| dc.description | The chlorophycean taxa are distinctive from other green algae in that their mitochondrial genome sizes are reduced and their mitochondrial ribosomal RNA genes are fragmented and scrambled. To understand mitochondrial genome evolution in the Chlorophyceae, which needs more information from other taxa, and to develop a system for green algal mitochondrial studies, this work undertook the characterization of the mitochondrial genome and ribosomal RNAs of Polytomella parva. This taxon's colorless and cell wall-less features facilitate the isolation of mitochondrial-enriched fractions free of thylakoid membranes. At least two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5-kb were detected. Sequences spanning 97 and 86% of these mtDNAs, respectively, revealed that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated non-synonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA genes, one tRNA gene, and six protein-coding genes). Based on DNA sequence data, it is proposed that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (= C. eugametos) as the outgroup, it is concluded that the non-synonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on average about 3.3 times that of the Chlamydomonas reinhardtii counterparts. RNA from a mitochondrial-enriched preparation was isolated, and the transcripts of the four small subunit (SSU) and eight large subunit (LSU) rRNA-coding regions were characterized by either Northern blot analysis or chemical sequencing. Secondary structure modeling of the SSU and LSU rRNA sequences was performed. The results show that (1) both the mitochondrial SSU and LSU rRNAs of P. parva are considerably shorter than their homologs from other green algae, although the main domains typical of conventional rRNAs are conserved, (2) the rRNA fragmentation pattern is different from that of other chlorophycean species but most similar to that of C. reinhardtii, among those that have been characterized, (3) three nucleotides are missing from the normally highly conserved GTPase centre of the LSU rRNA, and (4) post-transcriptional modification of the 3'-terminal region of the SSU rRNA is unusual; it has the "eubacterial" 3-methyluridine (corresponding to m3U at Escherichia coli 16S rRNA position 1498), but lacks the more highly conserved modifications at two adjacent A residues (corresponding to N6,N 6-dimethyladenosine at E. coli 16S rRNA positions 1518 and 1519). This research presents the first example of subgenomic mtDNAs of a green alga. The obtained data provide the necessary background for more direct functional studies of the chlorophycean mitochondrial ribosome and rRNAs, using P. parva as a model system. | en_US |
