THE GENE AND PROCESSED PSEUDOGENES OF THE RAT MITOCHONDRIAL SINGLE-STRAND DNA-BINDING PROTEIN - STRUCTURE AND PROMOTER STRENGTH ANALYSES

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping geno mic clones. The clones encompassed the 5' untranslated sequence and al l intron/exon junctions. The gene contained seven exons and six intron s. The first exon contained only 5' untranslated sequence. The 16-amin o 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 transcrip tion start site was determined by primer-extension analysis to be 69 b p upstream of the translation start codon. The upstream sequence lacke d TATA and CCAAT boxes at the expected locations, but did contain seve ral other potential regulatory elements including a GC box (Spl-bindin g site) and three NRF-2 sites, one of which was located precisely besi de the transcription start site. A 10 out of 12 imperfect NRF-1 site w as located within the first exon. The 5' flanking sequence (-546 to +3 0) was shown, to have strong promoter activity in transient transfecti on assays in primary rat hepatocytes and HepG2 cells. In addition, evi dence for the existence of several mtSSB processed pseudogenes was obt ained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5' upstream reg ion of one of the pseudogenes was analyzed and found to contain neglig ible promoter activity. (C) 1998 Elsevier Science B.V. All rights rese rved.