Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair.

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.

Transcriptional regulation of mouse delta-opioid receptor gene.

Three major types of opioid receptors, mu (MOR), delta (DOR), and kappa (KOR), have been cloned and characterized. Each opioid receptor exhibits a distinct pharmacological profile as well as a distinct pattern of temporal and spatial expression in the brain, suggesting the critical role of transcription regulatory elements and their associated factors. Here, we report the identification of a minimum core promoter, in the 5'-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line. In vitro protein-DNA binding assays and in vivo transient transfection assays indicated that members of both the upstream stimulatory factor and Sp families of transcription factors bound to and trans-activated the DOR promoter via the E box and GC box, respectively. Furthermore, functional and physical interactions between these factors were critical for the basal as well as maximum promoter activity of the DOR gene. Thus, the distinct developmental emergence and brain regional distribution of the delta opioid receptor appear to be controlled, at least in part, by these two regulatory elements and their associated factors.

Cloning and promoter mapping of mouse kappa opioid receptor gene.

We have isolated mouse kappa opioid receptor (KOR) genomic clones containing the complete 5'-untranslated region, a portion of the 3'-untranslated region, and its coding region. The mouse KOR gene spans at least 16 kb and contains at least 4 exons. Exon I encodes the major portion of 5'-untranslated sequence and transcription initiation is determined, by primer extension experiments, to reside at 3 major sites, located at 334, 340, and 716 nucleotides upstream from the translation initiation codon. No typical TATA or CAAT boxes can be identified within an approximately 1-kb upstream region. Exon II contains 271 nucleotides, including 14 nucleotides of 5'-untranslated sequence. Exon III contains 353 nucleotides and exon IV begins at amino acid position #204 and encodes the rest of the 3'-end sequence of the cloned cDNA. Based upon Northern blot analysis, KOR transcript is estimated to be approximately 6 kb in length.

Genomic structure of the mouse delta opioid receptor gene.

Using mouse delta opioid receptor (DOR) cDNA sequence to probe genomic libraries in bacteriophage lambda and P1 vectors, clones traversing the entire DOR coding sequence and 5' and 3' flanking regions were isolate. Genomic sequence encoding mature DOR message, including 5' and 3' untranslated sequence, is divided by two introns of 26 kb and 3 kb, resulting in the gene occupying 32 kb of chromosomal DNA. Multiple putative transcription initiation sites were located, by RNase protection assay, in TATA-less G+C rich sequence between 390 and 140 nucleotides upstream from the ATG translation start codon. A polyadenylation site was located 1.24 kb downstream from the TGA translation stop codon. Examination of 1.3 kb of 5'flanking sequence revealed potential binding sites for several known transcription factors including: Sp1, Ap-2, NF-kappa B, NF-IL6, and NGFI-B.

Genomic structure analysis of promoter sequence of a mouse mu opioid receptor gene.

We have isolated mouse mu opioid receptor genomic clones (termed MOR) containing the entire amino acid coding sequence corresponding to rat MOR-1 cDNA, including additional 5' flanking sequence. The mouse MOR gene is > 53 kb long, and the coding sequence is divided by three introns, with exon junctions in codons 95 and 213 and between codons 386 and 387. The first intron is > 26 kb, the second is 0.8 kb, and the third is > 12 kb. Multiple transcription initiation sites were observed, with four major sites confirmed by 5' rapid amplification of cDNA ends and RNase protection located between 291 and 268 bp upstream of the translation start codon. Comparison of the 5' flanking sequence with a transcription factor database revealed putative cis-acting regulatory elements for transcription factors affected by cAMP, as well as those involved in the action of gluco- and mineralocorticoids, cytokines, and immune-cell-specific factors.

Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion.

The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990). We have purified this protein to homogeneity by affinity chromatography and identified it as the E. coli factor for inversion stimulation (Fis). Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription. Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion. The previously reported negative regulatory nature of this site (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions. Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site. Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting. Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA. Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd.