Copyright 0 1996 by the Genetics Society of America Establishing Genetic Interactions by a Synthetic Dosage Lethality Phenotype Eugene S. Kroll,*?lKatherine M. Hyland,*” Philip Hieter” and Joachim J. Lit *Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and tDepartment of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0414 Manuscript received August 31, 1995 Accepted for publication February 1, 1996 ABSTRACT We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned “reference” gene is inducibly overexpressed in a set of mutant strains carrying potential“target” mutations. To test the specificity of the method,two reference genes,CTFl3, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ~$14-42(cbfl, ndclo), ~$17-61 (ch14), ctfl9-58 and ~$19-26.ORC6 overexpression caused synthetic dosage lethality in combination withcdc2-1, cdckl, cdcl4- 1, cdcl6-I and cdc46-1. These relationships reflectspecific interactions, as overexpression of CTF13caused lethality in kinetochore mutants and overexpressionof ORC6 caused lethality in replication mutants.In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrumof interacting mutations andis of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethalityis easily adapted to the study of cloned genes in other organisms. practical strategy for studying the function of a isolate synthetic lethal mutations, a strain containing the A protein of interest is to identify other proteins that reference mutation must harbor the wild-type copy of interact with it. This may lead to the isolation of new a gene on a counterselectable plasmid that covers the components participating in thesame pathway or iden- mutation of interest. After mutagenesis, synthetic lethal tification of previously characterized factors that can mutations are identified in strains that have become de- help elucidate the function of the protein understudy. pendent on theplasmid for viability. Dosagesuppression Identification of a genetic interaction between a muta- screens involve identification of wild-type genes that, at tion in the gene coding fora protein of interest and a increased copy number or when overexpressed, comple- mutation in an unlinked locus is a proven method for ment the phenotype caused by a reference gene muta- establishing functional links between proteins. A ge- tion. If the reference mutation is conditionally lethal, netic interaction can be manifested as a novel pheno- this canbe accomplished by transformation of a genomic type that is not attributable to either mutation alone. library on a high copy vector and selection of viable Typically, genetic interaction screens use a mutation in transformants at the nonpermissive condition. Overex- the gene of interest as a starting point and rely on pression of some gene products are known to cause detri- finding phenotypes caused by the combination of two mental phenotypes including the loss of viability (LIU et mutations. Several powerful genetic interaction screens al. 1992). These genes would not be recovered in a dos- have been developed that are based on either loss or age suppression screen. However, since only a fraction gain ofviability as a phenotype (ROSE et al. 1990). of genes in yeast are toxic upon amplification or overex- Among those most widely used are second site suppres- pressionin a wild-type strain background (LIU et al. sion (HARTMAN and ROTH 1973), synthetic lethality 1992), this is not generally a problem. (GUARENTE1993), and dosage suppression screens Various molecular genetic strategies lead to the isola- (RINE 1991). tion of a cloned wild-type gene of known sequence and Synthetic lethality screens, which identify two nonalle- unknown function. In this study, we have set up a ver- lic and nonessential mutations that arelethal for the cell sion of a synthetic lethality screen in which a cloned only when both are present, have proved to be very use- “reference” gene is overexpressed in a set of mutant ful in finding genes encoding interacting proteins. To strains carrying potential “target” mutations. This pro- vides a means for identifylng genetic interactions using Corresponding author: Philip Hieter, Department of Molecular Biol- a cloned wild-type gene (rather than a mutant) as the ogy and Genetics,Johns Hopkins Medical School, The Johns Hopkins University, 725 N. Wolfe St., Baltimore, MD 21205. starting point in a screen (Figure 1).We reasoned that E-mail: [email protected] although increasing the amountof activityof one factor I These