Fig. 1. Drosophila microcephalin. (A) Genomic organisation of mcph1. Schematic drawing illustrating exons (boxed), coding sequence (grey shading) and P{GawB}NP6229 insertion site (triangle). Translational start sites (arrows) are present in exon 1, and intron 1 (for transcripts with unspliced intron 1). Variable splicing in intron 7 results in a second premature stop codon (asterisk). Deleted regions in the mcph1^d1 and mcph1^d2 mutants, generated by imprecise excision of the P{GawB}NP6229 element are marked by black bars. The mcph1^d1 deletion is 1210 bp from the P insertion site to coding base pair 626 in exon 3. mcph1^d2 deletion extends 1649 bp from the P insertion site to intron 4. (B) Schematic drawings showing the organisation of the mcph1 transcripts and (C) proteins. The long (L) isoform contains three BRCT domains (black boxes), and the short (S) isoform contains a single complete BRCT domain near its N terminus. The human MCPH1 protein domain structure is shown for comparison. Transcripts with unspliced intron 1 (indicated by dashed lines) are predicted to utilise a second translational start site, resulting in proteins with the same domain structure as MCPH1-L and/or MCPH1-S, but 47 amino acids shorter at the N terminus. (N-terminally truncated proteins not shown.) (D) In situ hybridisation of mcph1 transcript in wild-type embryos using clone LD43341 as probe. (a-c) mcph1 sense probe. (d-f) mcph1 antisense probe, showing that Drosophila microcephalin transcript is present at its highest embryonic level during syncytial stages (d). (E) Immunoblot analysis of wild-type embryos (wt) and embryos from mcph1^d2/d2 females (d2) using an affinity-purified rabbit polyclonal anti-MCPH1 antibody raised against a polypeptide at the N-terminal end of MCPH1 (AA 1-130), indicated by the antibody symbol in C. The antibody detects Microcephalin at 115 kDa in wild-type but not in mcph1^d2/d2 embryos. Loading control, cyclin B. (F) Developmental western blot of MCPH1. The 115 kDa isoform of MCPH1 is highly expressed in embryos (0-2 hour AED) and ovaries, but is present at lower levels in third instar larval brain. (G) The 115 kDa endogenous protein migrates most similarly to the short isoform. Transgenically expressed epitope tagged (Myc x6) MCPH1(L) and MCPH1(S) isoforms compared with the lower 115 kDa endogenous MCPH1 protein present in all lanes except mcph1^d2/d2 embryos. Loading control -tubulin (-Tub). (H) RT-PCR amplification of mcph1 exons 6-8 in wild-type 0- to 2-hour embryos, late embryos and testis. Primer locations indicated by arrowheads in A. The upper band corresponds to transcripts containing the unspliced intron 7, and the lower band, fully spliced exon 6-8. Control, Ribosomal protein RP49 transcript.