MULTIPLE ALLELES OF Acanthamoeba NUCLEAR SMALL SUBUNIT RIBOSOMAL DNA GENE: FURTHER EVIDENCE OF POSSIBLE GENETIC EXCHANGE BETWEEN CLOSELY RELATED STRAINS

The nuclear small subunit ribosomal RNA (Rns) gene has been used in the last decade to establish genotypes of the genus Acanthamoeba based upon these sequences. In some cases direct sequencing of the Rns gene has resulted in mixed sequences, suggesting either a mixed culture of more than one Acanthamoeba strain or the presence of multiple alleles of this gene in a single Acanthamoeba strain. A previous study has established that multiple alleles in a single Acanthamoeba strain were possible. Here we examine an Acanthamoeba isolate from a keratitis case that produced mixed sequences when PCR products were directly sequenced. A single Acanthamoeba trophozoite was directly isolated from an agar plate to produce a clonal culture derived from this single troph. Following PCR amplification Rns products were cloned and sequenced. This produced two distinct sequences, indicating the presence of two alleles in this isolate. One allele, Rns 02-039/1, is identical to the Rns gene of A. sp. strain Galka. The second allele, Rns 02-039/2, has 99.7% similarity with the Rns from A. castellanii strain V042. Both of these isolates are genotype T4 based on Rns sequence analysis. Further, both are in subgenotype T4a identified by mitochondrial rns analysis, and both are in clusters of identical sequences in rns analysis. These data suggest that transfer of alleles (genetic exchange) may be possible between closely related strains of Acanthamoeba. Specifically, the results suggest a possible transfer of one allele (02-039/2) after the divergence of A. sp. Galka from A. sp. 02039. ______________________________ Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, Ohio, USA. Technological Institute of Sonora, Cd. Obregon, Sonora, Mexico, Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA. Corresponding Author: Gregory C. Booton Dept. of Evolution, Ecology, and Organismal Biology. The Ohio State University. 388 Aronoff Laboratory. 318 W. 12th Ave., Columbus, Ohio 43210. Phone: 614-292-4570. Email: [email protected]