Genetic mapping and phenotypic analysis of shotH.3.2 in Drosophila melanogaster

Abstract

Genetic screens are used to identify genes involved in specific biological processes. An EMS mutagenesis screen in Drosophila melanogaster identified growth control phenotypes in the developing eye. One mutant line from this screen, H.3.2, was phenotypically characterized using the FLP/FRT system and genetically mapped by complementation analysis and genomic sequencing by undergraduate students participating in the multi-institution Fly-CURE consortium. H.3.2 was found to have a nonsense mutation in short stop (shot), anortholog of the mammalian spectraplakin dystonin (DST). shot and DST are involved in cytoskeletal organization and play roles during cell growth and proliferation.

Figure 1. Characterization of shot^H.3.2 mutation by phenotypic analysis, complementation mapping, and genetic sequencing

FRT42D, Dark⁸² control mosaic eyes (A) exhibit an increased ratio of red (mutant) to white (wildtype) tissue compared to FRT42D, shot^H.3.2, Dark⁸²mosaic eyes (B). Mosaic eyes with shot^H.3.2, Dark⁸² clones display antennal defects (arrow) and disorganized wildtype ommatidia (arrowhead). (C) Map of deficiency lines that complemented (red bars) and failed to complement (highlighted in green) the H.3.2 mutation, resulting in a region of overlap of 2R:13,839,479..13,897,827 (between blue dashed lines). short stop (shot) is highlighted in yellow. Adapted from FlyBase using release FB2021_03 (Larkin et al. 2021). (D-E) The nucleotide location of H.3.2 was identified by sequence analysis. FRT42D, Dark⁸² control sequence with a single C peak at 2R:13,904,428 in the coding region of shot (D), compared to FRT42D, shot^H.3.2, Dark⁸² mutant sequence with a heterozygous double peak revealing a nucleotide change from C to T at this position (E, asterisk). (F) Amino acid alignment of wildtype (WT) and H.3.2 mutant Shot sequences. Amino acids 471 to 570 encode a spectrin-like repeat in Shot isoform H. The Trp548* mutation in the H.3.2 mutant allele is indicated (box).