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Korean J Parasitol > Volume 34(2):1996 > Article

Original Article
Korean J Parasitol. 1996 Jun;34(2):127-134. English.
Published online Jun 20, 1996.  http://dx.doi.org/10.3347/kjp.1996.34.2.127
Copyright © 1996 by The Korean Society for Parasitology
PCR and RFLP variation of conserved region of small subunit ribosomal DNA among Acanthamoeba isolates assigned to either A. castellanii or A. polyphaga
H H Kong and D I Chung*
Department of Parasitology, Kyungpook National University School of Medicine, Taegu 700-422, Korea.
Received January 30, 1996; Accepted April 30, 1996.

Abstract

Twelve isolates of Acanthamoeba spp. assigned to either A. castellanii or A. polyphaga, and type strains of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RNA gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellaii was 9.8% whereas that among the isolates assigned to A. polyphaga 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. castellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. polyphaga was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. castellanii and A. polyphaga (2.6%) which appeared between the Castellani (or CCAP 1501/2 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphaga. It is suggested that taxonomic validity of the isolates assigned to either A. castellanii or A. polyphaga should be reevaluated.

Figures


Fig. 1
PCR products of 16 isolates of Acanthamoeba analysed in this study. Numbers above the lanes refer to the strains of Table 1. Hae III digested Φ X174 DNA and PCR marker (promega. U.S.A.) were used ad the size marker.


Fig. 2
Schematic representation of PCR-RFLP patterns of Acanthamoeba rDNA conserved region obtained with eight kinds of restriction endonucleases. Numbers above th lanes refer to the strains of Table 1. Amplisize® (Biorad, U.S.A.) was used as a size marker.


Fig. 3
Phylogenetic tree of Acanthamoeba isolates based on genetic divergence estimates. The matirx of divergence estimates in Table 2 was used to construct this tree suing UPGMA.

Tables


Table 1
Sixteen isolates of Acanthamoeba analysed in this study


Table 2
Proportions of homologous fragments (values above the diagonal) and estimates of genetic divergencea)(values below the diagonal) among isolates

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