Copyright 0 1994 by the Genetics Society of America Formamide Sensitivity: A Novel Conditional Phenotype in Yeast Andres Aguilera Departamento de Genitica, Facultad de Biologia, Universidad de Sevilla, E-41012 Sevilla, Spain Manuscript received June 16, 1993 Accepted for publication September 28, 1993 ABSTRACT Yeast mutants unable to growin the presence of 3% formamide have been isolatedin parallel with mutants sensitive to either 37" or 6% ethanol. The number of formamide-sensitivemutations that affect different genes that can be identified from yeast cells is at least as large as the number of thermosensitive or ethanol-sensitive mutations. These mutations are of two types: those that are sensitive to formamide, temperature and/or ethanol simultaneously; and those that are specific for formamide sensitivity and show no temperature or ethanolsensitivity phenotype. Those genes susceptible to giving rise to formamide-sensitive alleles include the structural gene for DNA ligase, CDC9, and the structural gene for arginine permease, CANl. The results indicate that formamide sensitivitycan be used as a novelconditional phenotype for mutations on bothessential and nonessential genes. This work also confirms that ethanol-sensitivity can beused as a conditional phenotype._ to identify mutations in at least as many genes as those susceptible to temperature or formamide sensitive mutations. HE understanding of any biological process re- the differentbiological processes studied. Second, not T quiresthe identificationof theproteins and all amino acids of a protein are equally susceptible to genes involved in it. The contributionof genetic lead to athermosensitive phenotype (ALBERet al. methods to this understanding through the isolation 1987). Third, the identification and genetic analysis ofmutants is very important.However, there are of extragenic suppressors of thermosensitive muta- obvious limitations for the identification of mutations tions in essential genes require a second conditional in essential genes. Conditional mutations, that is mu- phenotype (MOIR and BOTSTEIN1982). And fourth, tations that only express the mutant phenotype under the use of different temperatures is not possible for some restrictive conditions (HOROWITZ1948), serve homeothermic eukaryotes. to overcome this problem. Recently BARTELand VARSHAVSKY(1988) used Temperature has been used extensively to isolate heavy water (D20) as a conditional mutant phenotype conditional mutantssince first proposedby HOROWITZ in yeast. Their work provides an important tool to and LEUPOLD(1 95 1). The mutant phenotype can be identify novel conditional mutations and open new expressed at eitherhigh (thermosensitive) or low (cry- ways to isolate conditional mutants in homeothermic osensitive) temperatures. This technique has allowed organisms. the identification of many genes involved in essential A characteristic that can also be used to isolate biochemical and cellular processes, such as DNA rep- conditional mutants is sensitivity to ethanol,which has lication, cell division, cytoskeleton structure, etc., been characterized at the genetic and physiological since the first thermosensitive mutants were reported level (AGUILERAand BENITEZ1986). A great many in phage (EDGARand LIELAUSIS1964; EPSTEINet al. genes can be mutated to produce anethanol-sensitive 1963), bacteria (EIDLICand NEIDHART1965, KOHI- phenotype. A number of these mutations also lead to YAMA et al. 1966) and yeast (HARTWELL, 1967). Dif- thermosensitivity. That study suggests that many ferentmutant proteins contain changes in charge, genes,not necessarily involved in glucose or lipid hydrophobicity, solvent structure and hydrogen bond- metabolism, are susceptible to mutations that lead to ing that influence its secondary or tertiary structure an ethanol-sensitivity phenotypefor growth. How- and that can confer a thermolability phenotype (AL- ever, the use of ethanol-sensitivity as a conditional BER et d. 1987). phenotype may berestricted to organisms such as There areseveral reasons to believe that alternative yeast that tolerate ethanol concentrationsof 6%. conditionalphenotypes are necessary. First, not all In this study, formamide sensitivity as a novel con- proteins are equally susceptible to aminoacid changes ditional phenotype in yeast was investigated for the that make them thermolabile; the extensive work of following reasons: (1) formamide is an ionizing solvent HARTWELL(1 967) on yeast suggested that the isola- that destabilizes noncovalent bonds such as hydrogen tion of thermosensitive mutants is not random among bonds; (2) formamide is not metabolized by cells and Genetics 136: 87-91 (January, 1994) 88 A. Aguilera there are no reasons to believe a priori that yeasts TABLE 1 have evolved as highly formamide-tolerant organisms Classification of 41 conditional mutants isolated as is the case for ethanol; and(3) formamideis a small molecule (HCONH2) that should enter cellseasily. Strains We have isolated some mutants that are simultane- Conditional