Discovery of new genes and deletion editing in Physarum mitochondria enabled by a novel algorithm for finding edited mRNAs

Gene finding is complicated in organisms that exhibit insertional RNA editing. Here, we demonstrate how our new algorithm Predictor of Insertional Editing (PIE) can be used to locate genes whose mRNAs are subjected to multiple frameshifting events, and extend the algorithm to include probabilistic predictions for sites of nucleotide insertion; this feature is particularly useful when designing primers for sequencing edited RNAs. Applying this algorithm, we successfully identified the nad2, nad4L, nad6 and atp8 genes within the mitochondrial genome of Physarum polycephalum, which had gone undetected by existing programs. Characterization of their mRNA products led to the unanticipated discovery of nucleotide deletion editing in Physarum. The deletion event, which results in the removal of three adjacent A residues, was confirmed by primer extension sequencing of total RNA. This finding is remarkable in that it comprises the first known instance of nucleotide deletion in this organelle, to be contrasted with nearly 500 sites of single and dinucleotide addition in characterized mitochondrial RNAs. Statistical analysis of this larger pool of editing sites indicates that there are significant biases in the 2 nt immediately upstream of editing sites, including a reduced incidence of nucleotide repeats, in addition to the previously identified purine-U bias.     

Assembly and annotation of full mitochondrial genomes for the corn rootworm species, Diabrotica virgifera virgifera and Diabroticabarberi (Insecta: Coleoptera: Chrysomelidae), using Next Generation Sequence data

Complete mitochondrial genomes for two corn rootworm species, Diabrotica virgifera virgifera (16,747 bp) and Diabrotica barberi (16,632; Insecta: Coleoptera: Chrysomelidae), were assembled from Illumina HiSeq2000 read data. Annotation indicated that the order and orientation of 13 protein coding genes (PCGs), and 22 tRNA and 2 rRNA sequences were in typical of insect mitochondrial genomes. Non-standard nad4 and cox3 stop codons were composed of single T nucleotides and likely completed by adenylation, and atypical TTT start codons was predicted for both D. v. virgifera and D. barberinad1 genes. The D. v. virgifera and D. barberi haplotypes showed 819 variable nucleotide positions within PCG regions (7.36% divergence), which suggest that speciation may have occurred ~ 3.68 million years ago assuming a linear rate of short-term substitution. Phylogenetic analyses of Coleopteran MtD genome show clustering based on family level, and may have the capacity to resolve the evolutionary history within this Order of insects.     

Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44–48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.     

The Complete Chloroplast and Mitochondrial Genomes of the Green Macroalga Ulva sp. UNA00071828 (Ulvophyceae,Chlorophyta)

Sequencing mitochondrial and chloroplast genomes has become an integral part in understanding the genomic machinery and the phylogenetic histories of green algae. Previously, only three chloroplast genomes (Oltmannsiellopsis viridis, Pseudendoclonium akinetum, and Bryopsis hypnoides) and two mitochondrial genomes (O. viridis and P. akinetum) from the class Ulvophyceae have been published. Here, we present the first chloroplast and mitochondrial genomes from the ecologically and economically important marine, green algal genus Ulva. The chloroplast genome of Ulva sp. was 99,983 bp in a circular-mapping molecule that lacked inverted repeats, and thus far, was the smallest ulvophycean plastid genome. This cpDNA was a highly compact, AT-rich genome that contained a total of 102 identified genes (71 protein-coding genes, 28 tRNA genes, and three ribosomal RNA genes). Additionally, five introns were annotated in four genes: atpA (1), petB (1), psbB (2), and rrl (1). The circular-mapping mitochondrial genome of Ulva sp. was 73,493 bp and follows the expanded pattern also seen in other ulvophyceans and trebouxiophyceans. The Ulva sp. mtDNA contained 29 protein-coding genes, 25 tRNA genes, and two rRNA genes for a total of 56 identifiable genes. Ten introns were annotated in this mtDNA: cox1 (4), atp1 (1), nad3 (1), nad5 (1), and rrs (3). Double-cut-and-join (DCJ) values showed that organellar genomes across Chlorophyta are highly rearranged, in contrast to the highly conserved organellar genomes of the red algae (Rhodophyta). A phylogenomic investigation of 51 plastid protein-coding genes showed that Ulvophyceae is not monophyletic, and also placed Oltmannsiellopsis (Oltmannsiellopsidales) and Tetraselmis (Chlorodendrophyceae) closely to Ulva (Ulvales) and Pseudendoclonium (Ulothrichales).     

