Fig. 1. Drosophila pinch is encoded by the steamer duck (stck) locus. (A) Protein sequence similarity between the LIM domains of human PINCH1 (LIMS1 – Human Gene Nomenclature Database) and Drosophila PINCH. (B) Northern blot analysis of staged RNA samples from different developmental timepoints. Embryonic stages are numbered and represent time collected after egg laying at 25°C. Larval samples (L) are from the three larval stages (instar); the third instar larval sample is represented twice to confirm the decrease in rp49 probe seen at that time of development (Borie et al., 1999). Pupal and adult samples are labeled P and A, respectively. RNA markers (not shown) indicated the size of the hybridizing band to be 1.4 kb. Northern blot quantitation is indicated below by the graph. (C) Sequence analysis of the stck alleles. The pinch transcription unit contains six exons, indicated by the blocks, with the initiating MET codon encoded by the second exon. Individual LIM domains are color coded. The pinch sequence in stck¹⁷ contains a 571 bp deletion encompassing nucleotides 2095-2664 (corresponding to 615-1066 of the published cDNA sequence; Accession Number AF078907). stck¹⁸ harbors a 2 bp deletion removing nucleotides 2309-2310 in the fifth exon (774-775 of the published cDNA sequence), resulting in a frame shift in the middle of the fourth LIM domain. (D) Western blot demonstrating reduction of PINCH protein in stck mutants. Each lane contains 10 µg of protein lysate from the following samples: stage 17 wild-type embryo (lane 1); stage 17 l(3)097 homozygous embryo (lane 2); stage17 l(3)097/stck¹⁷ embryo (lane 3); stage 17 l(3)097/stck¹⁷ embryo from stck¹⁷ germline clone (lane 4). The blot has been hybridized with the affinity-purified PINCH antiserum. The faint band present in lane 4 that migrates at 40 kDa is nonspecific immunoreactivity.