DETECTION OF STABLE PRE-RIBOSOMAL-RNA IN TOXIGENIC PSEUDO-NITZSCHIA SPECIES

Nucleotide sequence analysis of ribosomal DNA (rDNA) spacer regions is useful for taxonomic comparisons of closely related microorganisms. T hese regions have been less useful for routine microbial identificatio n and detection, partly because rRNA precursors (pre-rRNAs) in microbi al cells are assumed to be too labile to be detectable by high-through put probe hybridization methods. We characterized the sequence diversi ty and physiological stability of pre-rRNA in the toxigenic marine dia toms Pseudo-nitzschia australis, P. multiseries, and P. pungens. As wi th nucleotide sequences of the first internal transcribed spacer (ITS1 ) reported previously, sequences of ITS2 and the 5' external transcrib ed spacer (ETS1) exhibited considerable divergence among these species , including large insertions-deletions detectable by PCR-based spacer length analysis. In slot blot hybridization assays on RNA extracted fr om lysates of Pseudo-nitzschia cells, oligonucleotide probes directed to pre-rRNA spacers generated much stronger signals than did complemen tary probes directed to the coding strands of the rDNAs, indicating th at the pre-rRNA-targeted probes detected multicopy transcripts. A grou p of probes directed to a discrete 90-base region within the ITS1 pre- rRNA gave no detectable signal, suggesting that this region is degrade d early in the rRNA maturation pathway. Other pre-rRNA regions were al ways detectable and, in marked contrast to prokaryotic systems analyze d in this manner, were stable and abundant in both actively dividing a nd nondividing cells. Long, multilabeled RNA probes, which would exhib it considerable cross-reactivity if directed to mature rRNA sequences, detected species-specific pre-rRNA sequences from as few as 1,000 cel ls. Pre-rRNA is a potentially useful molecular target for detecting an d identifying Pseudo-nitzschia species and possibly other unicellular eukaryotes as well.