Figure 1.
Schematic representation of the D-loop regulatory region.
The names of regulatory motifs are indicated: the three conserved sequence blocks (CSB1, CSB2, and CSB3), light-strand promoter (LSP), heavy-strand promoter (HSP), the conserved termination-associated sequence (TAS), and origin of H-strand DNA replication (OH). Blue rectangles represent TFAM-binding sites and the red rectangle represents the mTERF binding site. The arrows pointing left and right show transcription orientation of H (H1 and H2) and L strands, respectively. The location of ribosomal RNA genes (12S and 16S) and the genes encoding tRNAs for phenylalanine, valine and leucine (F, V, and L, respectively) is indicated.
Table 1.
Natural genetic variation in selected mtDNA regulatory elements.
Figure 2.
Increased TFAM binding to oligonucleotide probes harboring the C295T mtDNA variant.
A. EMSA analysis of TFAM binding to double-stranded oligonucleotide probes containing the C295T mtDNA variant compared to its “wild-type” control (wt-295). Shown are autoradiograms of representative EMSA gels performed at a 10∶1 (TFAM 10×) and 25∶1 (TFAM 25×) TFAM∶probe molar ratio. The locations of the free and TFAM-bound (shifted) probe are indicated. B. Graphical representation and statistical analysis of the gels in A. The data of binding in the C295T variant is normalized to that of the wt-295, which was given a value of 1.0. The mean+/−one standard deviation (error bars) is shown as is the corresponding p-value.
Figure 3.
Run-off in vitro transcription assays preformed using mtDNA templates with or without TFAM binding site variants.
A. A schematic map of the mtDNA templates used in the run-off in vitro transcription assay, with the TFAM-binding sites (striped rectangles), light-strand promoter (LSP, bent arrow), and the location of the 242 and 295 variants (arrows) indicated. B. A representative in vitro transcription reaction using templates containing the indicated mtDNA variants. Equal amounts of the linear mtDNA templates (ethidium bromide-stained at the bottom) were used in the in vitro transcription assay with a partially purified POLRMT (i.e. mtRNA polymerase) fraction from HeLa cells. The 223-nt full-length run-off transcript and a second major, shorter transcript of ∼160 nt (see Discussion) are indicated. Quantification of the 223-nt, full-length transcript and the ∼160-nt truncated product from multiple independent experiments is shown in C. and D., respectively. Both mutant templates showed a trend toward increased LSP transcription activity when either the full-length or shorter product was analyzed, but only in the case of shorter, ∼160 transcript from the haplogroup J template (C295T) was this difference statistically significant (p-value 0.034).
Figure 4.
Relative steady-state levels of mtDNA transcripts in cybrids harboring different mtDNA haplogroups.
Each color represents the transcription level estimated by real time quantitative PCR (see Materials and Methods) for mtDNA-encoded genes in the mentioned cybrids. Briefly, the measured transcripts levels were normalized to the geometric mean of the three housekeeping genes: GAPDH, β-Actin and β2 microglobulin. Cybrids haplogroup assignment was verified by PCR RFLP of selected polymorphic sites and sequencing and HVR1 and HVR2: cybrids 3861 and 1106 belong to haplogroup J1 and J1b2, respectively, while cybrids CB, LR, NN, SB and TL belong to haplogroup H. Notice that the transcript level of ND1 and ND4L genes were significantly lower in one of the haplogroup J1 cybrids (3861), and that the ND3 transcript level was lower in one of haplogroup H transcripts (NN).
Figure 5.
Increased mtDNA copy number in haplogroup J cybrids compared to haplogroup H cybrids.
The copy number of mtDNA was measured using an mtDNA marker (ND2) and a nuclear DNA marker (18S rRNA gene). Data were obtained from three independent measurements for each of the tested cybrids, 2 of haplogroup J and 5 from haplogroup H. Standard deviation is shown for the mtDNA/nDNA ratios obtained from haplogroup J and haplogroup H cybrids.
Table 2.
List of double-stranded oligonucleotides containing the mtDNA TFAM-binding sites.
Table 3.
Primers used for the real time PCR-based estimation of transcript levels and mtDNA copy numbers.