authors contributed equally to this work. (encoded by the reference gene) may not produce a Genetics 143: 9.5-102 (May, 1996) 96 E. S. Kroll et al. a. VectoronlyVector + "Reference"gene upon overexpression of the RED3 gene (FRIEDMAN et al. 1994). Overproduction of the MCM3 gene product Transform "Target" mutant collection accentuates the defects caused by the mcm2 mutation under noninducing conditions (glucose) (YAN et al. 1991). Itis also interesting to note thatMCM2 overexpression suppresses the phenotype causedby the mcm3 mutation, and the mcm2 mcm3 double mutant is GALl REF ne nonviable (YAN et al. 1991). The goal of this work was to assess the feasibility of employing the synthetic dosage lethality phenotype for the identification of meaningful genetic interactions. This study was designed to determine the functional I- Target 1- mutationTarget specificitymutation of themethod by conditionallyoverex- pressing two known reference genes, CTFl3 and ORC6, Induce overexpression of in the context ofa large collection ofpreviously charac- "Reference" gene (galactose) terized target mutations, in this case a set of ctfand cdc mutants. We also determined the relative spectrum of interactions identified by synthetic dosage lethality (at permissive temperature) as opposed to dosage suppres- sion (at nonpermissive temperature) in a collection of previously characterized temperature-sensitive mutants. Our results indicate that screening for synthetic dosage lethality provides a convenient and relevant approach for the identification of genes encoding functionally interacting proteins. MATERIALSAND METHODS Yeast strains and media: Table 1 lists the genotypes of all Vectoronly EFaene Genetic interaction strains used in this study. The chromosome transmission fi- +I + None delity (ctf) mutants tested were all isogenicderivatives of YPH278,as described by SPENCERet al. (1990), cdc strains +/ - Syntheticdosage lethality were described by LI and HERSKOWITZ(1993). Standard yeast -1 + Dosagesuppression mediaused was as described (ROSE et al. 1990). Synthetic complete (SC) media lacking leucine and containing as a -1 - None carbon source either 2% glucose (SC glucose - Leu) or 2% FIGURE1.-The synthetic dosage lethality (SDL) assay. (a) galactose and 2% raffinose (SC Gal + Raf - Leu) were used. A collection of mutants carrying potential target mutations Standard yeast transformation procedures were utilized as de- scribed (ROSE et al. 1990). Yeastwere cultured at 25" and are transformed with two plasmids under noninducing condi- tions. One plasmid contains the cloned wild-type copy of a shifted to other temperatures as indicated. Plasmid constructs: Overexpression was achievedusing "reference gene" under the control of an inducible GALl promoter. The second plasmid is a vector-only control. (b) constructs with inducible GALl promoters. pJL749 contains the GALl promoter driving the expression of ORC6 in the Overexpression of the reference gene is accomplished by LEU2, 2-p based vector,pRS425 (SIKORSKIand HIETER1989), streaking on galactose plates. (c) Growth of four independent as previously described (LI and HERSKOWITZ1993). pJL772 is transformants was scored at the temperatures indicated (see identical to pJL749 except that it lacks the ORC6 sequence. Table 1).Lethality (or slow growth) well dosage suppres- as as pKF88 was constructed in the expression vector p415GEU2. sion were noted. growth; -, no growth. +, p415GEU2has a 460-bp GALl promoter fragment cloned into the KpnI site, two tandem copies of the El tag sequence, noticeable phenotype in a wild-type strain, when com- inserted into theBamHI site of pRS415 (SIKORSKIand HIETER bined with decreased or alteredactivity of another gene 1989) and the URA? promoter that serves as a transcription encoding an interacting protein a(in hypomorphic mu- terminator, and was obtained from pRS314GU (R. SIKORSKI, tant strain),a lethal phenotype might result that could personal communication). The GALl promoter directs tran- scription from its own ATG toward the polylinker. The CTF13 be conveniently detected in a screen. In fact, several OW was cloned into this expression vector to create pKF88, cases of synthetic dosage lethality or toxicity have pre- (as described for pKF80 by DOHENYet al. 1993) and was shown viously been reported. For example, overexpression of to complement a ctfl? deletion mutation. the ORC6 gene lowers the nonpermissive temperature Synthetic dosage lethality screen (Figure 1): Strains were