Genotyping Echinococcus granulosus from dogs from Western Iran

Cystic echinococcosis is a zoonotic infection caused by the dog tapeworm, Echinococcus granulosus. In the present study, adults of E. granulosus (n = 20) were collected from 71 dogs from Western Iran and were genetically characterized using DNA sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1). Consensus sequences were obtained for cox1 (366) and nad1 (471) genes. Phylogenetic analysis of concatenated nad1 and cox1 nucleotide sequence data was performed using Bayesian Inference approach. Overall, the dog isolates indicated nine different sequences in cox1 and seven in nad1 genes. Three genotypes (G1 [75%], G2 [10%] and G3 [15%]) were identified from the isolates. The G2 sequences indicated 100% homology with reference G2 sequence in both cox1 (Genbank accession number M84662) and nad1 (AJ237633) genes. G3 sequences showed 100% homology with G3 reference sequence in nad1 (AJ237633), but displayed two different cox1 profiles, each having 99% homology with reference G3 sequence (M84663). In the phylogenetic tree all of the isolates were grouped into a distinct cluster corresponding to the G1–G3 complex with relevant reference sequences. The presence of G1 genotype (sheep strain) of E. granulosus sensu stricto as dominant genotype in dogs is emphasized. To the best of our knowledge, this study established the first record of E. granulosus sensu stricto, G2 genotype in Iran.     

A point mutation in the core subunit gene of yeast mitochondrial RNA polymerase is suppressed by a high level of specificity factor MTF1

Analysis of a transcribed region in the mitochondrial genome of Oenothera revealed an open reading frame (ORF) of 577 codons (orf577) that is also conserved in carrot, here encoding a protein of 579 amino acids (orf579). RNA editing alters the mRNA sequence of orf577 in Oenothera with 46 C to U transitions, many of which improve sequence similarity with the homologous Marchantia gene orf509. The deduced polypeptides show significant similarity with the ccll-encoded protein involved in cytochrome c biogenesis in the photosynthetic bacterium Rhodobacter capsulatus. A highly conserved domain is also found in plastid ORFs, suggesting that these bacterial, chloroplast and mitochondrial genes encode polypeptides with analogous functions in assembly and maturation of cytochromes c.     

Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and <Emphasis Type="Italic">nad</Emphasis>5 genes

In the present study, samples representing three hard tick species and one soft tick species, namely Dermacentor marginatus, Haemaphysalis punctata, Ixodes ricinus and Argas persicus from southwestern Romania, and one hard tick, Haemaphysalis longicornis, from China were characterized genetically by a portion of mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) and a portion of nicotinamide adenine dinucleotide dehydrogenase subunit 5 gene (pnad5). The pcox1 and pnad5 were amplified separately from individual ticks by PCR, sequenced and analyzed. The length of pcox1 and pnad5 sequences of all samples was 732 and 519 bp, respectively. The intra-specific sequence variation in De. marginatus was 0.1–1.0% for pcox1 and 0.2–1.2% for pnad5, whereas in Ha. punctata it was 0.4–1.9% for pcox1 and 0.4–1.0% for pnad5. For the tick species examined in the present study, sequence comparison revealed that the inter-specific sequence differences were higher: 15.9–27.6% for pcox1 and 20.3–42.4% for pnad5. This suggests that the cox1 and nad5 sequences could provide useful genetic markers for the specific identification and genetic characterization of ticks in Romania and elsewhere.     

