Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae)

Abstract

Genomic DNA was extracted from three oocyst isolates of Hammondia triffittae from foxes and two oocyst isolates of Hammondia heydorni from dogs, as well as from cell culture-derived tachyzoites of Toxoplasma gondii (RH strain) and Neospora caninum (NC-Liverpool strain), and examined by PCR with primers targeting the cytochrome b (cytb) and the cytochrome c oxidase subunit I (cox1) genes in order to characterise both genes and, if possible, the remainder of the mitochondrial genome of these species. Several primers were designed and used in various combinations to amplify regions within and between both genes and to determine gene order. When certain forward primers targeting cytb were used in combination with certain reverse primers targeting cox1, two overlapping sequences were obtained for each species and isolate studied, which showed that a full-length copy of cytb was followed 36-37 bp downstream by a full-length copy of cox1, and these sequences are believed to represent the true mitochondrial genes and the gene order in the mitochondrial genome of the four species examined. The cytb of T. gondii, N. caninum, H. heydorni and H. triffittae comprised a total of 1,080 bp (359 amino acids) and used ATG and TAA as start and stop codon, respectively. The cox1 of these species also used TAA as stop codon, whereas the most likely start codon was ATG, resulting in a gene comprising 1,491 bp (496 amino acids). Pair-wise sequence comparisons based on either cytb or cox1 clearly separated T. gondii from N. caninum and both of these species from the two Hammondia species, whereas the latter two species were 100 % identical at cytb and shared 99.3 % identity at cox1. Phylogenetic analyses using the maximum-likelihood method confirmed these findings and placed T. gondii in a clade separate from the three other species and all four Toxoplasmatinae in a sister clade to Eimeria spp. PCR with other primers and/or primer pairs than those used to obtain the full-length mitochondrial genes yielded several types of about 1-1.5 kb long sequences, which comprised stretches of the primer-targeted genes at both ends and an intervening non-coding sequence of various length and composition. Thus, portions of cytb could be found both upstream and downstream from portions of cox1 and portions of the same gene could be found adjacent to each other (cytb→cox1; cox1→cytb; cytb→cytb; cox1→cox1). Sequence comparisons revealed that some of these gene fragments were truncated genes, whereas others included the putative start or stop codon of the full-length mitochondrial genes. From the nature of the gene fragments and/or their flanking sequences, they are assumed to be located on the chromosomes of the nuclear genome and to represent nuclear mitochondrial DNA segments (numts) or pseudogenes. In the four species examined, there were no nucleotide differences between the full-length mitochondrial copies of cytb and cox1 and their various incomplete nuclear counterparts. With a few exceptions, identical numt types and closely similar flanking seq...

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences

“…Thereby, as in other studies (GJERDE, 2013a, b), organellar genetic sequences can give a reliable estimate of the diversity among sarcocystid species. Gjerde (2013a), demonstrated unequivocally that mitochondrial markers have the ability to reconstruct evolutionary history within the Sarcocystidae family.…”

“…Thereby, as in other studies (GJERDE, 2013a, b), organellar genetic sequences can give a reliable estimate of the diversity among sarcocystid species. Gjerde (2013a), demonstrated unequivocally that mitochondrial markers have the ability to reconstruct evolutionary history within the Sarcocystidae family. The cox2 sequences of Toxoplasma gondii, Neospora caninum, Hammondia triffitti and Hammondia heydorni reported in the work above had nucleotide diversity of 0.06484, whereas the diversity in synonymous and non-synonymous sites was 0.21371 and 0.01569, respectively (data not shown).…”

“…A third lineage of Hammondia heydorni was recently reclassified as Hammondia triffitti, due to its molecular divergence from the other lineages and to its characteristic of only using foxes, and not dogs, as its definitive host. Isolates classified as H. triffitti have cytB fragments identical to those of the H. heydorni lineages used in this study (GJERDE, 2013a). A single synonymous nucleotide substitution differentiates the sequence of SF1 from that of SN138.…”

“…Cytochrome oxidase II and cytochrome B (cytB) coding sequences have been used for this purpose (GJERDE, 2013a, b). Similar to mitochondria, apicoplasts are organelles of maternal inheritance (FERGUSON et al, 2005) that contain conserved genes that are commonly used to reconstruct evolutionary histories (FEAGIN, 1994;KOHLER et al, 1997).…”

Genetic Evidence for Cytochrome b Q i Site Inhibition by 4(1 H )-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii

“…The sequence with accession number AF023246.1 was generated by 3= and 5= rapid amplification of cDNA ends (RACE) (17). The sequence with accession number JX473253.1 was generated by PCR from DNA using primers based on the sequence with accession number AF023246.1 (18).…”