Bindin, a major protein of the sea urchin acrosome granule, mediates the species-specific adhesion and binding of sperm to egg required to effect fertilization. We report the isolation and sequence of bindin cDNA clones prepared from Strongylocentrotus purpuratus testis RNA. The bindin gene appears to be productively expressed only in males and only in testes. The protein is produced from a 51-kDa precursor, which is subsequently processed to yield the mature 24-kDa bindin protein.Fertilization in echinoderms occurs by a multistep, speciesspecific process, in which each step may serve as a barrier to gene flow across species lines. The initial physiological event as the sperm approaches the egg is the acrosome reaction, which is induced by the fucose sulfate ofthe eggjelly coat and is mediated by sperm membrane receptors (1-3). This reaction results in exocytosis of the acrosomal granule and the extrusion of the acrosome process. The sperm binds to the egg vitelline layer by means of the acrosome process, and the plasma membranes of the sperm and egg then fuse. In some interspecific combinations heterologous egg jelly does not induce the sperm acrosome reaction, while in others heterospecific acrosome reactions occur, but even when this is observed, fusion of heterospecific sperm and egg membranes fails to take place (4-6). Vacquier and associates (7-10) demonstrated that the major protein of the acrosome granule, which they named bindin, is the molecular species responsible for the recognition reaction by which the acrosome process is bound to a glycoprotein receptor embedded in the vitelline membrane of the egg. Partial purification and characterization of the sperm receptor glycoprotein have been reported (10-12). On activation of the sperm, bindin is exposed by the eversion of the acrosome granule. Bindin molecules coat the external surface of the acrosomal process and have been detected in the electron microscope by immunocytological methods at the exact site of the spermegg bond (7). Bindin has been purified and characterized chemically, as reviewed by Vacquier (10). It contains no detectable carbohydrate, and a partial amino acid sequence has been derived (10).We report here the isolation of bindin cDNA clones and the complete sequence of the derived protein. This showed unexpectedly that bindin is synthesized from a much larger precursor. Bindin is among the few known proteins of nonimmunogenic origin, the sequence of which specifies a developmentally important interaction between different cell types. Comparison with a homologous fragment of bindin sequence available from another sea urchin species of the same genus shows a remarkable degree of divergence that may be of both current and evolutionary significance in preventing interspecific gene flow. MATERIALS AND METHODSConstruction of cDNA Library. Double-stranded cDNA was synthesized by the RNase H procedure of Watson and Jackson (13). Linker ligation, size selection of cDNA, and subsequent procedures are as described by Huynh et al. (14). Ab...