PG25, A PINEAL-SPECIFIC CDNA, CLONED BY DIFFERENTIAL DISPLAY PCR (DDPCR) AND RAPID AMPLIFICATION OF CDNA ENDS (RACE)

Synthesis of melatonin in the mammalian pineal gland is regulated by t he rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (S NAT) and other unknown factors. To screen for pineal specific mRNAs po tentially involved in melatonin synthesis and/or regulation, different ial display PCR (DDPCR) was employed. We used 80 primer pairs and exam ined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochem ical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the codi ng region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a rece ntly identified human lung endothelial cell-specific gene, ESM-1. Inte restingly, not only the amino acid sequences but also the cDNA sequenc es, including the long 3' untranslated regions, are highly similar. Th is suggests that the conserved 3' untranslated region may carry inform ation to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) leve l compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potenti ally full-length cDNAs with restricted expression in anatomically comp lex regions of the brain. This protocol combining several existing met hodologies is suitable for use with limited tissue sources and uses mi nimal amounts of isotopes. (C) 1997 Elsevier Science B.V.