Ethyl Methanesulfonate

Mutagenesis was carried out using VC2010, a local subculture of the standard laboratory strain N2 (Brenner 1974 (link)) with either ethyl methanesulfonate (EMS) (Sulston and Hodgkin 1988 ), N-ethyl-N-nitrosourea (ENU) (De Stasio and Dorman 2001 (link)), trimethylpsoralen/UV (UV/TMP) (Flibotte et al. 2010 (link)), or with an EMS/ENU cocktail. F1 animals were screened in 1% nicotine (Moerman and Baillie 1979 (link)) to ensure mutagenesis and propagated through F10, with single F10 animals used to establish strains for DNA isolation and frozen stocks as shown in Figure 1. Genomic DNA from mutant strains and 40 wild isolates (Supplemental Table 10) was extracted as described earlier (Flibotte et al. 2010 (link)), and libraries prepared for sequencing using a modified Illumina protocol using only one addition of Agencourt AMPure XP beads per sample through Y-adaptor ligation. Multiplexed libraries were sequenced with Illumina GAII/HiSeq technology, and clusters passing default quality filters were demultiplexed using a custom perl script. Raw FASTQ files from each strain were aligned to build WS230 of the C. elegans genome (www.wormbase.org) using the alignment program phaster (P Green, pers. comm.) or BWA version 0.5.9 (Li and Durbin 2009 (link)) for comparison. All reported variants were generated using phaster (see Supplemental Material for details).

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.