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Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools

  1. Parke A. Rublee
  1. *Institute of Human Virology and University of Maryland School of Medicine, Baltimore, MD 21201; ‡Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; §Biologisk Institutt, Universitetet I Oslo, N-0315 Oslo, Norway; Biology Department, University of North Carolina, Greensboro, NC 27402; ‖Department of Botany, North Carolina State University, Raleigh, NC 27695; and **Florida Marine Research Institute, Florida Department of Environmental Protection, St. Petersburg, FL 33701

  1. Communicated by Elisabeth Gantt, University of Maryland, College Park, MD (received for review February 26, 1999)

Abstract

The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA “signature” sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

Footnotes

    • ↵† To whom reprint requests should be addressed at: Institute of Human Virology, University of Maryland Biotechnology Institutes and University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201. E-mail: oldach{at}umbi.umd.edu.

    • Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF077055 and AF218805).

  • Abbreviations

    HMA,
    heteroduplex mobility assay;
    SSCP,
    single-stranded conformational polymorphism;
    SEM,
    scanning electron microscopy
    • Received February 26, 1999.
    • Accepted January 4, 2000.

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