Alignment of predicted menin polypeptides in Drosophila species. Multiple alignment of predicted menin protein sequences from seven species of Drosophila compared to human performed by ClustalW. Conserved residues (shaded) and Drosophila melnaogaster splice junctions (brackets). Human (gi|18860839, NP_000235.2) and Drosophila melanogaster (gi:28574051, NP_523498.2) sequences are from Genbank (Benson et al., 2005). Drosophila pseudoobscura sequence is from FlyBase (FlyBase, 2003). Remaining sequences are from draft annotations (V. N. Iyer, D. A. Pollard and M. B. Eisen, personal communication) of assemblies generated by Agencourt (D. Smith, personal communication) and Washington University (R. Wilson, personal communication) genome sequencing centers.

Map of mnn1 locus and description of mnn1 alleles. (A) Molecular map of the mnn1 genomic region showing the flanking gene milton (milt) and the CG31907 gene nested in mnn1. Big arrows indicate the 5′ to 3′ orientation of the genes. Genomic maps of mnn1 mutants are reported in scale below. The black filled triangle localizes the P{wHy}^30G01 insertion. Gaps in lines indicate the deleted genomic sequences described for each mnn1 mutant allele. (B) Molecular map of the mnn1 transcripts. Filled boxes show exons, thin lines show introns, black filling indicates the open reading frame, gray filling indicates untranslated regions and alternative poly-A. Mnn1-RA and mnn1-RB indicate the long and the short transcript annotated in FlyBase. Y sign indicates the antigenic sequence used to generate anti-Mnn1. Interrupted lines show genomic map of mnn1^Δ46 and mnn1^Δ79 alleles in scale with the mnn1 transcripts. Small arrows localize the sites and directions of RT-PCR primers. (C) Amino acid sequence of N-terminus of Mnn1 protein. Stars indicate alternative translational start sites. Filled circles indicate amino acid residues mutated in MEN1 patients. Sequences in gray show the amino acid residues missing in mnn1^Δ46 and mnn1^Δ79 due to the absence of the ATG (D) RT-PCR analysis of RNA extracted from mnn1⁺⁸⁴ precise-excision and mnn1^Δ46 and mnn1^Δ79 mutant strains, using a primer overlapping the ATG sequence (upper panel) or primers spanning intron 4 sequence (middle panel). Control for genomic DNA contamination is shown (lower panel). (E) Western blot anti-Mnn1 on bacterial expressed pure Mnn1 and protein extracts from wild type (mnn1⁺¹¹³ and mnn1⁺⁸⁴) and mnn1 mutant (mnn1^Δ46 and mnn1^Δ79) adult heads. All lanes are from a single blot. Expected mobility of Mnn1 and an anonymous (Anon) cross-reacting material is shown.

Anti-Mnn1 tissue staining in wild type, over-expressing and Mnn1 null mutants. (A–D) Anti-Mnn1 immunofluorescence (green) of third instar larval brain, in wild type (B, D) and protein null (A, C). Immunofluorescent (A, B) and differential contrast channels (C, D) of the same preparation are shown. Staining is observed in the brain hemispheres (arrow) and ring gland (arrow head). (E–H) Multiple channel view of magnified ring gland polyploid tissue. Mnn1 is stained in green (E), actin in red (F) and DNA in blue (G). Merge of the three channels (H) reveals weak but consistent nuclear staining of Mnn1. Nuclear staining appears non-uniform. (I–P) Mnn1 staining (green) in third instar larvae following UAS-mnn1 induction by AB1-GAL4 driver in salivary gland (I), insulin dilp2-GAL4 driver in brain lobe (K), How24-GAL4 driver in CNS (M), motor neuron OK6-GAL4 driver in CNS (O). (J, L, N, P) Magnification of groups of cells showing intense Mnn1 staining (empty boxes in upper panel). Double staining for DNA (blue) clearly shows Mnn1 in the nucleus of polytene salivary cells (J) and nuclei of groups of neurons (L, N, P). Weaker Mnn1 staining appears punctate (arrowheads in panel L) and may represent endogenous Mnn1 in surrounding cells.

Loss-of-function mnn1 phenotypes. Analysis of mnn1⁻ mutant (red lines) and mnn1⁺ control (black lines) for survival on standard media (A) and on 10 mM paraquat in sucrose (B). Genotypes are indicated. (A) Red vs. black lines show a significant survival difference (P < 3 × 10⁻⁴ K–S test) more evident in the first 30 days (P < 2 × 10⁻⁷ K–S test) while red vs. red lines are almost identical (P > 0.99 K–S test). (B) Red vs. black lines show a slight difference (P < 3 × 10⁻⁴ K–S test) in oxidative stress response.

Over-expression of mnn1 results in adult-pharate phenotype enhanced by jun dominant negative. (A) Wild type pupae dissected at developmental stage P14 corresponding to the time of death caused by mnn1 induction. (B) Adult-pharate phenotype observed when UAS-mnn1 is driven by How24-GAL4. Note the dark spot at the end of the proboscis (arrow). (C) The dark spot is detectable as early as pupal stage P7 (arrow), same genotype as panel B. (D) Pupae over-expressing mnn1 die later than (E) pupae over-expressing mnn1 and dominant negative jun contextually (UAS-mnn1 and UAS-jun^bZIP driven by How24-GAL4).

Thoracic over-expression of mnn1 and interactions between Mnn1 and Jun/Fos in the thorax. (A) Dorsal view of thorax in wild type. (B) Dorsal view of thorax when UAS-mnn1 is driven by pnr-GAL4. The thorax is reduced in the anterior–posterior dimension, the scutum is slightly enlarged, the scutellum is shortened, intrascutal and scutoscutellar sutures are less evident, scutellar bristles are curved and shorter. (D) SEM view of the thoracic dorsal midline showing an enlarged space between the dorsocentral bristles. (C) Wild type bristle distribution. (E) Absence of thoracic abnormalities in Jun defective flies (jra¹/+). (F) Thoracic defect observed when UAS-mnn1 is driven by pnr-GAL4 in the thorax. (G) Enhanced thoracic cleft in flies with reduced dose of jun and over-expressing mnn1. (H) Mild defect of thoracic closure produced by over-expression of a jun dominant-negative when UAS-jun^bZIP is driven by pnr-GAL4. (I) Enhanced thoracic cleft when UAS-mnn1 and UAS-jun^bZIP are driven simultaneously by pnr-GAL4. (J) Absence of thoracic defect in flies over-expressing a dominant negative fos (UAS-fos^bZIP) with the How24 driver. (K) Moderate thoracic defect detected in eclosed flies expressing fos dominant negative with reduced dose of mnn1. Absence of mnn1 in How24 > fos^bZIP flies results in pupae lethality (Table 2).

Interactions between Mnn1 and Fos in the eye. (A, D) Wild type eye showing the organization of normal ommatidia. (B, E) Small-rough eye phenotype resulting from the expression of fos dominant negative (UAS-fos^bZIP driven by the ey-GAL4 driver). The number and hexagonal shape of ommatidia, as well as the distribution of the mechanical bristles are altered. (C, F) Mnn1 over-expression reverts the phenotype when UAS-mnn1 and UAS-fos^bZIP are driven by ey-GAL4. (A–C) × 125; (D–F) × 1000.