(updated July 2021)
The ribosomal small subunit rRNA found in the mitochondrial genome became the second gene to be used extensively for phylogenetic analysis of Acanthamoeba. The gene has been referred to in several ways. The gene designation for the gene in the mitochondria is rns (with a lower-case r to distinguish it from the equivalent nuclear encoded Rns gene). The gene is also often referred to as the 16S-like rRNA, referring to its homology to the ribosomal small subunit 16S gene of prokaryotic organisms (and the fact that mitochondria evolved from a prokaryotic endosymbiont of the primitive eukaryotic ancestor).
The rns gene sequence for Acanthameoba was first reported in 1994 (Lonergan and Gray, 1994; J. Mol. Biol. 239: 476; accession # U03732), and subsequently as part of the complete Acanthamoeba castellanii Neff mitochondrial genome sequence reported by the same group (accession # U12386). The complete sequence in the Neff strain is 1541 bp in length. That sequence was used to provide information concerning potential PCR primers that were then used to analyze the mitochondrial 16S-like rRNA gene in a series of Acanthamoeba isolates, beginning with our own studies (Ledee et al., 2003; Invest. Ophthalmol. Vis. Sci. 44: 1142). The pattern with which 16S-like sequences from Acanthamoeba have been obtained and deposited into the DNA databases is shown in the figure below. It indicates that, unlike the nuclear 18S rRNA gene, the mitochondrial 16S-like rRNA gene has been sporadically studied, with the largest number of sequences corresponding to the study of Ledee et al, 2003, referenced above. Nevertheless, entries continue to be deposited, because the gene sequence does provide substantial use as a marker of Acanthamoeba.
As of January 2020, 154 mt-DNA rns sequences of at least 1475 bp in length had been obtained. These included 112 sequences that had been deposited directly in the DNA sequence databases. There are a further 25 sequences that come from genome sequences that have been deposited in the DNA databases. In addition, 17 sequences are available that had not been formally deposited.
With respect to partial sequences, 13 sequences from Acanthamoeba cultured isolates were also deposited, as were a series of 36 sequences from several different environmental surveys of DNA material from uncultured organisms. Some of these uncultured entries represent organisms labeled as “uncultured eukaryotes”. Another group of sequences are labeled as “Uncultured bacterium clone #” but actually appear to be short (usually slightly greater than 200 bp) fragments of an Acanthamoeba rns gene. References and accession numbers are provided on an accompanying page.
The distribution of sequence lengths for the mitochondrial 16S-like gene that exist on our database is shown in the figure below. Unlike the nuclear 18S rRNA gene sequences, 16S-like rRNA gene sequences are dominated in the upper modal group by almost-complete sequences (those of ~1500 bp and longer), with the smaller modal group representing those sequences obtained in environmental screening of uncultured organisms.
One unfortunate aspect of the data is the fact that a number of the isolates for which the rns sequence was obtained do not have an equivalent Rns sequence. This makes comparisons of the two genes less powerful than might have been possible. We encourage any investigator who wishes to use such sequences in the future to obtain information on both sequences.
We have reported some results at meetings from comparisons of the phylogenetic information for the nuclear and mitochondrial Rns and rns genes. In general they provide similar placements of isolates with respect to the overall Acanthamoeba phylogeny, although some isolates are placed in slightly different relationships. One aspect by which the mitochondrial 16S-like rRNA gene differs from the nuclear analogue is that the separation of the T4-A and T4-B subtypes seen in the nuclear 18S rRNA gene analysis is blurred, resulting in a single (large) group of sequences. Other sub-types of T4 seen in the 18S rRNA sequences continue to be supported in the mitochondrial data.
An additional aspect of the data for 16S-like rRNA sequences is that information from rns sequences has only been obtained from a sub-set of the sequence types identified by nuclear Rns sequences. Specifically, there continue to be no mitochondrial rns sequences that had been attributed to isolates from sequence types T6, T12, T13, T14, T15, T16, T18, T19 and T20. It is hoped that this will be remedied in the near future.
Watch this section for additional information, including the degree of variability of mitochondrial versus nuclear rRNA sequences, the specifics on the phylogenetic concordance of the two sequences and other aspects of the mitochondrial rns sequences. We intend to provide information on the distribution of relative variability within the gene, so as to guide future studies. This will include information on whether partial sequences of the rns gene contain essentially the same information as the complete sequence and whether particular regions are more or less informative than other parts of the gene.