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Dominant Negative Selection of Heterologous Genes

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Dominant Negative Selection of Heterologous Genes Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9410-9414, October 1992 Genetics Dominant negative selection of heterologous genes: Isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest MALCOLM WHITEWAY*, DANIEL DIGNARD, AND DAVID Y. THOMAS Eukaryotic Genetics Group, National Research Council of Canada, Biotechnology Research Institute, Montr6al, Qu6bec, Canada, H4P 2R2 Communicated by Ira Herskowitz, June 30, 1992 (receivedfor review December 2, 1991) ABSTRACT We have used a genomic library of Candida analysis of the pheromone response pathway. Recessive albicans to transform Saccharomyces cerevisiae and screened mutations leading to pheromone resistance (7, 8) have iden- for genes that act similarly to dominant negative mutations by tified genes encoding the a-pheromone receptor (9, 10), the interfering with pheromone-mediated cell cycle arrest. Six pheromone response G-protein fB subunit (11), two protein different plasmids were identified from 2000 transformants; kinases (12, 13), a transcription factor (14), and a product four have been sequenced. One gene (CZFI) encodes a protein apparently involved in regulating cyclin activity (8). Domi- with structural motifs characteristic of a transcription factor. nant mutations leading to pheromone resistance led to the A second gene (CCNI) encodes a cyclin homologue, a third isolation of a G1 cyclin (15). In addition, overexpression ofS. (CRLI) encodes a protein with sequence similarity to GTP- cerevisiae genes that reduce pheromone sensitivity has al- binding proteins of the RHO family, and a fourth (CEKI) lowed the identification of a G-protein a subunit (16) as well encodes a putative kinase of the ERK family. Since CEKI as the KSS1 protein kinase (17). confers a phenotype similar to that of the structurally related We have used C. albicans as the source of potential S. cerevisiae gene KSSI but cannot complement a KSSI defect, interfering sequences. A variety of C. albicans genes can be it is evident that dominant negative selection can identify expressed in S. cerevisiae, yet the C. albicans sequences proteins that complementation screens would miss. Because diverge significantly from their S. cerevisiae homologs at the dominant negative mutations exert their influence even in protein level (18). In addition, although it does contain signal wild-type strain backgrounds, this approach should be a gen- transduction components, including a putative G-protein a eral method for the analysis of complex cellular processes in subunit (19), C. albicans is apparently an asexual diploid organisms not amenable to direct genetic analysis. organism (18) and thus does not contain a pheromone re- sponse pathway analogous to that in S. cerevisiae. We Complementation of defective genes in the yeasts Saccha- anticipated that C. albicans could provide a source of pro- romyces cerevisiae and Schizosaccharomyces pombe has teins structurally related to elements of the S. cerevisiae been used to identify functionally related or homologous mating response pathway, yet not normally functioning in a genes from a number of organisms, including humans (e.g., mating response pathway; overexpression of such proteins refs. 1-3). The complementation approach is powerful, but it could potentially interfere with proper pheromone-mediated has limitations since heterologous proteins may often not be cell cycle arrest. Our results show that this selection of able to fully complement a phenotype and replace a missing interfering functions using libraries from heterologous orga- activity, even if expression levels are high. However, be- nisms is efficient and suggest that this approach could pro- cause in some situations high-level expression of partially vide a strategy for the identification of genes acting in a defective proteins can interfere with the function of the variety of cellular pathways in otherwise genetically intract- corresponding wild-type protein, some heterologous genes able organisms.t may act as novel examples of dominant negative mutations (4). Expression of such foreign proteins could potentially interfere with cellular functions in different ways; for exam- MATERIALS AND METHODS ple, the foreign protein could be (i) a dysfunctional compo- Recombinant Techniques. Standard protocols were used nent of a multimeric complex that inactivates the entire for yeast and Escherichia coli (20-22) and for DNA sequenc- complex, (ii) a negatively acting element that cannot be shut ing (23). off, or (iii) a component that competes for some limiting Strains and Plasmids. S. cerevisiae strains used were cofactor or