*3.3.5. Stop codon depletion in antisense strand and stop codon translation: ND4l and ND5*

Annotations in GenBank for *Aleurodicus dispersus*' ND4l and ND5 do not match proteins homologous to the corresponding NADH dehydrogenase subunits. For ND4l, the peptide translated from frame 0 of the antisense of that gene (this frame includes five stops) yields a short alignment with the regular protein in *Aleurodicus dugesii* (24 residues, 87% similarity, e value 8.5, not shown). Hence, the annotated ORF in *Aleurodicus dispersus* is probably a stop-codon-depleted antisense sequence (the corresponding frame in *Aleurodicus dugesii* has four stops), which does not code for the regular ND4l gene. This stop codon depletion in *Aleurodicus dispersus* introduced five stops in the sense strand frame that apparently codes for ND4l according to the above-described alignment.

For ND5, the peptide translated from the +1 frame of the antisense of the GenBank-annotated sequence is homologous over its complete length to NADH dehydrogenase subunit 5 of *Aleurodicus dugesii* (89% similarity, e value 0, not shown). This frame has a single stop codon that aligns with serine in *Aleurodicus dugesii* (see discussion of insertion of serine at stops in previous section).

> codon percentages decrease in 11 among 13 protein coding genes of *Aleurodicus dispersus*, as compared to *Aleurodicus dugesii*. This is a significant majority of cases according to a one tailed sign test using a binomial distribution and assuming equal probability of getting more or less stop codons in any of these mitogenes (P = 0.00562). This overall stop codon depletion occurs in all seven "recoded" genes. Stop codon depletion occurs qualitatively in four among the six genes with regular, unchanged coding structure. This tendency is not statistically significant for this subgroup of genes when using the robust, but blunt nonparametric sign test. A paired t test between mean percentages of stop codons averaged across frames indicates also for these six genes a statistically significant decrease in stops in *Aleurodicus dispersus*, as compared to *Aleurodicus dugesii*. This result suggests that stop codon depletion occurred across all or at least most of this genome, and for most frames, not only for genes whose coding structure was altered, and not only for frames

> **Table 1.** TAR stop codons in the six frames of mitogenome-encoded genes of *Aleurodicus dugesii* and *Aleurodicus*

Directed Mutations Recode Mitochondrial Genes: From Regular to Stopless Genetic Codes

http://dx.doi.org/10.5772/intechopen.80871

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Presumably, unknown mechanisms associated with replication depleted stop codons in this species' mitogenome, perhaps cumulatively over several replication or DNA edition cycles. Total stop codon depletion in some frames produced new ORFs. Natural selection against stop codons presumably enhanced unknown enzymatic phenomena, eliminating stop codons in these frames. It seems plausible that these frames in usual mitogenomes code for proteins translated by stop suppressor tRNAs. Specific unknown conditions in *Aleurodicus dispersus* may favor enhanced expression of peptides coded by frames that usually include stops in other mitogenomes, such as *Aleurodicus dugesii*. These constraints would have ultimately caused genomic stop codon depletion in *Aleurodicus dispersus*. In regular mitogenomes, stop codon translation downregulates expression of these unusual peptides in favor of proteins coded by regular ORFs, but in *Aleurodicus dispersus*, this hierarchy may be inexistent (when two stopless ORFs occur in a gene) or reversed (as in several mitogenes of *Lepidochelys olivacea* [9]), with translation of the unusual peptide not necessitating stop codon suppression, and translation of regular mitochondrial proteins requiring tRNAs with anticodons matching stop codons.

who became ORFs.

*dispersus*.

#### *3.3.6. Stop codon depletion in antisense strand, frameshift, and stop translation: ND4*

A further mitochondrial gene for which the GenBank annotation does not produce the expected protein for *Aleurodicus dispersus* is ND4. Alignment analyses detect peptides homologous with regular NADH dehydrogenase subunit 4 when translating frames +1 and +2 of the antisense of the GenBank-annotated ND4 gene. Blastp alignment analyses detect homology with the regular ND4-encoded protein of *Aleurodicus dugesii*; the regular protein is encoded in antisense frame +1 until residue 297 (89% similarity, e value 3 × 10−126, not shown). This alignment includes a single stop, matching tyrosine in *Aleurodicus dugesii*. Part of the remaining protein is coded by a stopless stretch of antisense strand frame +2, where residues 341–427 align with the regular protein from *Aleurodicus dugesii* (73% similarity, e value 2 × 10−21). Hence, the annotated ORF in *Aleurodicus dispersus* is a stop-codon-depleted antisense frame that codes for an apparently different protein, the actual NADH dehydrogenase subunit 4 is encoded by two frames, one containing a stop, on the opposite strand.
