Cloning and functional analysis of the rat ornithine decarboxylase-encoding gene

Abstract

We have isolated a functional gene ( ODC) encoding rat ornithine decarboxylase (ODC; EC 4.1.1.17) from a partial rat liver genomic DNA bank. The entire gene is located on a 7776-bp BamHI fragment and was shown to comprise twelve exons, of which ten encode the ODC protein (exons III–XII). Introduction of the BamHI fragment into an ODC-deficient hamster cell line restores ODC activity, indicating that the gene is functional. Comparison of the structure and nucleotide (nt) sequence of the rat ODC gene with recently reported mouse ODC genes, reveals that the gene is highly conserved. Primer extension analysis and RNA sequencing demonstrates that the transcription start point of rat ODC mRNA is located 303 nt upstream from the A residue in the start codon. Compared with our previously published sequence of the rat ODC cDNA, this indicates that a short sequence at the extreme 5′ end of our cDNA clone represents a cloning artefact. The correct 5′ leader of ODC mRNA, which is very G + C rich (62%), can be folded into a highly stable secondary structure, which may play a role in the translational control of ODC activity. Like in mouse, the promoter region of rat ODC is also extremely rich in G + C, and contains a TATA box and several putative SP1-binding sites. Possible binding sites for other transcription factors, like AP-1, AP-2 and CREB, can also be observed in the promoter region and, moreover, in the first intro. With the help of constructs containing different portions of the promoter region fused to the bacterial chloramphenicol acetyltransferase-encoding gene ( cat), the function and boundaries of the rat ODC promoter were analyzed. For transient expression in Rat-1 cells, sequences between 78 and 105 bp upstream of the cap site were found to be essential. In this region, only a putative CAAT box (CCGAT) is observed. Deletion of 4 nt within this sequence motif clearly diminished the promoter activity, thereby lending further support to its functional significance.