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. 2009 Sep 24:2:192.
doi: 10.1186/1756-0500-2-192.

A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis

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A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis

Joachim E M Jacob et al. BMC Res Notes. .

Abstract

Background: 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.

Findings: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Overview of the organization of the circular mt DNA of R. similis. The arrow indicates direction of transcription. Genes and non-coding regions are indicated: in white, the protein-coding and rRNA genes, in gray, the tRNA genes called by their amino acid symbol (S1: Ser-AGN, S2: Ser-UCN, L1: Leu-CUN, L2: Leu-UUR). Bold and italic numbers indicate non-coding and overlapping nucleotides between neighboring genes, respectively. The pattern-filled part represents the large non-coding region, divided in five regions as explained in the text. The repeat region of 302 bp is filled with large checkers and the 26 bp repeat region is filled with small checkers. The black lines at the inner periphery of the ring represent EST sequences, with UAG stop codons indicated by little black triangles. The colored bar code-like circles represent the nucleotide content of the coding strand, differing intensities corresponding to different content (red: thymine, blue: adenine, green: guanine, purple: cytosine). The positions of secondary structures depicted in figure 4 are indicated by light-gray balks perpendicular to the circle. The predicted origins of replication for the heavy (OriH) and light strand (OriL) are indicated (see figure 5).
Figure 2
Figure 2
Predicted secondary structure of the 16S rRNA gene of R. similis. Watson-Crick base pairing and G:U base pairing is indicated by a line and a dot, respectively. Numbering of helices is according to De Rijk et al. [29] and other published nematode mt rRNA structures. Binding sites for the amino-acyl of tRNAs (A), peptidyl-transferase (P), or both (AP) as defined by Noller [30] and Hu [6] are indicated. Compared to other nematode 16S rRNAs, shaded nucleotides are conserved in at least 90% of the cases, while arrows indicate remarkable deletions.
Figure 3
Figure 3
Northern blot on mt RNA of R. similis. A. cox1 antisense probe. Hollow arrowhead indicates 2.2 kb. Expected length is 1.6 kb. B. nad5 antisense probe. Expected length is 1.5 kb. C. cytB antisense probe. Black arrowhead indicates 1.4 kb. Expected length is 1.0 kb. Probing with sense probes gave no detectable signals in all cases (data not shown).
Figure 4
Figure 4
Summary of potential secondary structures in the non-coding regions. C and G nucleotides are shaded. A. GC-rich patch located before the short repeat region (9108..9335); B. large stem loop with A-rich 'bulge', with EST evidence indicated by dashed line (arrow: 5' start) (8906..9036); C. The sextuple C motif located in the sequence between the two repeat regions (10498..10729); D. AT-rich non-coding region between tRNAMet and tRNAThr (12611..12675); E. local GC-rich region, surrounded by AT-rich sequences, located in the sequence between the two repeat regions (10855..10963). A stretch of 8 A nucleotides at the 3' end is similar as described by Hu [6] and Jex [7].
Figure 5
Figure 5
Nucleotide composition analysis of the mt genome of R. similis. A. Based on a cumulative GC-skew (G-C/G+C) graph, the offset (vertical bar) from the fitted model (dashed line) corresponds to the location of the origin of replication of the light strand (OriL) [22]. B. A linear representation of the circular mt genome, with rRNA and PCGs as indicated. Black bars and checkers represent tRNAs and repeat regions, respectively. The predicted OriL is located near the sextuple C motif. The prediction of OriH is explained in C and D. C. Due to deaminations preferably occurring during single-strandness, the ratios T/C (right y-axis), G/A (left y-axis) and T+G/C+A (left y-axis) differ. From these curves, in which 1 is the mean value, predictions about OriL and OriH can be made. The minimum of the T/C curve corresponds to the OriL, while for G/A the minimum is located in the tRNAs preceding nad4. These differences could be explained by different kinetics for both deamination processes [21]. The T+G/C+A measure (covering both processes) reaches a maximum at the start of cox2 and a minimum at the predicted OriL. From this, two replication origins could be concluded, with OriH located at the start of cox2. If OriL is at 11,000 and OriH at 4,000, both replicases meet around 1,000 (assuming similar speeds), leaving this region single-stranded for a short period, causing a local minimum (star). D. The average T+G/A+C (left y-axis) and G/A (right y-axis) of the nucleotides at third codon positions. From cox2 on, both measures decrease with a minimum at nad6. The G/A ratio shows a pattern corresponding to that of T+G/A+C, as opposed to graph C, where the kinetics of the A to G mutations [21] and the natural variation cause a high level of noise in the G/A ratio.

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