Research Communication Massive Mitochondrial DNA Content in Julius Lukes1,2*† Richard Wheeler3† Diplonemid and Kinetoplastid Protists Dagmar Jirsová1 Vojtech David4 John M. Archibald4* 1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceské Budejovice (Budweis), Czech Republic 2Faculty of Science, University of South Bohemia, Ceské Budejovice (Budweis), Czech Republic 3Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 4Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada Summary The mitochondrial DNA of diplonemid and kinetoplastid protists is known 260 Mbp of DNA in the mitochondrion of Diplonema, which greatly for its suite of bizarre features, including the presence of concatenated cir- exceeds that in its nucleus; this is, to our knowledge, the largest amount cular molecules, extensive trans-splicing and various forms of RNA edit- of DNA described in any organelle. Perkinsela sp. has a total mitochon- ing. Here we report on the existence of another remarkable characteristic: drial DNA content ~6.6× greater than its nuclear genome. This mass of hyper-inflated DNA content. We estimated the total amount of mitochon- DNA occupies most of the volume of the Perkinsela cell, despite the fact drial DNA in four kinetoplastid species (Trypanosoma brucei, Trypano- that it contains only six protein-coding genes. Why so much DNA? We plasma borreli, Cryptobia helicis,andPerkinsela sp.) and the diplonemid propose that these bloated mitochondrial DNAs accumulated by a Diplonema papillatum. Staining with 40,6-diamidino-2-phenylindole and ratchet-like process. Despite their excessive nature, the synthesis and RedDot1 followed by color deconvolution and quantification revealed maintenance of these mtDNAs must incur a relatively low cost, consider- massive inflation in the total amount of DNA in their organelles. This was ing that diplonemids are one of the most ubiquitous and speciose protist further confirmed by electron microscopy. The most extreme case is the groups in the ocean. © 2018 IUBMB Life, 00(00):1–8, 2018 Keywords: DNA content; kinetoplast DNA; mitochondrial DNA; protist INTRODUCTION fact the boundaries of this diversity are to be found among plants and the unicellular eukaryotes (protists) belonging to the Mitochondrial genomes come in all shapes and sizes. Much of supergroup Excavata (1). The mitochondrial genomes of flower- our detailed knowledge of the diversity and evolution of this ing plants range from ~200 kb to over 10 Mb, have very low gene endosymbiotically derived organelle stems from Metazoa, but in densities, undergo frequent rearrangements associated with the gain or loss of intergenic regions (2–4) and are capable of acquir- Abbreviations: AT, adenine and thymine; bp, base pairs; CNE, constructive ing, via horizontal gene transfer, whole mitochondrial genomes neutral evolution; DAPI, 40,6-diamidino-2-phenylindole; GC, guanine and cyto- from other organisms (5). The mitochondrial genomes of exca- sine; gRNA, guide RNA; kDNA, kinetoplast DNA; mtDNA, mitochondrial DNA vate protists are unusual in other ways. While jakobids harbor © 2018 International Union of Biochemistry and Molecular Biology Volume 00, Number 00, Month 2018, Pages 1–8 the most gene-rich mitochondrial DNA known (6), it is the mito- — *Address correspondence to: Julius Lukes, Institute of Parasitology, Biology Cen- chondrial genomes of Euglenozoa a diverse group of aerobic tre, Czech Academy of Sciences, CeskeBud ejovice (Budweis), Czech Republic. excavates composed of photosynthetic Euglenida, heterotrophic Tel: +420 38 777 5416. Fax: +420 38 531 0388. E-mail: [email protected] free-living Diplonemea, and parasitic Kinetoplastea—that exhibit or a range of unique features. The mitochondrial DNA John M. Archibald, Department of Biochemistry and Molecular Biology, Dal- (or kinetoplast DNA) of the human parasite Trypanosoma brucei housie University, Halifax, Canada. Tel: +1 902 494 2536. Fax: +1 902 494 is amongst the best-studied non-nuclear genomes. Composed of 1355. E-mail: [email protected]†Julius Lukes and Richard Wheeler con- tributed equally to this work. thousands of mutually interlocked minicircles and dozens of Received 24 May 2018; Accepted 12 June 2018 maxicircles, this single densely packed kDNA network requires DOI 10.1002/iub.1894 highly complex machinery for its maintenance and expression Published online 00 Month 2018 in Wiley Online Library (7). The transcripts of most maxicircle protein-coding genes (wileyonlinelibrary.com) undergo RNA editing in the form of uridine insertions and/or IUBMB Life 1 IUBMB LIFE deletions (8, 9). These post-transcriptional events are specified (10 mM TrisÁHClpH8.0,50mMNaCland1mMEDTA)at100nM, by hundreds of minicircle-encoded guide (g) RNAs, which