Manipulation of cloned yeast DNA

A major advantage of working with yeast is the ability to replace the wild-type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This technique unavailable in most other eukaryotes allows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative transformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniques integrative disruption and one-step gene disruption generate either insertion or deletion mutations. The third technique transplacement is more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one-step integrative replacement, and also conditional alleles by a copper-inducible double-shutoff procedure.