Subtractive analysis of S. franciscanus and S. purpuratus ovary mRNA: what kinds of genes determine species-specificity?

Abstract

Our goal is to identify and understand points of regulation in sperm–egg recognition as well as in steps of early development. These processes are species-specific and are the key to understanding speciation. In both vertebrates and invertebrates, the interaction of the sperm and egg displays a wide range of species-specificity. The questions we would like to answer are: What kinds of molecules determine the specificity and control the fertilisation process? Are early steps in development regulated in a species-specific manner?As an approach to identifying genes that determine species-specificity, in two different species, S. purpuratus (S. p.) and S. franciscanus (S. f.), we used a new subtractive hybridisation method known as RDA (representational difference analysis) (Lisitsyn et al., 1993; Hubank et al., 1994). Several species-specific clones were isolated from S. f. ovary mRNA by this method using mRNA from another species but the same genus of sea urchin, S. p. Four different clones were obtained and the species-specificity of the sequence was confirmed by hybridisation. One of them has four tandem EGF repeats and is homologous to the S. p. EGF-II gene (Yang et al., 1989) and A. crassispina EGIP (exogastrula inducing protein) gene (Ishihara et al., 1982). The first three EGF repeats (EGF 1–3) have 60% similarity among these species, but the fourth EGF domain (EGF 4) is highly divergent. The EGF-II protein is believed to be involved in the signalling events of early development, because purified EGF causes exogastrulation when it is added to the seawater prior to gastrulation (Ishihara et al., 1982). Recombinant EGF 3 from S. f. induces exogastrulation in both S. f. and S. p.