Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn

Authors


  • This paper is the result of work aiming to develop simple and robust methods for studying microbial eucaryotes. Martin Embley and Pat Dyal are working in collaboration with Dr Bland Finlay (Institute of Freshwater Ecology, England) to study ciliate phylogeny and ecology, with particular emphasis on the evolution of anaerobic ciliates. Dave Roberts and Sue Hope study ciliate morphology and ultrastructure.

Tel.: 071 9388760. Fax: 071 9388754. E-mail: tme@nhm.ac.uk

Abstract

A two-stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary-otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto-fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.

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