Transcript Mapping and Genome Annotation of Ascidian mtDNA Using EST Data

Abstract

Mitochondrial transcripts of two ascidian species were reconstructed through sequence assembly of publicly available ESTs resembling mitochondrial DNA sequences (mt-ESTs). This strategy allowed us to analyze processing and mapping of the mitochondrial transcripts and to investigate the gene organization of a previously uncharacterized mitochondrial genome (mtDNA). This new strategy would greatly facilitate the sequencing and annotation of mtDNAs. In Ciona intestinalis, the assembled mt-ESTs covered 22 mitochondrial genes (∼12,000 bp) and provided the partial sequence of the mtDNA and the prediction of its gene organization. Such sequences were confirmed by amplification and sequencing of the entire Ciona mtDNA. For Halocynthia roretzi, for which the mtDNA sequence was already available, the inferred mt transcripts allowed better definition of gene boundaries (16S rRNA, ND1, ATP6, and tRNA-Ser genes) and the identification of a new gene (an additional Phe-tRNA). In both species, polycistronic and immature transcripts, creation of stop codons by polyadenylation, tRNA signal processing, and rRNA transcript termination signals were identified, thus suggesting that the main features of mitochondrial transcripts are conserved in Chordata.

Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species

“…Indeed, among bivalves atp8 has been annotated only in Hiatella (Heterochonchia; Dreyer and Steiner, 2006) and Lampsilis (Palaeoheterodonta; Serb and Lydeard, 2003) but we have identified this gene also in the M and F types of both Venerupis and Inversidens species. Similarly, atp8 was not annotated in the first published tunicate mtDNA of Halocynthia roretzi (Yokobori et al, 1999), although a very short form of ATP8 is actually mitochondrially encoded in all tunicates (Gissi and Pesole, 2003;Yokobori et al, 2005;Iannelli et al, 2007a). In spite of these annotation difficulties, atp8 could be considered a real 'dispensable' mitochondrially encoded gene, as it is absent in several distant metazoan lineages.…”

Exploring the transcriptome of the burrowing nematode Radopholus similis

“…Depending on the cDNA library construction method, a remarkably high fraction of ESTs can be of mitochondrial origin and these EST can even be used as a guideline for the sequencing of the mitochondrial genome (Gissi and Pesole 2003). We searched our unigene dataset for ESTs most likely derived from the mitochondrial genome, and found Wve unigenes with signiWcant similarity to various nematode mitochondrial genes (see Table 4).…”

Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes

“…Although EST projects are used primarily to gather structural and functional information for nuclear transcripts, a relatively large proportion of sampled cDNA clones correspond to mitochondrial transcripts. As such, EST projects simultaneously provide information about the nuclear and mitochondrial transcriptomes (Gissi and Pesole, 2003). Such information is essential for correctly annotating gene boundaries and understanding transcriptional processes unique to the mitochondrial genome (Edqvist et al, 2000;Barth et al, 2001).…”

“…Unfortunately, genomic mtDNA does not reveal posttranscriptional modifications that determine the boundaries of protein-coding regions. For example, several mitochondrial protein-coding genes do not contain a full stop codon, but instead use polyadenylation of the terminating T nucleotide to complete the stop codon sequence (Gissi and Pesole, 2003). Identification of the correct terminating T nucleotide may not always be straightforward when comparing mtDNA sequences, but it is when mRNA sequences are available in the form of ESTs (Gissi and Pesole, 2003).…”

“…For example, several mitochondrial protein-coding genes do not contain a full stop codon, but instead use polyadenylation of the terminating T nucleotide to complete the stop codon sequence (Gissi and Pesole, 2003). Identification of the correct terminating T nucleotide may not always be straightforward when comparing mtDNA sequences, but it is when mRNA sequences are available in the form of ESTs (Gissi and Pesole, 2003). For example, in C. intestinalis the Cytb reading-frame could potentially end at a TAG stop codon located within the adjacent tRNA PRO gene.…”