Expression of a reporter gene interrupted by the Candida albicans group I intron is inhibited by base analogs.
We previously reported the identification of an intron (CaLSU) in the 25S ribosomal RNA of some Candida albicans yeast strains. CaLSU was shown to self-splice and has the potential to adopt a secondary structure typical of group I introns. The presence of CaLSU inC. albicans strains correlates with a high degree of susceptibility to base analog antifungal agents, 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). Cell death, resulting from addition of base analogs to growing cultures, precluded demonstration of a causal relationship between CaLSU presence and susceptibility to base analogs. In the present study, CaLSU was inserted in a non-essential lacZ reporter gene and expression was examined in Saccharomyces cerevisiae. Different mutations affecting in vitro self-splicing also had similar effects on reporter gene expression in vivo. This indicates that in vivo removal of CaLSU from the reporter gene occurs through the typical self-splicing mechanism of group I introns. Base analogs inhibited expression of the reporter gene product in a concentration-dependent manner upon their addition to the cultures. This supports a model in which disruption of intron secondary structure, consecutive to the incorporation of nucleotide analogs, is a major factor determining the susceptibility of C.albicans cells to base analogs.
Mercure
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Département de Microbiologie et Immunologie, Université de Montréal, PO Box 6128, Station Centre-Ville, Montréal, Québec H3C 3J7, Canada.
Cousineau
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Montplaisir
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Belhumeur
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Lemay
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D000935:Antifungal Agents / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D002176:Candida albicans / Q000737:chemistry*; D002455:Cell Division / Q000187:drug effects; D005437:Flucytosine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007438:Introns / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009690:Nucleic Acid Conformation; D012326:RNA Splicing / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000935:Antifungal Agents; D012335:RNA, Ribosomal; C065039:RNA, ribosomal, 25S; D005437:Flucytosine; D001616:beta-Galactosidase; D005472:Fluorouracil