A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis
© Jacob et al; licensee BioMed Central Ltd. 2009
Received: 28 July 2009
Accepted: 24 September 2009
Published: 24 September 2009
Mitochondria (mt) contain their own autonomously replicating DNA, constituted as a small circular genome encoding essential subunits of the respiratory chain. Mt DNA is characterized by a genetic code which differs from the standard one. Interestingly, the mt genome of nematodes share some peculiar features, such as small transfer RNAs, truncated ribosomal RNAs and - in the class of Chromadorean nematodes - unidirectional transcription.
We present the complete mt genomic sequence (16,791 bp) of the plant-parasitic nematode Radopholus similis (class Chromadorea). Although it has a gene content similar to most other nematodes, many idiosyncrasies characterize the extremely AT-rich mt genome of R. similis (85.4% AT). The secondary structure of the large (16S) rRNA is further reduced, the gene order is unique, the large non-coding region contains two large repeats, and most interestingly, the UAA codon is reassigned from translation termination to tyrosine. In addition, 7 out of 12 protein-coding genes lack a canonical stop codon and analysis of transcriptional data showed the absence of polyadenylation. Northern blot analysis confirmed that only one strand is transcribed and processed. Furthermore, using nucleotide content bias methods, regions for the origin of replication are suggested.
The extraordinary mt genome of R. similis with its unique genetic code appears to contain exceptional features correlated to DNA decoding. Therefore the genome may provide an incentive to further elucidate these barely understood processes in nematodes. This comprehension may eventually lead to parasitic nematode-specific control targets as healthy mitochondria are imperative for organism survival. In addition, the presented genome is an interesting exceptional event in genetic code evolution.
Nematodes are one of the largest phyla of multicellular animals on earth with over 20,000 described species. The burrowing nematode Radopholus similis infects numerous (sub)tropical crops and is considered as one of the most damaging pests on banana. Recently transcriptomic sequence data of this parasite were studied and several 'expressed sequence tags' (ESTs) originated from genes of the mitochondrial (mt) genome . Mitochondria are found in all eukaryotic cells and provide the cell with energy through the process of oxidative phosphorylation. Originating from an ancestral endosymbiotic α-proteobacterial species , they still contain a haploid, autonomously replicating genome of relatively short length, in nematodes ranging from 12.5 kb to 26 kb . To date, complete mt genomes of 31 nematode species are available in GenBank: 9 of the Enoplean class and 22 (mostly comprising animal-parasitic nematodes) of the Chromadorean class. The mt gene products are usually 2 ribosomal RNAs, 22 transfer RNAs and 12-13 intronless protein-coding genes which encode crucial subunits in respiratory complexes I, III, IV and V. Mt genomes of nematodes differ in some aspects from other metazoan mt genomes. The compact nematode mt genomes usually lack ATPase subunit 8, and contain shortened rRNA molecules and truncated tRNAs. Nearly all nematode mt tRNAs lack one arm, either the TΨC arm which is replaced by the 'TV-replacement loop', or the DHU arm which is replaced by D-replacement loops [3, 4]. In addition, in nematodes of the class Chromadorea (containing most important parasitic nematodes), all mt genes are unidirectionally transcribed from one strand.
Results and discussion
Relative synonymous codon usage (RSCU) and number of codons per 1000 codons (NC1000) in the protein coding genes of the mitochondrial genome of R. similis.
Mt DNA replication is an asymmetrical process, in which the heavy strand replication initiation precedes that of the light strand. Hence some sequence parts of mt genomes spend more time single stranded, a state in which the DNA is more prone to deaminations of cytosine to uracil, and to a lesser extent of adenine to hypoxanthine . The subsequent accumulation of T and G (at the expense of C and A, respectively) in these regions can provide information about the origins of replication . In this way, the light strand origin of replication is predicted to be located in secondary structures surrounding the sextuple C motif (figure 1 and 5). Analysis of the relative amount of T and G versus A and C, on complete sequence as well as on nucleotides of the third codon positions, showed a pattern that could only be explained by assuming two different origins of replication . Based on these observations, we hypothesize that the origin of the heavy strand is located in the cox2 gene (figure 5). Although for nematodes nothing is known on this issue, our data suggest a similar mode of action for mt replication as other Metazoa, with origins of replications correlated to secondary structures in the DNA. The predicted OriL is located at the region harboring the sextuple C motif and surrounding secondary structures, and the predicted OriH lies in the vicinity of two tRNA genes, which could function as replication origins as observed in Vertebrates . However, no clear similarity between the secondary structures reported for other nematodes (see for example ) and those of R. similis was found.
