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12.2: Complementation is often used to isolate transformants

  • Page ID
    17567
  • The DNA used for transformation must carry a selectable marker whose presence can be detected by screening. Following a transformation, cells are plated on selective media that will allow transformed, but not untransformed, cells to grow. All the pYES2.1-derived plasmids that we are using carry a normal copy of the yeast URA3 gene, as well as the URA3 promoter, so the gene is regulated much like a normal chromosomal gene. Our yeast deletion strains were derived from strain BY4742, which has the ura3∆0 allele (Winzeler et al., 1999) Complementation will occur because the plasmid carries a functional copy of the gene that is defective in the mutant host strain. The Ura3p protein produced from the plasmid-encoded URA3 gene compensates for the ura3 deletion in the yeast chromosome, allowing transformed cells to grow in the absence of uracil, as shown below. Because of its reliability, many yeast transformation schemes rely onURA3 complementation to isolate transformants.

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    Transformation and plasmid complementation
    Competent ura3 yeast cells are transformed by incubating cells with a plasmid containing the yeast URA3gene at an elevated temperature.
    Transformed cells are selected on media that does not contail uracil.