Fig. 1. Amphiphysin gene organization and characterization of mutant alleles. (A) Amphiphysin consists of 10 exons extending over 18 kb of DNA, including a large 13 kb second intron that contains no detectable genes. The EP(2)2175 transposon is inserted 412 bp from the AUG encoding the first methionine of the cDNA; imprecise excision of the EP transposon produced the amph²⁶ and amph⁵⁴ null alleles. In amph²⁶ there is a 3428 bp deletion (breakpoints –1925/1502) that removes the entire first exon; in amph⁵⁴ there is a 1834 bp deletion (breakpoints –581/1253) that removes the entire first exon but does not remove the first exon of Sin3A. All phenotypes are observed with either mutant. The Sin3A and Gq-3 genes are adjacent to the amph gene, but neither amph mutation affects the coding sequences of Sin3A or Gq-3 genes, and the Sin3A and amph²⁶ mutations complement each other (A. Razzaq, personal communication), consistent with amph²⁶ having no effect on Sin3A function. (B) Western analysis of Drosophila extracts from Amphiphysin mutant and precise excisions (amph⁺¹). In each of the putative null mutants, amph²⁶ and amph54 (data not shown), there is no immunoreactivity detected. (C) Schematic representations of the four Amph isoforms. In each case, the alternative splicing occurs in exon 8, reducing or eliminating the central domain of Amphiphysin (top); sequence analysis of the four isoforms from the end of exon 7 to the start of exon 9 (bottom). (D) Drosophila Amphiphysin does not interact with Drosophila Dynamin: western analysis and Coomassie staining of Dynamin binding to the SH3 domain of Amphiphysin. Drosophila extract was mixed with three different fusion proteins: GST, GST fused to the SH3 domain of Amph (GST-SH3) and GST fused to a mutated form of the SH3 domain (GST-SH3*). In each case, we could not detect any binding of Dynamin to the GST proteins. The asterisks indicate the GST proteins.