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1.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 4. Mitochondrial uptake of DNA is an active process. (A) DNA uptake requires the ΔΨ potential. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of a constant concentration of succinate and increasing concentrations of malonate. (B) DNA uptake requires the proton motive force. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of the protonophore CCCP. (C and D) DNA uptake is inhibited by KCl and MgCl2. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of KCl (C) or MgCl2 (D). Migration of the incorporated plasmid (2.3PL) is indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.
2.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 6. Uptake of DNA is not related to MPT. (A) DNA uptake is inhibited by CaCl2. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of CaCl2. (B) DNA uptake is inhibited in oxidative conditions. Incorporation of labeled maize 2.3 kb plasmid was tested with mock-treated mitochondria (Cont) and with mitochondria pretreated with H2O2 and FeSO4 (Fenton’s reagent, Fent). (C) DNA uptake is inhibited by thiol oxidation. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the absence (Cont) and presence (Dia) of 3 mM diamide. Migration of the incorporated plasmid (2.3PL) is indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.
3.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 5. Mitochondrial uptake of DNA is likely to involve VDAC/ANT complexes. (A) DNA uptake requires surface-accessible protein(s). The labeled maize 2.3 kb plasmid was incubated in the presence of mock-pretreated (Cont) or trypsin-pretreated (Tryp) potato mitochondria. (B) DNA uptake is inhibited by Ruthenium Red. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of Ruthenium Red (RuR). (C) DNA uptake is inhibited by antibodies against the VDAC. Incorporation of labeled maize 2.3 kb plasmid was tested in the presence of mock-treated mitochondria (Cont), mitochondria pretreated with BSA, or mitochondria pretreated with a polyclonal antiserum against the VDAC, NAD9 or alanyl-tRNA synthetase (AlaRS). As the antisera used contained 50% (v/v) glycerol, a control assay was also run in the presence of the relevant amount of glycerol (Glyc). (D) DNA uptake is inhibited by ADP. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of increasing concentrations of ADP and a constant concentration (10 µg/ml) of oligomycin (+). (E and F) DNA uptake is inhibited by atractyloside, but enhanced by bongkrekic acid. Mitochondrial incorporation of labeled maize 2.3 kb plasmid was tested in the presence of atractyloside (E) or bongkrekic acid (F). Migration of the incorporated plasmid (2.3PL) is indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.
4.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 2. Mitochondrial uptake of different DNA substrates. (A) Single-strand DNA is not taken up by plant mitochondria. The labeled linear maize 2.3 kb plasmid was incubated in its native form (Nat) or heat- denatured form (Denat) in the presence of potato mitochondria (Imp). Incubation time for uptake was 30 min. Aliquots of the initial native or denatured DNA probe were also loaded on the gel (Cont), showing the migration of the double-strand (ds2.3PL) or single-strand (ss2.3PL) plasmid. The migration shift of the incorporated DNA versus the initial probe was due to the presence of the bulk of the mitochondrial nucleic acids, as shown by mixing a probe aliquot with a nucleic acid sample obtained from an import assay run without substrate. (B) DNA uptake is not sequence specific. Labeled linear DNA corresponding to the pBluescript vector (BLSc), the A.thaliana threonyl-tRNA synthetase (ThrRS), the maize 2.3 kb plasmid cloned in pBluescript (BLSc 2.3PL), as well as labeled circular pBluescript DNA (BLSc circ), were incubated in the presence of potato mitochondria (Imp). Incubation time for uptake was 30 min. Aliquots of the initial DNA probes were also loaded on the gel (Cont). The size and migration of the probes are indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.
5.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 1. Plant mitochondria can incorporate large size DNA. (A) Organization of the 2.3 kb maize mitochondrial plasmid (2.3PL). The plasmid contains two copies of a 170 nt inverted repeat (IR), a 885 nt open reading frame (orf1) and chloroplast-like genes for a tRNAPro (P) and a tRNATrp (T). (B) Time course of DNA uptake. Labeled linear maize 2.3 kb plasmid was incubated for different times in the presence of purified potato tuber mitochondria prior to DNase digestion. Mitochondrial nucleic acids were subsequently extracted, fractionated by agarose gel electrophoresis and transferred onto a nylon membrane, which was autoradiographed. (C) Stability of the incorporated DNA. Uptake of labeled maize 2.3 kb plasmid into potato mitochondria was run for 30 min. Following DNase treatment and washing, a sample was kept for direct analysis of the 30 min import (Imp), whereas the rest of the mitochondria was further incubated (Post-inc) for 3 or 16 h in the absence of added DNA. (D) DNase protection of incorporated plasmid is resistant to outer membrane breaking. Following incorporation of labeled maize 2.3 kb plasmid for 15 or 30 min, mitochondria were mock treated (Mt) or submitted to osmotic shock (Mpl) before DNase treatment. (E) DNA uptake is pH dependent. Incorporation of labeled maize 2.3 kb plasmid in the presence of potato mitochondria was run with a series of potassium phosphate buffers with different pH values. Incubation time for uptake was 30 min. Migration of the incorporated plasmid (2.3PL) is indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.
6.

