Fig. 2. Vibrator alleles and their relative strengths. (A) Schematic representation of P-element insertions within the vibrator gene. The gene spans a region of 8908 bp and is composed of nine exons and eight introns. The predicted gene model (shown in blue) comprises 1178 bp, the open reading frame (red) encodes a protein of 272 aa. The position of P-element insertions is represented by inverted triangles. Sequencing indicated that the insertion vib^j7A3 (P-element insertions `A' in panels A and B) lay within the second intron of the gene; the vib<sup>j5A6</sup> insertion (P-element insertions `D' in panel A) was within the first intron; the insertions vib^S110416 (P-element insertions `B' in panels A-D), vib<sup>EP513</sup> (P-element insertions `C' in panels A and B) and vib^S045002 (P-element insertions `E' in panels A and 2B) all lay within the first exon. The insertion vib<sup>EP651</sup> (P-element insertions `F' in panels A and B) was 222 bp upstream. Chromosomes carrying vib^EP513 and vib^EP651 had second site mutations, causing early embryonic lethality (Table 1). An additional P-element-insertion site in vib^S045002 at 77B;78A was uncovered by Df(3L)ri-79c (77B;78A) (Deak et al., 1997). Work presented here suggests that vib^S110416 carries a second site mutation (see footnote of Table 3). Consequently, most analyses were preformed on hemizygotes against the deficiency Df(3R)Dl-BX12. The lethal stage of hemizygous alleles (see Results) indicated the allelic series: vib^j7A3>vib^S110416>vib^EP513 vib^j5A6>vib^S045002>vib^EP651. (B) Levels of vibrator protein are diminished in P-element-mediated mutants. Anti-vibrator antibody recognises a band at an expected molecular mass of 35 kDa in western blots. Lanes 1-4, vibrator protein levels in hemizygous and homozygous vib^j7A3 second instar larvae, compared with the balanced stock. The extracts analysed in lanes 1 and 2 or 3 and 4 are from two or four larvae, respectively. Lanes 5-8, extracts of early pupae of the indicated genotypes, representing a series of alleles, all as transheterozygotes with vib^S110416. Protein levels reflect mutant strength in the allelic series and are lowest in vib^j7A3, at intermediate levels in vib^EP513 and vib^S045002, and highest in vib^EP651. Actin is shown as a loading control. Df stands for the deficiency (Df(3R)Dl-BX12); TM6C is a balancer chromosome. (C) Protein levels are dramatically reduced in vib^S110416 / Df(3R)Dl-BX12 testes. Protein extracts of three pairs of wild-type or mutant third instar larval testes are loaded in each lane. A band at the expected mass of 35 kDa is present in Oregon R testes but is absent in the hemizygous mutant. Actin is shown as loading control. (D) Protein levels show little diminution in homozygous vib^S110416 larval neuroblasts. Protein extracts of Oregon R or mutant larval neuroblasts were loaded in each lane. Levels of vibrator protein at 35 kDa are not significantly different between mutant and wild-type. -tubulin is shown as the loading control.