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.