Dataset Information

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

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.

SUBMITTER: Inacio J

PROVIDER: S-EPMC154520 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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

Inácio João J Behrens Sebastian S Fuchs Bernhard M BM Fonseca Alvaro A Spencer-Martins Isabel I Amann Rudolf R

Applied and environmental microbiology 20030501 5

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 pr ...[more]

PMID: 12732564

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Project description:In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083(T). Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.

Dataset Information

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

Publications

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

Similar Datasets

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