The gene and processed pseudogenes of the rat mitochondrial single-strand DNA-binding protein: structure and promoter strength analyses

Abstract

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5' untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns. The first exon contained only 5' untranslated sequence. The 16-amino acid mitochondrial targeting presequence, encoded by the second and third exons, was precisely bisected by intron 2. All intron donor and acceptor sites were consistent with the GT/AG consensus. The transcription start site was determined by primer-extension analysis to be 69bp upstream of the translation start codon. The upstream sequence lacked TATA and CCAAT boxes at the expected locations, but did contain several other potential regulatory elements including a GC box (Sp1-binding site) and three NRF-2 sites, one of which was located precisely beside the transcription start site. A 10 out of 12 imperfect NRF-1 site was located within the first exon. The 5' flanking sequence (-546 to +30) was shown to have strong promoter activity in transient transfection assays in primary rat hepatocytes and HepG2 cells. In addition, evidence for the existence of several mtSSB processed pseudogenes was obtained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5' upstream region of one of the pseudogenes was analyzed and found to contain negligible promoter activity.