Mitochondrial Genome of Spirometra theileri Compared with Other Spirometra Species

This study was carried out to provide information on the taxonomic classification and analysis of mitochondrial genomes of Spirometra theileri. One strobila of S. theileri was collected from the intestine of an African leopard (Panthera pardus) in the Maswa Game Reserve, Tanzania. The complete mtDNA sequence of S. theileri was 13,685 bp encoding 36 genes including 12 protein genes, 22 tRNAs and 2 rRNAs with absence of atp8. Divergences of 12 protein-coding genes were as follow: 14.9% between S. theileri and S. erinaceieuropaei, 14.7% between S. theileri and S. decipiens, and 14.5% between S. theileri with S. ranarum. Divergences of 12 proteins of S. theileri and S. erinaceieuropaei ranged from 2.3% in cox1 to 15.7% in nad5, while S. theileri varied from S. decipiens and S. ranarum by 1.3% in cox1 to 15.7% in nad3. Phylogenetic relationship of S. theileri with eucestodes inferred using the maximum likelihood and Bayesian inferences exhibited identical tree topologies. A clade composed of S. decipiens and S. ranarum formed a sister species to S. erinaceieuropaei, and S. theileri formed a sister species to all species in this clade. Within the diphyllobothridean clade, Dibothriocephalus, Diphyllobothrium and Spirometra formed a monophyletic group, and sister genera were well supported.     

Evolution of monoblepharidalean fungi based on complete mitochondrial genome sequences

We have determined the complete mitochondrial DNA (mtDNA) sequences of three chytridiomycete fungi, Monoblepharella15, Harpochytrium94 and Harpochytrium105. Our phylogenetic analysis based on concatenated mitochondrial protein sequences confirms the placement of Mono blepharella15 together with Harpochytrium spp. and Hyaloraphidium curvatum within the taxonomic order Monoblepharidales, with overwhelming support. These four mtDNA sequences encode the standard fungal mitochondrial gene complement and, like certain other chytridiomycete fungi, encode a reduced complement of 7–9 tRNAs, some of which require 5′-tRNA editing to be functional. Highly conserved sequence elements were identified upstream of almost all protein-coding genes in the mtDNAs of Monoblepharella15 and both Harpochytrium species. Finally, a guanosine residue is conserved upstream of the predicted ATG or GTG start codons of almost every protein-coding gene in these genomes. The appearance of this G residue correlates with the presence of a non-canonical cytosine residue at position 37 in the anticodon loop of the mitochondrial initiator tRNAs. Based on the unorthodox features in these four genomes, we propose that a 4 bp interaction between the CAUC anticodon of these tRNAs and GAUG/GGUG codons is involved in translation initiation in monoblepharidalean mitochondria. Intriguingly, a similar interaction may also be involved in mitochondrial translation initiation in the sea anemone Metridium senile.     

Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.     

Distribution patterns and introduction pathways of the cosmopolitan brown alga Colpomenia peregrina using mt cox3 and atp6 sequences

Colpomenia peregrina is an annual brown macroalga found in temperate waters worldwide. To understand population differentiation and to reconstruct pathways of colonization/introduction, we analyzed variation in two mitochondrial protein-coding genes, cytochrome c oxidase subunit III (cox3) and ATP synthase F0 subunit 6 (atp6), and cp RuBisCO spacer. A total of 359 cox3, 342 atp6, and 38 RuBisCO spacer sequences from Colpomenia peregrina were obtained for samples collected at 28 sites from 12 countries. The combined cox3?+?atp6 sequences (1,231 bp) revealed 99 polymorphic sites and 69 haplotypes. An mt haplotype network revealed four distinct groups, separated by 7 to 26 mutation steps. NW Pacific populations were present in each group (but dominant in one), whereas SW Pacific and the Atlantic populations each were present in one group. The network and phylogenetic analyses, along with patterns of genetic diversity, suggested a NW Pacific center of origin, expanding first to the SW Pacific, then the NE Pacific, and most recently to the north Atlantic. A generalized skyline plot revealed a dramatic population expansion of the species ca. 20 kya.