23S RNA-derived replicon as a 'molecular tag' for monitoring inoculated wine yeast strains
2008
Esteban, Rosa | Rodríguez-Cousiño, Nieves
In this study we have developed a useful method to identify a particular yeast strain within a mixture of strains during must fermentation, based on the presence or absence of a stable genetic element derived from Saccharomyces cerevisiae 23S RNA autonomous replicon. 23S RNA is a natural virus-like RNA replicon present in some S. cerevisiae strains, which encodes only its own RNA-dependent RNA polymerase named p104. A modified version of 23S RNA (23S-tagged RNA) was generated after transformation of S. cerevisiae wine strains with a launching plasmid, where six nucleotides were changed in the 23S RNA cDNA sequence without modifying the amino acid sequence of p104 RNA polymerase. Once generated, the 23S-tagged RNA can replicate autonomously (without the plasmid), is very stable, is present in high copy number in stationary phase or nitrogen-starved cells and confers no phenotype to the host, like the endogenous 23S RNA replicon. However, it can be distinguished from endogenous 23S RNA by reverse transcription followed by polymerase chain reaction (RT-PCR) with specific oligonucleotide primers. 23S RNA-derived replicon can be used to tag wine yeast strains in order to monitor easily their prevalence over endogenous strains during wine fermentation.
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