Fluorescent oligonucleotide rDNA probes that specifically bind to a common nanoflagellate, Paraphysomonas vestita

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Abstract
Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as ‘phylogenetic stains’ in the identification of bacteria. In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, Paraphysomonas vestita. The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique P. vestita sequences 18-21 nucleotides in length. Five sequences in different parts of the SSU rRNA gene were used to design 5 -fluorescently labelled oligonucleotide probes. Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent. All five probes targeted to P. vestita proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria. These probes provide a means of quantitatively measuring the proportion of P. vestita cells in samples of mixed protists.