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. 2003 May;69(5):2899-905.
doi: 10.1128/AEM.69.5.2899-2905.2003.

In situ accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-labeled oligonucleotide probes comprising the D1 and D2 domains

João Inácio  1 , Sebastian Behrens, Bernhard M Fuchs, Alvaro Fonseca, Isabel Spencer-Martins, Rudolf Amann
Affiliations

In situ accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-labeled oligonucleotide probes comprising the D1 and D2 domains

João Inácio et al. Appl Environ Microbiol. 2003 May.

Abstract

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455(T). The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.

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Figures

FIG. 1.
FIG. 1.
Fluorescence intensities of all oligonucleotide probes, standardized to that of the brightest probe (D-223), indicated in a model of the S. cerevisiae 26S rRNA secondary structure in which the D1 and D2 domains (delimited by the NL1 and NL4 primer target sites) are enlarged. The color coding indicates differences in the level of Cy3 probe-conferred fluorescence. The secondary structure is adapted from the European rRNA database (http://rrna.uia.ac.be).
FIG. 2.
FIG. 2.
Comparison of in situ hybridization signals for S. cerevisiae and other yeast species. The probes selected fall into different accessibility classes in the S. cerevisiae 26S rRNA D1 and D2 domains and have an identical target site in that region for all of the yeasts indicated.
FIG. 3.
FIG. 3.
Comparison of the relative in situ accessibilities (black line) of the S. cerevisiae 26S rRNA D1 and D2 domains and the average nucleotide substitution rates (gray) in yeasts.
FIG. 4.
FIG. 4.
Comparison of the accessibilities of homologous regions in S. cerevisiae 26S rRNA and E. coli 23S rRNA to Cy3-labeled probes.
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