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To isolate strains harboring gle1 synthetic lethal mutations, we treated two parental strains with ethylmethane sulfonate to ∼25% viability and plated them for growth at 23°C [SWY1791 (MATα gle1-P380L ade2-1 ade3::HISG ura3-1 his3-11,15 leu2-3,112 TRP1 lys2 can1-100 pGE1/ADE3/URA3 pNUP116/HIS3) anf SWY1792 (MATa gle1-P380L ade2-1 ade3::HISG ura3-1 his3-11,15 leu2-3,112 trp1-1 LYS2 can1-100 pGLE1/ADE3/URA3 pNUP116/ HIS3)]. Approximately 200,000 total colonies were screened using an ADE3-dependent color sectoring assay (22). Nonsectoring colonies were isolated, and 28 strains passed tests for dependence on pGLE1/URA3 and lack of gle1Δ alleles. We divided 24 alleles into four complementation groups (Fig. 1A), with 4 remaining unassigned. On the basis of growth defects, the gsl alleles were ranked in relative order of increasing severity and penetrance. For characterization experiments, the most severe allele was chosen (for example, gsl2-6 or plc1Δ, gsl1Δ, gsl3-3, gle1-4). Exceptions include the synthetic lethal screen (gle1-2) and cloning of GSL genes (gsl1-4, gsl2-2), in which cases the adele used was that most readily complemented by GLE1 in a colony sectoring assay. Allele-specific differences were noted in the IP profiles of gsl3-1 and gsl3-3, and likely represent distinct mutations.
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6 in gsl1Δ cells. Protein A-Ipk1p was not visible when expressed on a centromere plasmid or integrated. In subcellular fractionation experiments, the majority of the centromere expressed protein A-Ipk1p was isolated with nuclei, suggesting the results in Fig. 5 reflect that of endogenous Ipk1p.
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note
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We thank J. Datto for technical help, J. Thorner for sharing unpublished data, and K. Weis and D. Goldfarb for plasmids. Supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (J.D.Y.), a Whitehead Scholar Award (J.D.Y.), Duke University Medical Scientist Training Program (A.R.O.), an NIH Training Grant for predoctoral trainees (R.M. and E.B.I.), and grants from the American Cancer Society (Junior Faculty Research Award) (S.R.W.) and the National Institutes of General Medical Science (S.R.W.).
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