Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in Drosophila

The structural organization of the Drosophila melanogaster gene encoding mi tochondrial single-stranded DNA-binding protein (mtSSB) has been determined and its pattern of expression evaluated during Drosophila development. The D, melanogaster mtSSB gene contains four exons and three small introns, Th e transcriptional initiation site is located 22 nucleotides upstream from t he initiator translation codon in adults, whereas several initiation sites are found in embryos. No consensus TATA or CAAT sequences are located at ca nonical positions, although an AT-rich sequence was identified flanking the major transcriptional initiation site. Northern analyses indicated that th e mtSSB transcript is present at variable levels throughout development, In situ hybridization analysis shows that maternally deposited mtSSB mRNA is distributed homogeneously in the early embryo, whereas de novo transcript i s produced specifically at an elevated level in the developing midgut, Tran sfection assays in cultured Schneider cells with promoter region deletion c onstructs revealed that the proximal 230 nucleotides contain cis-acting ele ments required for efficient gene expression. Putative transcription factor binding sites clustered within this region include two Drosophila DNA repl ication-related elements (DRE) and a single putative E2F binding site. Dele tion and base substitution mutagenesis of the DRE sites demonstrated that t hey are required for efficient promoter activity, and gel electrophoretic m obility shift analyses showed that DRE binding factor (DREF) binds to these sites, Our data suggest strongly that the Drosophila mtSSB gene is regulat ed by the DRE/DREF system, This finding represents a first link between nuc lear and mitochondrial DNA replication.