(updated January 2020)
This category represents a heterogeneous collection of sequences, with length dependent upon the PCR primers that were used in the initial amplification and subsequent sequencing of the product. Almost 90% of the ~ 3700 Acanthamoeba 18S rRNA gene sequences in the DNA databases are partial sequences. This is likely to remain the case, or even increase, because relatively few almost complete sequences are now being obtained.
With respect to the distribution of sizes of partial sequences, 41 of the partial 18S rRNA sequences are between 1501 – 2000 bases in length, 138 between 1001 – 1500 bases, 218 between 501 – 1000 bases, 2006 are between 351 – 500 bases in length, 876 are between 101 – 350 bases in length and 28 are shorter than 100 bases (27 between 70 – 99 bases). Note that these sizes exclude the parts of a small number of sequences that have been found to have an intron sequences inserted into some part of the 18S rRNA gene.
Before 2000, most Acanthamoeba sequences were obtained from genetically cloned material (either as a single cloned segment, or more often as two verlapping cloned segments). Beginning in 1999, PCR became the primary approach for obtaining sequences, either “almost complete” or partial. A number of potential PCR primers were evaluated for their ability to produce partial products of the 18S rRNA gene for rapid and accurate diagnosis of infections involving Acanthamoeba. During this period, rapid diagnostic procedures were a major focus of our research group. In 2001, we reported the development and use of a series of PCR primers, some of which have become widely used in the analysis of both clinical and environmental samples of Acanthamoeba (Schroeder et al, 2001, J. Clin. Micro). More details will be provided below.
SEGMENTS OF THE 18S rRNA GENE OF ACANTHAMOEBA
The most widely analyzed and deposited sub-sequences of the 18S rRNA gene are those that correspond generally to a region of the gene originally described in the paper from our lab by Dyková et al. (J. Parasitol 1999). We used two PCR primers, designated JDP1 and JDP2, that produced a product of approximately 400-460 bases from all isolates of Acanthamoeba that were being analyzed. The amplimer, which was ultimately designated ASA.S1, for “Acanthamoeba-specific amplimer S1″, showed interstrain sequence variation sufficient to distinguish the sequence types of 18S rDNA genotypes that we had earlier described (Stothard et al., 1998).
Two amplimers within the 18S rRNA gene of Acanthamoeba (ASA.S1 and GTSA.B1) are shown diagrammatically in the figure below.
The 18S rDNA sequence of the Neff strain of A. castellanii (GenBank acc # M13435) was used as the reference for all base-pair coordinates shown in the figure. (Figures from Schroeder et al,. 2001).
In some cases, to differentiate individual Acanthamoeba keratitis (AK) genotypes, we have also used a set of additional PCR primers that produced a larger amplimer designated GTSA.B1, standing for the “genotype-specific amplimer B1”. This region is shown in the lower figure above. The GTSA.B1 amplimer can then be subdivided into three variable fragments designated DF1, DF2 and DF3 which can be independently sequenced (represented by the three segments in the diagram of GTSA.B1, above. DF3 represents a region of the gene that is also within the amplimer ASA.S1. This subsegment of the ASA.S1 amplimer can be analyzed using the 892C primer as a sequencing primer, yielding a sequence approximately 200-250 bases in size that includes most of the highly variable portions of the ASA.S1 region. This segment, designated DF3, is equivalent to the right-most region in GTSA.B1 in the figure above.
PHYLOGENETIC INFORMATION CONTAINED IN PARTIAL SEQUENCES
The segments ASA.S1 or DF3 contain a significant portion of the phylogenetic signal that exists in the entire 18S rRNA molecule. However, use of only the partial sequences for analysis can lead to greater uncertainty about differences between sequence types. In some cases, the partial sequence can be misleading with respect to phylogenetic relationships, especially in parts of the Acanthamoeba phylogeny that contains fewer reported isolates. Where sufficient numbers of “almost complete” sequences have been determined, placement within the genetic diversity of the genus Acanthamoeba of a new isolate, based on a partial sequence, can usually be performed very accurately. Nevertheless, phylogenetic analysis of the genus should rely on “almost complete” 18S rRNA sequences. In fact, a multigene approach is clearly more appropriate and is discussed on other pages of this site.
PRIMERS AND AMPLIMERS:
SSU1 – a general eukaryotic specific primer . The sequence within the square brackets represents a cloning site that will allow the fragment to be cloned into a plasmid for further analysis. — 5′-[CCGCGGCCGCGT]CGACTGGTTGATCCTGCCAGTAG-3′
Primers used to amplify ASA.S1
JDP1 — (5’-GGCCCAGATCGTTTACCGTGAA-3′) [beginning at position 936 of the Neff strain sequence acc #M13435] ; and JDP2 — (5’-TCTCACAAGCTGCTAGGGGAGTCA-3’) [ending at position 1402]
Primers used to obtain sequences for region DF3:
892 — (5’-CCAAGAATTTCACCTCTGAC-3’) and 892C — (5’-GTCAGAGGTGAAATTCTTGG-3’) [both at position 1121 in Neff ]
Sequencing primers used for single direction sequencing (see GTSA.B1 diagram above) include:
373 (5′-TCAGGCTCCCTCTCCGGAATC-3′) for bp 178 to 355
570C (5′-GTAATTCCAGCTCCAATAGC-3′) for bp 705 to 926
Additional primers are listed in the following table (taken from Schroeder et al, 1998, Current Genetics):
An additional set of primers was reported by Mathers et al. 2000. These primers have been referred to as the Nelson primers and produce a product of approximately 280-290 bp in a region 3′ to DF3. The primers are:
Nelson forward: 5′ – GTTTGAGGCAATAACAGGT – 3′ at position 1835 in Neff #M13435 Nelson reverse: 5′ – GAATTCCTCGTTGAAGAT – 3′ , ending at position 2063 of Neff