DNA sequences derived from the 5' region of a gene coding for the 50-kD skeletal matrix protein (SM50) of sea urchin embryo spicules were linked to the CAT reporter gene and injected into unfertilized eggs. CAT mRNA and enzyme were synthesized from these fusion constructs in embryos derived from these eggs, and in situ hybridization with a CAT antisense RNA probe demonstrated that expression is confined to skeletogenic mesenchyme cells. A mean of 5.5 of the 32-blastula-stage skeletogenic mesenchyme cells displayed CAT mRNA (range 1-15), a result consistent with earlier measurements indicating that incorporation of the exogenous injected DNA probably occurs in a single blastomere during early cleavage. In vitro mutagenesis and deletion experiments showed that CAT enzyme activity in the transgenic embryos is enhanced 34-fold by decreasing the number of SM50 amino acids at the amino-terminus of the fusion protein from 43 to 4. cis-regulatory sequences that are sufficient to promote lineage-specific spatial expression in the embryo are located between -440 and +120 with respect to the transcriptional initiation site
