2 ISOFORMS OF ORTHODENTICLE-RELATED PROTEINS (HPOTX) BIND TO THE ENHANCER ELEMENT OF SEA-URCHIN ARYLSULFATASE GENE

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene , expressed specifically in aboral ectoderm, contains a 229-bp enhance r in its first intron that is required for the activation of HpArs gen e expression. Deletion analysis shows that a tandem repeat of orthoden ticle-related protein (Otx) binding sites are responsible for the acti vity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift as says, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at th e blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stag e. Two distinct types of HpOtx cDNA clones have been isolated from cDN A libraries of unfertilized eggs and gastrulae. Nucleotide sequences o f the homeobox and downstream regions are well conserved in the two ty pes of HpOtx cDNAs, while the region upstream from the homeobox has di fferent nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Resul ts from Northern blot analysis confirm the presence of two types of Hp Otx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms. (C) 1997 Acade mic Press.