Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria

Abstract

We have devised an efficient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modified genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT-PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a first step towards true in vivo engineering of mammalian mitochondrial genomes.

Figure 1

Cloning of mouse mtDNA by an in vitro transposition reaction. (A) Schematic representation of three mouse mtDNA clones and sequences of the transposon insertion sites. A synthetic transposon (1.5 kb) was constructed containing the γ-ori from plasmid R6K, Cm^R and two hyperactive 19 bp mosaic end sequences adjacent to SalI and SmaI restriction enzyme sites. The transposons were inserted into the ND1, COXII and ND5 genes on mouse mtDNA, respectively, and all insertions generated 9 bp duplications of the target DNA (upper case). (B–D) Restriction enzyme analyses of transposon-inserted mouse mtDNA clones. Plasmids pMusMtTN-ND1 (B), pMusMtTN-COXII (C) and pMusMtTN-ND5 (D) were digested with one or two unique restriction enzymes at 37°C for 2 h and were electrophoresed on 1% agarose gels. Lane M, λ DNA HindIII digest; lanes 1, 6 and 11, DNA digested with SalI; lanes 2, 7 and 12, DNA digested with SalI + MluI; lanes 3, 8 and 13, DNA digested with SalI + BspEI; lanes 4, 9 and 14, DNA digested with SalI + SphI; lanes 5, 10 and 15, DNA digested with SalI + BglII.

Figure 2

Effect of plasmid copy number on the stability of cloned mouse mtDNA. (A) Stability of the mouse mtDNA in a low copy number E.coli strain. Plasmid pMusMtTN-ND5 was transformed into DH5α λatt::pirwt and was re-isolated after culturing in LB medium containing Cm (12.5 µg/ml) at 30 or 37°C for regular time intervals (24–72 h). Restriction patterns after SalI and SmaI digestion were compared on a 1% agarose gel. The top arrow points to the 16.3 kb mouse mtDNA and the lower to the band of the 1.5 kb γ-ori+Cm^R transposon. Lane M, λ DNA HindIII digest. (B) Stability of the mouse mtDNA in a high copy number E.coli strain. Mouse mtDNA clones were transformed into DH5α λatt::pir116 and were re-isolated from the recombinant E.coli after culturing in LB medium containing Cm at 37°C for 20 h. Restriction patterns after SalI digestion were compared on a 1% agarose gel. Lane M, 1 kb plus DNA ladder (Life Technologies); lanes 1–10, DNAs from DH5α λatt::pir116 strain; lane C, DNA from DH5α λatt::pirwt strain.

Figure 3

Identification of mtDNA fragments that serve as transcriptional promoters in E.coli. (A) Schematic outline of the approach used to clone fragments from the mouse mitochondrial genome with promoter activity. AluI-, HaeIII- and Sau3AI-digested mouse mtDNA fragments were cloned 5′ of the promoter-less CAT (Cm^R) gene in the pANTSγ-Cm vector and recombinant clones expressing Cm resistance were selected on LB-Cm plates. (B) Comparisons of CAT activities generated from mtDNA promoter fragment clones at low and high copy numbers. The recombinant plasmid clones obtained from the promoter screen were grown in each of two E.coli strains that replicated them at either low copy numbers (pirwt) or at high copy numbers (pir200), and the CAT activities of both cultures were assayed. The black and gray bars shown for each clone indicate the relative CAT activities from low copy and high copy strains, respectively, with the value of the low copy vector-only control arbitrarily set to 1.

Figure 4

DNA transcription assay by in organello RNA synthesis using isolated mouse mitochondria. (A) A schematic representation of p2CγSSB, a control plasmid containing the prokaryotic CAT gene and the D-loop region, L-strand promoter and H-strand promoter (LSP and HSP) sequences from mouse mtDNA. This plasmid was designed so that the CAT gene is transcribed by the HSP promoter when it is introduced into transcriptionally active mitochondria. (B and C) RT–PCR analyses for CAT transcripts in electroporated mouse mitochondria. Plasmid p2CγSSB was electroporated into the isolated mouse wild-type (B) and ρ⁰ (C) mitochondria and then in organello RNA syntheses of those electroporated mitochondria were performed in incubation buffer for 2 h at 37°C. After isolating total RNA from the mitochondria, RT–PCR was carried out using CAT-specific primers CmR-F and CmR-R (see Materials and Methods). Expected band size of the RT–PCR CAT products was 470 bp. Lane M, 100 bp DNA ladder (Life Technologies); lanes 1 and 2, no electroporation control with plasmid; lanes 3 and 4, 12 kV/cm electroporation; lanes 5 and 6, 14 kV/cm electroporation. Control lanes in which reverse transcriptase (RT) was omitted are indicated below each panel by minus signs.

Figure 5

DNA transcription assay of a recombinant mitochondrial genome by in organello RNA synthesis using isolated mouse ρ⁰ mitochondria. The full mouse mtDNA clone, pMusMtTN-COXII, was electroporated into isolated ρ⁰ mitochondria and in organello RNA syntheses were performed for 2 h at 37°C. RT–PCR was carried out using 16S rRNA-specific (A) or ND6-specific (B) primers. 16S rRNA-R and ND6-R primers were used as gene-specific primers for the first strand synthesis of 16S rRNA and ND6 transcription, respectively, and the 16S rRNA-F and MusMt-MluIA primer set or ND6-F and ND6-R primer set was used for subsequent PCR amplification, respectively (see Materials and Methods). Expected band sizes of the RT–PCR products were 232 bp for 16S rRNA (see panel A) and 123 bp for ND6 (see panel B), respectively. Lane M, 100 bp DNA ladder; lanes 1 and 2, 12 kV/cm electroporation; lanes 3 and 4, 16 kV/cm electroporation; lanes 5 and 6, no electroporation control with plasmid. Control lanes in which reverse transcriptase (RT) was omitted are indicated below each panel by minus signs.