A Motor Function for Drosophila Gemin3 Figure 2. In vivo association of Drosophila Gemin3 and SMN. Western blot probed for CFP::Gemin3 fusion protein (,143 kDa) using mouse anti-GFP antibody after immunoprecipitation (IP) with rabbit anti-GFP antibody (positive control), rabbit anti-SMN (test) or rabbit non-specific serum (NSS; negative control). Protein extracts were derived from larval muscles ubiquitously expressing CFP::Gemin3. CFP::Gemin3 co-purifies with SMN, but is absent in the negative control lane. Input was one-tenth volume of the whole tissue lysate. doi:10.1371/journal.pgen.1000265.g002 Several studies were pursued to demonstrate that the recessive lethality of both transposon insertions is specific to gemin3 disruption, thereby confirming that gemin3 is an essential gene. First, complementation crosses revealed that both gemin3 alleles retain their recessive lethality in trans to each other and to a chromosomal deficiency that completely eliminates the gemin3 gene (Df[3L]ED4457). Second, a re-mobilisation screen of the P- element in the gemin3R allele, which is the only transposon that could be excised, recovered homozygous viable precise excision alleles or revertants. Third, both low ubiquitous gemin3 and CFP::gemin3 transgenic expression driven by 1032-GAL4 [25] rescued the lethality of gemin3R homozygotes and gemin3R/gemin3W Figure 3. Alleles of gemin3. (A) Schematic showing the location of the gemin3 alleles and the span of the gemin3DN transgene. The gemin3W allele is a transposon insert within the gemin3 gene promoter, specifically at 92 nucleotides upstream of the transcription start site. The transposon insert generating the gemin3R allele is located 108 and 35 nucleotides downstream of the transcription and translation start site, respectively, thereby generating several premature stop codons. The gemin3DN transgene lacks 424 amino acid residues from the N-terminal of Gemin3 and is thus devoid of the helicase core. (B) RT-PCR analysis of gemin3 expression in wild-type, gemin3R/gemin3W transheterozygotes and gemin3R heterozygotes. Compared to wild-type, gemin3 mRNA was detected in low amounts in gemin3R heterozygotic adults. Importantly, gemin3 mRNA expression levels were drastically reduced in gemin3 heteroallelic mutant larvae. The housekeeping Tat-binding protein-1 (Tbp-1) RT-PCR product served as an internal control. Developmental stages at 25uC: F = adult females, M = adult males, L2 = 48 hrs after egg laying (AEL) 2nd instar larvae, L3a = 72 hrs AEL 3rd instar larvae, and L3b = 96 hrs AEL 3rd instar larvae. doi:10.1371/journal.pgen.1000265.g003 PLoS Genetics | www.plosgenetics.org 3 November 2008 | Volume 4 | Issue 11 | e1000265