Cloning Yeast Genes By Complementation
Published 2001 · Biology, Medicine
This unit presents a generalized protocol and describes the principles involved in cloning yeast genes by complementation in yeast. The protocol is presented using a hypothetical mutation of yeast, the cdc101‐1 mutation. This mutation was isolated as a cell cycle mutant and is both recessive and temperature‐sensitive for growth: it can grow relatively normally at 30oC but is unable to make a colony at 37oC. A genomic DNA clone that complements this mutation will be isolated by transforming the cdc101‐1 strain with a yeast genomic library and subsequently screening for temperature‐resistant colonies. Once isolated, two steps are necessary to prove that the insert present on the plasmid contains the wild‐type CDC101 gene. First, segregation of the complementing plasmid must result in co‐loss of both the plasmid‐borne selectable marker and the complementing phenotype, demonstrating that the observed complementation is plasmid‐specific and is not due to reversion of the cdc101‐1 mutation. Second, it must be ruled out whether the cloned gene encodes a phenotypic suppressor of the mutation, rather than the wild‐type gene. This is done via a complementation test, which demonstrates whether or not a disruption of the cloned gene that is integrated into the genome can complement the original mutation.