DNA extraction and LD-PCR
R. similis was cultured as decribed in . On 500 ng of total phenol/chloroform extracted DNA from approximately 10,000 nematodes, LD-PCR was performed using a combination of Expand Long Range dNTP Pack (Roche, Mannheim, Germany) and Phusion High-Fidelity DNA Polymerase (Finnzymes, Espoo, Finland) with gene specific primers based on EST sequences  (see additional file 6). PCRs were done following the manufacturer's instructions in 50 μL reaction mixtures, containing 6% DMSO. All fragments were directly sequenced.
Extraction of mitochondria
Approximately 400 μL packed nematodes (approximately 200,000 nematodes) were washed with sterile demineralized water, and brought into 1 mL sucrose buffer (300 mM sucrose, 30 mM Tris, and 10 mM EDTA, pH7.9). With the aid of a Teflon pestle and some sand, a nematode homogenate was obtained and centrifuged for 5 min at 500 g. The supernatant was further purified by additional centrifugation for 5 min at 1000 g. A pellet of mitochondria was obtained by centrifuging the supernatant at 15,000 g for 45 min. Obtained mitochondria were used immediately.
Mitochondrial RNA extraction
The mitochondria were dissolved in 200 μL Trireagent (Sigma, St. Louis, MO, USA), and incubated for 1 h at RT. The solution was three times sonicated at the lowest setting for 2 sec while keeping on ice (Branson Sonifier 250, Danbury, USA). After incubation for 15 min at room temperature, 40 μL chloroform was added and the tube was vigorously shaken by hand. Further RNA extraction steps were performed following the manufacturer's instructions.
Mt RNA was circularized using T4 RNA ligase (Fermentas, St. Leon-Rot, Germany), using 1 μg mt RNA in a total volume of 20 μL, following the manufacturer's instructions. After incubation for 1 h at 37°C, the volume was adjusted to 200 μL with demineralized water, and RNA purified by standard phenol/chloroform extraction. After resuspension in 20 μL demineralized water, the circularized RNA was used for the reverse transcriptase reaction using SuperScript II (Invitrogen, Carlsbad, CA, USA) following manufacturer's instructions and a reverse-oriented gene-specific primer located at the 5' end of the gene (see additional file 4 and 7). The resulting cDNA was subjected to nested PCR using gene-specific primers. PCR products were ligated into the pGEM-T vector (Promega, Wisconsin, USA) and transformed into E. coli DH5α cells. Positive clones were selected on LB plates supplemented with carbenicilin (100 μg/ml) and inserts were sequenced.
Mt RNA was separated on a 1× MOPS 1% formaldehyde agarose gel and blotted (downward capillary) onto Hybond-N+ membrane (Amersham, Uppsala, Sweden). RNA probes were generated from plasmids containing fragments of cox1, cytB and nad5 as template (see additional file 7 for primers) using Riboprobe kit (Promega) in presence of 32P-labeled nucleotides. Hybridization was done overnight at 56°C, and after washing, signals were visualized using a fluor imager FLA-5100 (Fujifilm, Tokyo, Japan).
1.2 μg total DNA was digested with SpeI and XmnI restriction enzymes, and separated by inversed field electrophoresis in 0.5 × TBE 1% agarose gel. After capillary blot, hybridization was performed using the High Prime DIG labeling Starter Kit (Roche) following manufacturer's instructions with a probe constructed using primers RsNADHD_2 and RsCOXI_7 (see additional file 7) covering 3,504 bp of the mitochondrial genome. The chemiluminescent signal was detected on a fluor imager FLA-5100 using standard settings (Fujifilm).
Annotation and nucleotide composition analysis
JJ has a grant of Ghent University (BOF).
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