Genetic mapping of Uba3 O.2.2 , a pupal lethal mutation in Drosophila melanogaster

Abstract

An EMS mutagenesis screen was conducted in Drosophila melanogaster to identify growth control mutants. The multi-institution Fly-CURE consortium phenotypically characterized the O.2.2 mutant using the FLP/FRT system which displayed a mutant lethal phenotype with reduced head development, and darkened ocular tissue. Complementation mapping was conducted to identify the affected gene. A failure to complement was identified in Uba3 , resulting in the identification of the novel allele, Uba3 ^O.2.2 . Uba3 is a known disruptor of the cell cycle and our data are consistent with early larval/embryonic lethality displayed in numerous species.

Figure 1. Characterization of the lethal Uba3 ^O.2.2 mutation by phenotypic analysis and complementation mapping.

FRT42D, Dark ⁸² control (A-B) display a mosaic eye of red (mw ⁺ ) (mutant) to white (mw ^- ) (wildtype) tissue in a viable pupa. (C-D) FRT42D, Dark ⁸² , Uba3 ^O.2.2 mutant was pupal lethal, displaying a reduced head and darkened ocular tissue. (E) Sanger sequencing analysis of FRT42D, Dark ⁸² control and (F) FRT42D, Dark ⁸² , Uba3 ^O.2.2 mutant indicating a heterozygous peak at 2R:13,859,838 (C to T). (G) The smallest region the O.2.2 mutant failed to complement was 2R:13,839,479..13,897,827 as defined by the leftmost breakpoint of Df(2R)BSC383 and the rightmost breakpoint of Df(2R)Exel7128. All genes with an “X” were tested and complemented O.2.2 with only the Uba3 ^SH2028 failing to complement. (H) Amino acid alignment of FRT42D, Dark ⁸² control (WT Uba3 ) to FRT42D, Dark ⁸² , Uba3 ^O.2.2 mutant ( O.2.2 Uba3 ) displaying the nonsense mutation in Uba3 ^O,2,2 . Image adapted from flybase.org (Gramates et al., 2017).