J. Rice et al., FLUORESCENT OLIGONUCLEOTIDE RDNA PROBES THAT SPECIFICALLY BIND TO A COMMON NANOFLAGELLATE, PARAPHYSOMONAS-VESTITA, Microbiology, 143, 1997, pp. 1717-1727
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.