The Identification of Transposon - Tagged Mutations in Essential Genes That Affect Cell Morphology in Saccharomyces cer & ae Kristin T . Chun

The yeast Saccharomyces cermisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphologyelongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis. A basic problem in biology is to understand the molecular mechanisms that generate specialized cell shapes. The yeast Saccharomyces cerevisiae provides a simple system in which to study this process. Early in the cell division cycle, the microtubule-organizing center (spindle pole body) is duplicated, and a ring of chitin forms in the wall of the mother cell ( PRINGLE and HARTWELL 1981). Cell wall growth is localized to the region within this ring, resulting in the formation of a small bud. A ring of 10-nm filaments appears in the cytoplasm next to the cell membrane between the mother cell and the bud and, as the cell cycle progresses, the bud continues to grow and eventually becomes the daughter cell. As the bud increases in size, DNA replication occurs, the spindle pole bodies separate, and the spindle is formed. The nucleus then migrates to the neck between the mother and daughter cells, the spindle elongates, and nuclear division takes place. Finally, with the formation of a cell membrane and cell wall barrier between the mother and daughter cells, cytokinesis physically separates the mother and daughter cells. Mutations in a number of essential genes cause cells to form abnormally shaped buds, indicating that the products of these genes are required to make a normal bud. At the restrictive temperature, cdc4, cdc34, and cdc53 mutants arrest late in G1-phase of the cell division cycle before the beginning of DNA replication (BYERS and GOETSCH 1974; BYERS 1981; PRINGLE and HARTCorresponding author: Mark G. Goebl, Department of Biochemistry and Molecular Biology, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46202-5122. E-mail: [email protected] Genetics 142: 39-50 (January, 1996) WELL 1981; ADAMS 1985). In these mutants, the spindle pole body duplicates, but the poles fail to undergo the separation required for spindle formation. Although these mutants initiate bud emergence, they proceed to form abnormal (multiple and elongated) buds. Mutations that increase the activity of Clnlor Cln2(G1 cyclins) associated Cdc28 kinase or decrease the activity of Clblor Clb2(Rtype cyclins) associated Cdc28 kinase also cause cells to form elongated buds (LEW and REED 1993). Moreover, a mutation in any of the genes that encode the three subunits of the replication factorA homologue in yeast (RFAl, RFA2, and RFA3) causes cells to arrest with single or multiple elongated buds (BRILL and STILLMAN 1991). Although the protein products of these genes are involved in a variety of functions, they are all required during the same period of the cell division cycle, late in the GI-phase, and may function in related pathways. Mutations in several other genes whose products are required later in the cell division cycle also cause the formation of aberrant, elongated buds. The products of the genes WC3, CDClO, W C l l , and CDCl2 are needed to assemble the ring of 10-nm filaments at the neck between the mother and daughter cell, and a mutation in any of these genes prevents cytokmesis (HAARER and PRINGLE 1987; FORD and PRINGLE 1991; PRINCLE and HARTWELL 1981; KIM et al. 1991). These mutants continue to bud, replicate their DNA, and proceed through nuclear division in the absence of cytokinesis, thus forming cells with multiple, elongated buds and multiple nuclei. Deletion mutations of either CDC3 or CDC12 are lethal (J. PRINGLE, personal communication). 40 K. T. Chun and M. G. Goebl To gain further insight into the process of bud morphogenesis, we set out to identify additional genes required for this process. Previously, collections of conditional mutations have been screened to identify genes required for essential functions, but genes that are not susceptible to the generation of mutations that cause a conditional phenotype would not have been identified in such screens (HARTWELL 1967; MOIR et al. 1982; KABACK et al. 1984; BARTEL and VARSHAVSKY 1988; RILES and OLSON 1988). However, a screen of mutants created by gene disruption could lead to the identification of a more comprehensive collection of essential genes, including those required for proper bud formation. This approach is not limited by the requirement that the mutated gene cause a conditional phenotype. In this report, we describe the use of transposon mutagenesis to generate a large collection of disruption mutations in essential genes and the identification of mutants that die with an aberrant bud morphology. The mutations in 33 of these mutants identify three previously uncharacterized genes as essential and suggest a role for their products in bud morphogenesis. MATERIALS AND METHODS Reagents: All chemical reagents, including those used for the preparation of media, were obtained from Sigma, Difco, or Boehringer Mannheim. All restriction endonucleases and other DNA modification enzymes were obtained from Boehringer Mannheim. Glusulase was obtained from NEN Research Products. Media and growth conditions: Yeast strains were grown in yeast extract, peptone, dextrose (YPD) medium (ROSE et al. 1990) at 30". To identify auxotrophic markers, yeast were grown on synthetic complete "dropout" medium (ROSE et al. 1990) in which all of the commonly encountered auxotrophies were supplemented except for those being used in the genetic selection. This medium was also supplemented with 2% glucose (SD medium). Presporulation medium consists of 1% yeast extract, 3% peptone, 5% glucose, and 2% agar. Sporulation media was prepared as described previously (SHERMAN 1991). Liquid cultures were grown with aeration at 30". Escherichia coli strains were grown at 36" in LB medium (DAVIS et al. 1980) supplemented with one or several of the following: ampicillin (50 pg/ml), kanamycin (80 pg/ml), streptomycin (100 pg/ml), and chloramphenicol (30 pg/ml). Strains and plasmids: The S. cereuisiae strains used in this study are listed in Table 1. A yeast strain lacking 2 pm plasmid DNA (KC107) was isolated using the method of DOBSON et al. (1980) as follows. Strain FY23 was transformed ( SCHIESTL and GIETZ 1989) with pJDB219 (kindly provided by B. FUTCHER), and Leu+ transformants were selected on synthetic complete medium lacking leucine. A single transformant was picked and grown at 30" for 6 days in 25 ml of synthetic complete medium lacking leucine, with a 1 : lOOO dilution into fresh medium each day. The final culture was then diluted 1 : lOOO into fresh W D medium and grown at 30" for five more days, again with a daily 1: lOOO dilution into fresh medium. Cells from this culture were then spread onto solid W D medium, grown at 30°, and replica-plated onto synthetic complete medium with and without leucine to identify Leuclones. Three Leuclones were identified, and Southern blot analysis (Genius I DNA labeling and detection kit, Boehringer TABLE 1