metabolite. M200-6C (MATa ura3 adel sstl sst2 ilv3) (24), EY965 (MATa We have tested the efficiency of a dominant negative ade2 his3 leu2 trpl ura3 can] fus3::LEU2 kssl::HIS3), a gift selection scheme for the identification of heterologous pro- from E. Elion (Harvard University), and YNN19 (MATa ura3 teins that interfere with the mating response pathway in S. leu2 his3 trpl lys2 fusi :lacZ LEU2), a gift from K. Matsu- cerevisiae. This pathway can be considered a model for signal moto (Nagoya University). Strain 1316-S5C (MATa cinlA transduction systems (for review, see refs. 5 and 6), and it cln2A cln3A sstl trpl ura3 adel his2 leu2::pGAL CLN3- provides a very good assay for a dominant interference LEU2), with deletions ofthe three CLN genes and expressing selection because the pathway has many potential targets and CLN3 under was from F. Cross a sensitive positive selection (pheromone resistance) to allow galactose-inducible control, detection of interfering functions. This pathway has been (Rockefeller University). Plasmids other than those identi- extensively studied by more conventional means, and the fied in the library screening were YEp352, a URA3 2-,um selection of strains insensitive to pheromone-mediated cell vector containing the M13mpl8 polylinker (25), and pBC50, cycle arrest has proved to be an important tool for the Abbreviation: aa, amino acid(s). *To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge tThe sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" GenBank data base [accession nos. M76585 (CEKI), M76586 in accordance with 18 U.S.C. §1734 solely to indicate this fact. (CZFI), M76587 (CCNI), and M83991 (CRLI)]. 9410 Downloaded by guest on September 26, 2021 Genetics: Whiteway et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9411 which contains KSSJ under control of the GAL] promoter (Fig. 1B). The transformants containing plasmids M152p8 and was provided by W. Courchesne (University ofNevada). and M153pll did not undergo the morphological changes in Assays. Pheromone treatment was as described (11) with the presence of a-factor that characterized cells containing a-factor. 83-Galactosidase activity was assayed essentially as the control plasmid YEp352. Transformants with plasmid described (21). M166p16 had many cells that underwent morphological changes in response to pheromone, but they also contained frequent cells that were dividing at a smaller than normal size. RESULTS The other plasmids did not dramatically alter the behavior of Isolation of Interfering Sequences. A high-copy library of the cells; after pheromone treatment the cellular morphology genomic sequences from C. albicans strain WO1 was gener- was similar to that of the strain containing YEp352. ated from DNA partially digested with Sau3A1, size- We also examined the effect of the interfering plasmids on fractionated to 5-10 kilobases, and cloned into the BamHI the induction of FUSI. The expression of this gene is highly site of YEp352. The library contains 30,000 independent induced by pheromone treatment and thus serves as a good plasmids, of which half contain inserts (C. Boone, personal marker for the transcriptional response to pheromone. Strain communication). This library was introduced into M200-6C, NYY19a, which contains a fusl::lacZ fusion and thus ex- a pheromone-supersensitive strain of S. cerevisiae defective presses P-galactosidase under control ofthe FUSI promoter, in both SSTJ (gene required for a-factor degradation) and was transformed with various plasmids. 3-Galactosidase SST2 (gene required for adaptation to pheromone). Trans- expression was measured with and without a-factor addition. formants were then screened on plates spread with 5 ,ug of None of the plasmids dramatically interfered with FUSI synthetic a-factor. Use of the highly pheromone-sensitive induction: plasmids M153pll and M157p4 reduced FUSI sstl sst2 double mutant host strain allowed even minor induction to about 80%o of the control, whereas M167pll increases in pheromone resistance to be detected. reduced it to 70%o ofthe control, and the other plasmids tested Approximately 2000 transformants were tested for those had no effect (data not shown). which conferred enhanced resistance to the S. cerevisiae a Characterization of Interfering Plasmids Containing CEK1. pheromone. Twelve resistant colonies were detected, but 3 Initial restriction analysis suggested that two of the interfer- were not dependent on the plasmid for the resistance and thus ing plasmids, M152p8 and M161pll, defined the same gene. presumably represented sterile mutants, which can arise Comparison of the two sequences revealed that the plasmids frequently (24). The remaining transformants contained plas- encoded proteins that were identical except for the
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