FLUORESCENT OLIGONUCLEOTIDE RDNA PROBES THAT SPECIFICALLY BIND TO A COMMON NANOFLAGELLATE, PARAPHYSOMONAS-VESTITA

Nanoflagellates are ecologically important, but morphological identifi cation requires techniques which are not practicable for use in quanti tative studies of populations; alternative methods of accurate recogni tion of nanoflagellate species in mixed populations are therefore desi rable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as 'phyl ogenetic stains' in the identification of bacteria. In this paper we d escribe the preparation and application of probes which specifically h ybridize with a common nanoflagellate species, Paraphysomonas vestita. The sequence of nucleotides in the SSU rRNA gene of this flagellate w as determined and compared with those of related species to select uni que P. vestita sequences 18-21 nucleotides in length. Five sequences i n different parts of the SSU rRNA gene were used to design 5'-fluoresc ently labelled oligonucleotide probes. Published sequences were used t o make probes that hybridized with all eukaryotes (EUK) or any cellula r organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all ca ses, UNI probes hybridized with the cells, but CON probes were only bo und to a limited extent. All five probes targeted to P. vestita proved to be species-specific; they hybridized well with this species, but n ot with three other species of the same genus, nor with three more dis tantly related flagellate species, nor with a ciliate, nor with bacter ia, These probes provide a means of quantitatively measuring the propo rtion of P. vestita cells in samples of mixed protists.