IDENTIFICATION OF EVOLUTIONARY CONSERVED REGULATORY SEQUENCES IN THE 5'-UNTRANSCRIBED REGION OF THE NEURAL-SPECIFIC UBIQUITIN C-TERMINAL HYDROLASE (PGP9.5) GENE

The structure at the 5' end of the gene encoding neural-specific prote in gene product 9.5 (PGP9.5) has been compared between two evolutionar y distant species: the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identit y was found across a 1-kb stretch of 5' untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identi fied a 233-bp CpG-rich minimal promoter. Truncation mutagenesis reveal ed the presence of essential positive cis-acting regulatory sequences within the region -182 to -123 relative to the transcription initiatio n site, Sequence alignment analysis of the human and Monodelphis promo ter sequences showed 76% identity in this 59-bp region of the gene. A perfectly conserved 12-bp sequence (PSN) located within this region ac ts as a non-cell-specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detect ed 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 conserv ed 16-bp sequence located at -356 (motif 5) was able to reduce the act ivity of a heterologous minimal promoter specifically in HeLa cells. I n conclusion, we have shown that expression of the PGP9.5 gene is regu lated by evolutionary conserved positive and negative cis-acting seque nces located in the untranscribed region of the gene.