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Transcriptional initiation under conditions of anoxia-induced quiescence in mitochondria from Artemia franciscana embryos

Brian D. Eads^1,2,* and Steven C. Hand^1,

¹ Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
² Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80303-0334, USA
^* Present address: Department of Zoology, University of Wisconsin, Madison, WI 53706, USA

View larger version (41K): [in a new window]   Fig. 1. Schematic of the rationale and methodology used to calculate the contribution of in vitro (de novo) initiation to mitochondrial transcription in Artemia franciscana (after Gaines and Attardi, 1984). Transcription initiation site of the H strand lies 5' to the 12S rRNA (Carrodeguas and Vallejo, 1997). (A) Unfilled boxes represent 12S rRNA fragments labeled in organello with [32P]UTP and protected from nuclease digestion by antisense riboprobes. The filled box represents the unprotected portion of the 12S rRNA not measured by the assay. The sizes and UTP contents of the fragments are shown, and RNA polymerase distributions are assumed to be random, which would give the labeling patterns illustrated in (B). Under the assumption of no in vitro initiation, the radioactivity in the 5' fragment would be a function of the length of this labeled, protected piece multiplied by the number of uridines it contains. Similarly, the radioactivity in the 3' fragment would be a function of the average length of the labeled fragment plus the length of the 12S rRNA upstream of it (which accounts for the polymerases upstream at the time that label is added) multiplied by the number of uridines. However, if all labeling is due to de novo initiation, the labeling of 5' and 3' fragments would occur simply in proportion to their U content (86 and 34, respectively). Using these assumptions, the proportion of labeling in the protected fragments of 12S rRNA due to de novo initiation or elongation can be calculated by solving the following two equations: (1) radioactivity in 5' fragment=0.55X+2.52Y and (2) radioactivity in 3' fragment=X+Y, where X represents counts due to elongation, Y represents counts due to de novo initiation, 2.52 is the ratio of 86:34 uridines, and 0.55 is calculated from the equation: 5' radioactivity/3' radioactivity=(285/2)x86U/(110/2+600)x34U=0.55. In other words, the ratio of 2.52Y to 0.55X gives the ratio of radioactivity from initiation to that from elongation in vitro in the 5' fragment. (C—G) Transcriptional initiation decreases under conditions of acidic pH for A. franciscana mitochondria. 12S rRNA products from ribonuclease protection assays were labeled in organello, separated by gel electrophoresis and quantified with a phosphorimager. Panel C, control mitochondria (normoxia, pH 7.9); panel D, normoxia at pH 6.4; panel E, in vitro anoxia at pH 6.4; panel F, in vitro anoxia at pH 7.9; and panel G, in vivo anoxia assayed aerobically at pH 7.9. Lanes are experimental replicates. Arrows indicate migration distance of standards.

View larger version (12K): [in a new window]   Fig. 2. Transcription by mitochondria isolated from Artemia franciscana embryos is depressed by anoxia and low pH. (A) Control mitochondria (normoxia, pH 7.9; circles) are compared with organelles isolated under anoxia from anoxic embryos and assayed anoxically at pH 6.4 (triangles). (B) Control mitochondria (circles) are compared with organelles isolated under anoxia from anoxic embryos and assayed anoxically at pH 7.9 (triangles). (C) Control mitochondria (circles) are compared with normoxic mitochondria incubated at pH 6.4 (triangles). Note different timescales along the x-axis. All values are means ± S.E.M. (N=3).

View larger version (12K): [in a new window]   Fig. 3. Regions of the mitochondrial genome of Artemia franciscana footprinted with dimethylsulfide (DMS). Light grey boxes are segments where DNA-protein contacts were localized (indicated by arrows below). The dark grey box depicts the putative H-strand initiation region (HSP) where no contacts were seen. tRNA (L, tRNAleu; V, tRNAval; M, tRNAmet) and rRNAs (12S and 16S) are identified. Approximate primer positions are noted by small arrows (above). The locations of the strong H-strand promoter (HSP; larger left-facing arrow), the weak HSP (smaller left-facing arrow), the L-strand promoter (LSP; right-facing arrow) and the termination region (octagon) are given. Drawing is not to scale.

View larger version (63K): [in a new window]   Fig. 4. Footprinting of A. franciscana mitochondria reveals DNA-protein contacts. Footprints were detected in the termination region within tRNAleu (C) and the control region (D), but no protein binding was detected immediately upstream of the 12S rRNA (A and B). For panels A and B, primers HSP1 and HSP2 were used for the heavy and light chains, respectively (see Table 1 for details). Panel C depicts primer extension of the light chain using the primer LEU 1, and panel D used the primer LSP 1 (genome positions numbered according to Valverde et al., 1994a). Lane assignments are as follows: panels A and B, 1, naked DNA; 2, in organello control (normoxia, pH 7.9); 3, in organello anoxia, pH 7.9; 4, in organello anoxia, pH 6.4; 5, normoxia, pH 6.4. Panels C and D: 1, normoxia, pH 6.4; 2, in organello anoxia, pH 6.4; 3, in organello anoxia, pH 7.9; 4, in organello control (normoxia, pH 7.9); 5, naked DNA. Relative to naked DNA, overmethylated residues are filled squares and undermethylated residues are unfilled squares.

View larger version (12K): [in a new window]   Fig. 5. Sequences of footprinted regions observed in Fig. 4. (A) Footprinting in the termination region of tRNAleu. The conserved binding sequence of mTERF (Valverde et al., 1994b) is boxed. (B) The sequence in the control region located approximately 75 nt 5' of the tRNAmet gene in an area thought to contain the L-strand promoter (Carrodeguas and Vallejo, 1997). The L strand was footprinted in both cases. Primers used were LEU 1 for A and LSP 1 for B (see Table 1 for primers). Genomic positions are indicated. Relative to naked DNA, overmethylated residues are filled squares and undermethylated residues are unfilled squares.

View larger version (42K): [in a new window]   Fig. 6. Footprinting of the H-strand promoter region using different primers. The control region 5' to the 12S rRNA was footprinted using the primers HSP 3, HSP 4 and HSP 5 for panels A—C, respectively. With naked DNA as a reference, undermethylated residues are noted by unfilled squares and overmethylated residues are noted by filled squares. Lane assignments were as follows. Panels A and B: 1, in organello anoxia, pH 6.4; 1a, in organello anoxia, pH 6.4; 2, normoxia, pH 6.4; 3, in organello anoxia, pH 7.9; 4, in organello control (normoxia, pH 7.9); 5, naked DNA. Panel C: 1, normoxia, pH 6.4; 2, in organello anoxia, pH 6.4; 3, in organello control (normoxia, pH 7.9); 4, naked DNA. Genomic positions are indicated by arrows.

View larger version (26K): [in a new window]   Fig. 7. Sequences of footprinted regions in the putative H-strand promoter. With naked DNA as a reference, undermethylated residues are noted by unfilled squares and overmethylated residues are noted by filled squares. Genomic positions are indicated. Panels A, B and C correspond to the gels A, B and C shown in Fig. 6.

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