Allele shuffling: conjugational transfer, plasmid shuffling and suppressor analysis in Saccharomyces cerevisiae.
Trans-acting suppressor analysis represents a powerful genetic technique capable of revealing interactions among biochemical pathways in vivo. Suppressor characterization in Saccharomyces cerevisiae has traditionally utilized meiotic segregation for the requisite manipulation of strain genotypes. Meiotic segregation is not compatible with all yeast genotypes and can be prohibitively labor intensive when examining large collections of suppressors. To facilitate rapid phenotypic analysis of suppressor mutations, we have devised a novel genetic strategy called 'allele shuffling'. This plasmid-based method should in principle identify allele-specific, allele-dependent and bypass suppressors. A centromere vector (YCp) was developed that can be directly transferred from Escherichia coli to yeast via 'trans-kingdom' conjugation. Suppressors of a thermolabile cdc23 allele, cdc23-39, were isolated in the background of a yeast host strain harboring the mutant cdc23-39 gene positioned on a counterselectable plasmid. CDC23 or cdc23-39 genes cloned into a mobilizable YCp vector were then transferred directly from E. coli cultures to each suppressed yeast strain on the surfaces of agar plates. Plasmid shuffling of the cdc23-39 allele transconjugants segregated away the original cdc23-39 gene present during mutagenesis, allowing the intra- or extragenic nature of suppression to be determined. Phenotypes (if any) produced by suppressor mutations were revealed in those transconjugants receiving the wild-type CDC23-containing episome. The allele shuffling method should be generally applicable to the analysis of suppressors of any essential yeast gene.
Sikorski
R S
RS
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
Michaud
W A
WA
Tugendreich
S
S
Hieter
P
P
D000483:Alleles; D064173:Anaphase-Promoting Complex-Cyclosome; D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; D018797:Cell Cycle Proteins; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D043743:Ubiquitin-Protein Ligase Complexes
D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; C577441:CDC23 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome