Regulation of the neurone-specific protein geneproduct (PGP)9.5 gene

Trowern, Angus Robert (1998) Regulation of the neurone-specific protein geneproduct (PGP)9.5 gene. University of Southampton, Doctoral Thesis.

Abstract

The tissue-specific expression of PGP9.5 was demonstrated via Western blot using polyclonal and monoclonal antibodies raised against human PGP9.5. The monoclonal antibody 13C4 showed greater specificity, detecting PGP9.5 as a single band of 27kDa present at high levels in the zebrafish and rat brain. The polyclonal antiserum, by contrast, detected multiple tissue-specific proteins in the rat and fails to detect PGP9.5 in the zebrafish. An ELISA was also developed to analyse cellular PGP9.5 expression with both antibody reagents and demonstrated the ability to detect reduced levels of PGP9.5 with low background.

The sequences of 1kb of 5' untranslated and untranscribed DNA were compared between two evolutionary distant species, the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene this region only displayed 48% homology between the species. The alignment did reveal two short, perfectly conserved sequences. The 12bp PSN element (-150 to -161), which acts as a non cell-specific activator of transcription and the 16bp 5 (-356 to 371) which was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. The alignment also revealed a 400bp CpG island present in both genes that extended to position -200 of the respective promoters.

Promoter studies on the 5' flanking DNA of the human PGP9.5 gene identified a 233bp minimal promoter incorporating nucleotides +51 to -182. Truncation and deletion mutagenesis revealed that a 59bp region (-123 to -182) was crucial to promoter function. This sequence was thus termed the 59bp activator. Sequence alignment of the human and Monodelphis promoters showed 76% identity within the activator region and also included the PSN element. Analysis of the 59bp activator by electromobility shift assays using the sense coding strand of the activator as a probe revealed a neurone-specific single-stranded DNA-binding protein.

In conclusion, this thesis has demonstrated that PGP9.5 expression is regulated by evolutionary conserved positive and negative acting cis-acting sequences located in the untranscribed region of the gene and may include a novel neurone-specific element.

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