Mitochondrial LSU rRNA of Diplonema. (A) Northern blot hybridization. Lane 1, in vitro transcription product of rnl module 1 (540 nt); lane 4, in vitro transcription product of rnl module 2 (359 nt; synthetic RNAs are 6 and 7 nt longer than the corresponding modules); lanes 2, 3, 5 and 6, total RNA (∼5 μg); lanes 7 and 8, poly(A) RNA (∼0.5 μg) extracted from whole cells. RNA in lanes 2 and 5 is from one preparation; that in lanes 3 and 6 is from an independent preparation. Blotted RNA was probed with radioactively labeled oligonucleotides dp216 (lanes 1–3) and dp218 (lanes 4–8) that target module 1 and module 2 of rnl, respectively. Bands represent the mature mt-LSU rRNA (∼900 nt), mono-module 1 transcripts (∼550 nt; the weak band in lane 3 is clearly visible on the original image), mono-module 2 transcripts (∼450 nt) and presumptive end-processing intermediates of single-module transcripts. The size markers are indicated on the left. The signal ratio of mt-LSU rRNA versus mono-module 1 transcripts varies noticeably from one preparation to another; it is 100:1 in lane 2 and 60:1 in lane 3. The signal ratio of mt-LSU rRNA versus mono-module 2 transcripts (lanes 5 and 6; total RNA) is ∼20:1. This ratio is ∼1:5 to ∼1:17 in poly(A)-enriched RNA (lanes 7 and 8), a variation depending on the particular oligo(dT) pull-down experiment. Notably, the steady-state of mono-module 1 transcript is lower than that of mono-module 2. The same is seen in RNA-Seq experiments (see Figure 4). (B) Upper part, schematic sequence of mtLSU rRNA. The U-tract between modules 1 and 2 (black box) is not encoded by mtDNA, but added post-transcriptionally. Regions with which northern hybridization probes dp216 and dp218 anneal are indicated. Lower part, coding regions of mt-LSU rRNA on mitochondrial chromosomes. Modules 1 and 2 are contained in cassettes of B-class chromosomes, but oriented in opposite direction relative to the chromosome’s constant region [indicated as B(+) and B(−), see text]. Non-coding regions within the cassettes (‘unique flanking regions’) are shown in dark gray. The constant region of chromosomes (light gray) is ∼95% identical across all B-class chromosomes (7). The black part of the constant region is also present in A-class chromosomes (‘shared constant region’).