Ontogenic expression of a Cyl actin fusion gene injected into sea urchin eggs

The 5' terminus of the Cyl actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2-5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the Cyl gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was microinjected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous Cyl gene first appear. The exogenous Cyl • CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient Cyl • CAT genes. The amounts of CAT enzyme produced by embryos bearing Cyl • CAT deletions that lack various regions of the Cyl sequence were measured. As little as 254 nucleotides of upstream Cyl sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining <850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete Cyl • CAT fusion construct.