along mixed 1:1 with the appropriate reverse strand, and annealed into with numerous large protein complexes produce a mere dozen double stranded DNA by denaturation at 95 C for 2 min followed mitochondrial proteins (10). Recent genomic and transcriptomic bycoolingto25 C over 45 min in a polymerase chain reaction evidence confirms earlier predictions (11) that uridine-indel RNA machine.ThedoublestrandedDNAwasdilutedto25nMand × × editing is present in all Kinetoplastea (12), with the notable mixedwith18nMDAPIor1 RedDot1 (supplied as a 200 stock, of fi exception of petite mutants of T. brucei,wholackthemitochon- unspeci ed concentration). Fluorescence with 358 nm excitation drial genome as such (13). and 461 nm emission (DAPI) and 651 nm excitation and 694 nm fl While the kinetoplast DNA of T. brucei and all related para- emission (RedDot1) was measured using a SpectraMax M5 uores- sitic trypanosomatids exists as a periflagellar densely packed cence plate reader (Molecular Devices). Background signal from an network, the mitochondrial DNA of parasitic and free-living unstained sample was subtracted from the measurements. bodonids, a sister group of trypanosomatids, exists in various Cells were washed by phosphate buffered saline (PBS) solu- forms (14). Electron microscopy and molecular studies have tion and centrifuged at 800g for 3 min; this step was repeated μ shown that it is usually dispersed throughout much of the mito- three times. Cells were then resuspended in 150 L of PBS and chondrial lumen, either uniformly or condensed in multiple foci. the mixture was transferred to slides, which were left for 30 min Its structure also varies, ranging from free circles and small at room temperature (RT) to allow cells to adhere. Due to the catenanes to long linear or circular molecules. Invariably, how- marine origin of Perkinsela and D. papillatum, 70% seawater ever, the bodonids carry much more DNA in their mitochondria was used instead of PBS in these steps. The remaining solution fi than do the trypanosomatids (12, 15–17). was washed off by PBS and slides were xed in 4% paraformal- The mitochondrial DNAs of the Euglenida and Diplonemea, dehyde for 30 min at RT. All paraformaldehyde was washed − which are sister groups of kinetoplastids, are strikingly different. down by PBS and slides were immersed in 20 Cmethanoland While the free-living alga Euglena gracilis contains a small mito- incubated for 4 h in a freezer. After this step, cells were rehy- chondrial genome with a canonical set of protein-coding genes drated in PBS for 10 min. For RNA treatment, slides were incu- μ whosetranscriptsareapparentlynot post-transcriptionally modi- bated with RNAse A (50 g/mL) for 2 h at RT and RNAse A was fied (18), the mitochondrial DNA of the heterotrophic D. papillatum subsequently gently removed by incubation in PBS. Slides were is truly bizarre. It is composed of thousands of DNA circles, which stained by RedDot1 diluted 1:1,000 in PBS for 30 min according ’ possess protein-coding genes broken into tens of fragments. In to the manufacturer s instructions. Fluorescent dye was washed order to produce functional mRNAs, the products of these broken and slides were mounted with mounting solution (Vectashield) genes must be trans-spliced into contiguous sequences (often joined containing DAPI. Representative areas in the mitochondrial or by stretches of post-transcriptionally added uridines) and heavily kinetoplast DNA and nuclear DNA of each species were selected edited by nucleotide substitutions (19–21). In other diplonemid spe- manually as the reference values for color deconvolution. This cies, the complexity of trans-splicing and RNA editing appears even organellar DNA was more AT-rich than nuclear DNA in all spe- higher than in D. papillatum (22, 23). The kinetoplastids and diplo- cies except D. papillatum. The AT content of the C. helicis kineto- nemids appear to illustrate parallel evolution of extremely complex plast DNA was only slightly more AT-rich than the nuclear DNA, mitochondrial RNA processing machineries not seen in other limiting the quality of color deconvolution. Relative quantity of eukaryotes. Here we show that these two lineages are also remark- kinetoplast DNA and nuclear DNA were measured from the inte- fl able in the total amount of DNA found in their mitochondria. grated RedDot1 uorescence intensity using manual area sec- tions based on the color deconvolution result. For Perkinsela this measurement is likely an underestimate of the proportion of kinetoplast DNA; out-of-focus and scattered light from the imme- MATERIALS AND METHODS diately adjacent large kinetoplast DNA will have contributed to Trypanosoma brucei, Trypanoplasma borreli, Perkinsela sp., and fluorescence signal