Abstract:The structure at the 5′ end of the gene encoding neural‐specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human andMonodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1‐kb stretch of 5′ untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233‐bp CpG‐rich minimal promoter. Truncation mutagenesis revealed the presence of essential positivecis‐acting regulatory sequences within the region −182 to −123 relative to the transcription initiation site. Sequence alignment analysis of the human andMonodelphispromoter sequences showed 76% identity in this 59‐bp region of the gene. A perfectly conserved 12‐bp sequence (PSN) located within this region acts as a non‐cell‐specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16‐bp sequence located at −356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negativecis‐acting sequences located in the u