Total phenotype" AYW3-3A AYW3-4b mutants ously formamide, ethanol and temperature sensitive for growth, andsome others that areexclusively form- fs ts es 3 6 9 amide sensitive. We have also identified formamide- fs ts 2 3 5 0 6 6 sensitive alleles of the CDC9 and CAN1 genes. The fs es fs 7 5 12 genetic analysis of the formamide sensitive mutants ts es 0 0 0 isolated in this study suggests that any type of gene, ts 4 1 5 whether essential or not, is susceptible to formamide es 1 3 4 sensitive mutations. Total 17 24 41 "3:sensitivity to 3% formamide; ts: sensitivity to 37°C; es: sensitivity to 6% ethanol. Sensitivity is defined in this table as the MATERIALS AND METHODS inability to form colonies. Strains: The yeast strains usedin this study arethe congenic strains W303-1B (MATa ade2-1 leu2-3, I12 ura3- AYW3-4B were mutagenized with nitrosoguanidine 1 trpl-1 his3-11, 15 canl-loo), AYW3-3A (MATa ade2 leu2- as described in MATERIALS AND METHODS. A total of 3, 112 ura3 trpl his3), and AYW3-4B (MATa ade2 leu2-3, 2635 surviving colonies (1887 from strain AYW3-4B 112 ura3 trpl his3), 5a-P3631 (MATa his7-1 lys2-18), and and748 from AYW3-3A) were picked onto new C9Y-1B (MAT& leu2 his3 cdc9-I). Twelve other strains car- YEPD plates andafter 2 days at 30°, they were rying 1 1different thermosensitive mutations in 10 cell cycle genes were used.These mutations were hpr6 (a CDC2 allele), replica-plated onto YEPD-3% formamide, YEPD-6% cdc4, cdc5, cdc6,cdc8-I, cdc9-1, cdcl3-I, cdcl4, cdcl7-I, hpr3 ethanol and YEPD. The first two groups of plates (a CDCl7 allele) and cdc28. The cdc alleles used are original were cultured at 30O , and the YEPD plates at 37 " C, from L. Hartwell, C.Newlon and H. Klein laboratories. in orderto scorefor a formamide-sensitive (The allele number is not given when it is not known.) (fs), ethanol-sensitive (es) or thermosensitive (ts) pheno- Media and growth conditions: Standard media such as rich medium YEPD, synthetic complete medium with bases type for growth. After three rounds of scrutiny, a and amino acids omitted as specified, and sporulation me- total of 41 mutants (17 from strain AYW3-3A and 24 dium were prepared according to published procedures from AYW3-4B) with an fs, es or ts phenotype were (SHERMAN,FINK and HICKS1986). L-Canavanine sulfate was selected. From Table 1 two results can be highlighted: added to synthetic medium at concentrations of 60 pg/ml. Formamide or ethanol were added to the medium at the of the 41 conditional mutants isolated 9 were simul- indicated concentrations after autoclaving. All yeast strains taneouslyfs, es and ts; and of 32fs mutants isolated, were grown at 30" with horizontal shaking for liquid cul- 12 were exclusively fs, and the other 20 were also ts tures. or es or both. These results indicate that: first, the Mutagenesis: Yeast strains were grown overnight in 5-ml number of mutations leadingto a formamide-sensitive YEPD. Cells in early stationary phase were resuspended in Tris-maleate pH 7.8 to a density of 10' cells/ml and muta- phenotypearose at afrequency similar tothat of genized with a final concentration of 20 rg/ml N-methyl- mutations leading to a ts phenotype or an es pheno- N'-nitro-N-nitrosoguanidine (nitrosoguanidine) for 15 min type;second, mutations conferring a fs phenotype according to CALDERONand CERDA-OLMEDO(1983). Muta- could lead simultaneously to a ts and/or es phenotype; tions were allowed to segregate by culturing the mutagen- ized cells in liquid YEPD for 6 hr at 30" prior to plating. and third, many mutations were specific for an), es The viability of the cells after mutagenesis was approxi- or ts phenotype. mately 30%.Mutagenized cells were plated on YEPD plates Genetic analysis of formamide-sensitive mutants: to isolatesingle colonies and on SC-can and SC-can-2% Twenty formamide-sensitive mutants (1 2 from strain formamide plates to select for CanR colonies in either the AYW3-4B and 8 from AYW3-3A) were selected for absence or the presence of 2% formamide, respectively. After 2 days at 30°,colonies from YEPD plates were replica- further genetic studies. All of them were crossed with plated onto YEPD-3% formamide, YEPD-6% ethanol and either strain AYW3-3A or AYW3-4B to determine YEPD. Those plates containing formamide or ethanol were the recessivity or dominance of the fs phenotype. In cultured at 30" and the simple YEPD plates at 37". all cases, diploid strains were able to grow on YEPD- Genetic analysis:Genetic analysis was performed accord- like ing to SHERMAN,FINK and HICKS(1 986). 3% formamide the wild-type haploid parental strains, indicatingthat all 20 mutations were recessive. That most of the mutations were monogenic was RESULTS confirmed by a genetic analysis of 12 of the selected Isolation of formamide-sensitive mutants: Wild- fs mutants(10 from strain AYW3-4B and 2 from type cells grow very poorly at formamide concentra- strain AYW3-3A). Between 9 to 14 tetrads were dis- tions above 3% v/v and not at all at a concentration sected and subjected to genetic analysis.