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. 2000 Apr;11(4):1471-85.
doi: 10.1091/mbc.11.4.1471.

Rearrangements of human mitochondrial DNA (mtDNA): new insights into the regulation of mtDNA copy number and gene expression

Y Tang  1 , E A Schon, E Wilichowski, M E Vazquez-Memije, E Davidson, M P King
Affiliations
Free PMC article

Rearrangements of human mitochondrial DNA (mtDNA): new insights into the regulation of mtDNA copy number and gene expression

Y Tang et al. Mol Biol Cell. 2000 Apr.
Free PMC article

Abstract

Mitochondria from patients with Kearns-Sayre syndrome harboring large-scale rearrangements of human mitochondrial DNA (mtDNA; both partial deletions and a partial duplication) were introduced into human cells lacking endogenous mtDNA. Cytoplasmic hybrids containing 100% wild-type mtDNA, 100% mtDNA with partial duplications, and 100% mtDNA with partial deletions were isolated and characterized. The cell lines with 100% deleted mtDNAs exhibited a complete impairment of respiratory chain function and oxidative phosphorylation. In contrast, there were no detectable respiratory chain or protein synthesis defects in the cell lines with 100% duplicated mtDNAs. Unexpectedly, the mass of mtDNA was identical in all cell lines, despite the fact that different lines contained mtDNAs of vastly different sizes and with different numbers of replication origins, suggesting that mtDNA copy number may be regulated by tightly controlled mitochondrial dNTP pools. In addition, quantitation of mtDNA-encoded RNAs and polypeptides in these lines provided evidence that mtDNA gene copy number affects gene expression, which, in turn, is regulated at both the post-transcriptional and translational levels.

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Figures

Figure 1
Figure 1
Rearranged mtDNAs. (A) Maps of the mtDNA species observed in cybrids from patient 1. The duplicated mtDNA (Dup-mtDNA) is composed of a full-length wild-type mtDNA (WT-mtDNA; thick arc) into which a subgenomic segment of wild-type mtDNA (i.e., the duplicated region) has been inserted (thin arc). On the deleted mtDNA (Δ-mtDNA, which is identical to the duplicated region), the protruding pie section denotes the deleted region. Only the COX II (solid box) and Cyt b (open box) genes involved in the rearrangement are shown (the breakpoint is indicated by a dashed line). Nucleotide positions associated with the rearrangement straddle the pie slices. Also shown are the origins of replication (OH and OL; solid arrows), the promoters of transcription (HSP and LSP; open arrows), the locations of the BamHI (B) and PvuII (P) restriction sites, and probes 1 (open circle) and 2 (solid circle) used in the Southern blot analyses. (B) Representative autoradiograms of Southern blot analyses of transmitochondrial cell lines from patient 1, containing 100% wild-type (WT; clone KAF4EB12.22), duplicated (Dup; KAF4EB12.28), and deleted (Δ; KAF4EB12.49) mtDNAs after digestion with PvuII (P) or BamHI (B) and hybridization with probe 1 or 2. The identity of each hybridizing fragment and its size (in kilobases) is indicated at right. The 8.8-kb band represents either linearized full-length Δ-mtDNA (in deleted lines) or the duplicated region in dup-mtDNA (in duplicated lines). U, uncut mtDNA. Lanes are numbered at the bottom. (C) Identification of dimeric species in deletion lines. Total DNA from deletion line KAF4EB12.49 from patient 1 was digested with successively smaller quantities of PvuII (units indicated above each lane) before Southern blot analysis. Other notations are as in B. (D) Representative Southern blot analysis of wild-type (206/F4 3.13.44) and deleted (206/F4 3.13.41) cybrid lines from patient 2 (map of the deleted mtDNA above autoradiograms). U1 and U2, two different topological conformations of the uncut deleted monomeric mtDNA that migrated at two different positions in the gel. Other notations are as in B. Marker (λ DNA digested with HindIII) sizes, in kilobases, are at left.
Figure 2
Figure 2
Biochemical function in homoplasmic cybrids from patient 1. (A) Rates of oxygen consumption per cell of the indicated cybrid cell lines (the digits under each bar denote the specific KAF4EB12.xx line analyzed; see Table 1) are shown (average of six measurements). (B) Rates of ATP synthesis of the indicated transmitochondrial cell lines are shown (average of four measurements per line). Each error bar represents 1 SD.
Figure 3
Figure 3
Representative growth curves. The indicated cybrids were grown in high-glucose DMEM in the presence (open squares) or absence (solid circles) of both uridine and pyruvate (A, C, and E), in DMEM (no glucose) plus galactose in the presence (open squares) or absence (solid circles) of both uridine and pyruvate, and in the presence of either uridine (open circles) or pyruvate (solid squares) alone (B, D, and F).
Figure 4
Figure 4
Quantitation of mtDNA. A representative dot blot analysis is shown. Duplicated serial dilutions of purified HeLa cell mtDNA (numbers indicate pg mtDNA spotted) and cell lysates from control ρ0 cells and from wild-type, duplicated, and deleted lines from patient 1 (cybrid clones12.xx from KAF4EB12; see Table 1) and patient 2 (cybrid clones 13.xx from 206/F4 3.13; see Table 1) were spotted according to the scheme shown in C. Multiple wells identified by the same cell line number denote samples that were collected at different cell passages. Each test well contained DNA from the lysis of 2500 cells. The membrane was hybridized first with probe 2 (B), stripped, and then hybridized with probe 1 (A). The dots were quantitated, and the amount of mtDNA in the various lines was calculated using the standard curve drawn from the HeLa cell data (D). TE, 1× Tris-EDTA buffer control for background.
Figure 5
Figure 5
Mitochondrial translation in representative homoplasmic cybrid clones (notation as in Figure 2). (A) Fluorogram of mitochondrial translation products. The predicted mtDNA-encoded polypeptides are shown at left. (B) Nucleotide and deduced amino acid sequences across the rearrangement breakpoint straddling the authentic COX II and Cyt b genes (see Figure 1). Numbers denote map positions inside and outside the deleted region. Note that the conceptual translation of the portion of the fusion mRNA located beyond the breakpoint is out of frame, resulting in a premature termination codon.
Figure 6
Figure 6
Northern blot analyses. (A) Linearized map of wild-type mtDNA. The COX II/Cyt b fusion gene and the predicted size of its transcript, are shown below the map, as are the in and out probes (bold) used to detect the authentic and fusion mRNAs. The four tRNAs examined in this study, and the direction of their transcription (leftward, L-strand encoded; rightward, H-strand encoded), are also shown. (B) Northern blots of representative cybrid lines (notation as in Figure 2) showing transcription in the breakpoint region, using the in and out probes. The expected sizes of the indicated transcripts are shown at right.
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