned to six complementation groups by standard complementation tests. The complementation group representing the β spectrin gene was identified by rescue with the P[βspec^T3I] transgene.
Balanced stocks of each mutant over a FM7[Kruppel-GFP] chromosome (Casso and Kornberg 1999) were used to recover β-spec⁻ embryos in all experiments. Embryos carrying the balancer chromosome express green fluorescent protein (GFP) with the characteristic Kruppel pattern (Gaul et al. 1987). Embryos were collected from each β-spec⁻/FM7[Kr-GFP] × FM7[Kr-GFP]/y line for 2 h at 25°C. Embryos were aged overnight at 22°C, dechorionated in 50% bleach, and transferred to microscope slides to score GFP expression by fluorescence microscopy. β-spec⁻ male embryos were identified by their lack of GFP expression. The mutants and their wild-type siblings were separately transferred to apple juice agar plates for further development at 22°C.