In situ accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-labeled oligonucleotide probes comprising the D1 and D2 domains.
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.
Centro de Recursos Microbiológicos, Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal.
João
J
Inácio
Sebastian
S
Behrens
Bernhard M
BM
Fuchs
Alvaro
A
Fonseca
Isabel
I
Spencer-Martins
Rudolf
R
Amann
D001483:Base Sequence; D002232:Carbocyanines; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013045:Species Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; C089187:cyanine dye 3