Figure. From: Plant mitochondria actively import DNA via the permeability transition pore complex.

Fig. 3. The incorporated DNA is transcribed and is a template for DNA synthesis in plant mitochondria. (A) Organization of the construct used as an import substrate for transcription and DNA synthesis studies. The tRNAPro gene present in the wild-type 2.3 kb maize plasmid (2.3PL) was replaced by the GFP gene (gfp) driven by the promoter of the potato mitochondrial 18S ribosomal RNA gene (Pr) in plasmid 2.3PLPrGFP. (B) GFP gene transcription in plant mitochondria as shown by RT–PCR. Potato mitochondria were incubated with PCR-amplified unlabeled 2.3PL or 2.3PLPrGFP DNA. Following DNA uptake, transcription was run for 2 h. Mitochondrial nucleic acids were isolated, treated with DNase and used for RT–PCR (+RT) with primers specific for the gfp sequence; amplification controls were run in identical conditions only omitting the reverse transcriptase enzyme in the corresponding medium (–RT). The expected DNA fragment (nucleotides 98–539 of the GFP gene) was also PCR-amplified directly from the initial construct (GF). (C) ORF1 and GFP gene transcription in plant mitochondria as shown by Southern hybridization. Potato mitochondria were incubated in the absence (Cont) or presence (2.3PLPrGFP) of PCR-amplified unlabeled 2.3PLPrGFP DNA. Following DNA uptake, transcription was run for 3 h in the presence of [α-32P]UTP. Mitochondrial nucleic acids were isolated from the different samples and identical amounts of radioactive transcripts were hybridized to Southern blots carrying DNA probes for the potato mitochondrial cob gene (cob), the ORF1 of the maize 2.3 kb mitochondrial plasmid (orf1) and the GFP gene (gfp). (D) DNA synthesis in plant mitochondria as shown by Southern hybridization. Potato mitochondria were incubated in the absence of exogenous DNA (Cont) or in the presence of PCR-amplified unlabeled 2.3PLPrGFP DNA (2.3PLPrGFP) or A.thaliana threonyl-tRNA synthetase encoding DNA (ThrRS). Following uptake, DNA synthesis was run for 1.5 h in the presence of [α-32P]dCTP. Mitochondrial nucleic acids were isolated from the different samples and identical amounts of radioactive DNA were hybridized to Southern blots carrying the (cob), (orf1) and (gfp) probes as in panel (C), or a threonyl-tRNA synthetase probe (thr). Fragment sizes determined with a DNA ladder [(La) in panel (B)] are indicated.

Milana Koulintchenko, et al. EMBO J. 2003 March 17;22(6):1245-1254.

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