Mutant alleles of <i>dVMAT</i> reduce expression of DVMAT-A and -B.

<p>(A) <i>dVMAT-A</i> and <i>-B</i> share a common translational start site (indicated as “A, B start”) and a common N-terminus, but diverge at their C-termini. Coding exons in the <i>dVMAT</i> gene that are common to both <i>dVMAT</i>-<i>A</i> and-<i>B</i> are shown as gray boxes, introns as black lines. To generate alternative carboxy termini, the indicated genomic sequence (magenta box) is spliced out from <i>dVMAT-A</i> and retained in <i>dVMAT-B</i>. The <i>in situ</i> probe for <i>dVMAT-B</i> contains the first 260 nucleotides of this sequence. The P element insert in the <i>dVMAT</i> mutant allele <i>dVMAT</i>^P1 (black arrowhead) disrupts the coding sequence of both <i>dVMAT-A</i> and <i>-B</i>. (B,C) <i>In situ</i> hybridization of head sections shows transcription of the <i>dVMAT-B</i> gene in a layer between the retina and lamina (B). Magnified view of (B) shown in (C). (D) Cartoons showing the predicted topology of DVMAT with lumenal domains above and cytoplasmic domains below the parallel gray lines representing the vesicle membrane. Open circles indicate domains shared by DVMAT-A and -B. Filled, magenta circles indicate the C-terminal domain specific for DVMAT-B. The P element insertion site in the last exon of the <i>dVMAT</i>^P1 (marked with “P” and a black arrowhead) functionally deletes transmembrane domains 11 and 12 and the C-terminus (shaded gray). The imprecise excision allele <i>dVMAT</i>^Δ14 results in an insertion of 51 base pairs, and 17 amino acids in-frame with the original downstream codons (marked as “Δ14” with a blue arrowhead, with blue circles indicating the inserted residues). (E) Western blot using the N-terminus antibody directed against both DVMAT-A and -B splice variants shows an absence of DVMAT protein in <i>dVMAT</i>^P1 homozygotes (P/P), and dramatically reduced levels in <i>dVMAT</i>^Δ14 (Δ14/Δ14), compared with heterozygous controls (P/CyO and Δ14/CyO). The plasma membrane associated protein Late Bloomer (indicated as “lb”) was used as a loading control. Re, retina; La, lamina; Me, medulla. Bars: (B) 50 microns, (C) 20 microns.