Cell, Vol 52. 935-941, March 25, 1988. CopyrIght 0 1988 by Cell Press

Hypersensitivity to Heavy Water: A New Conditional Phenotype

Bonnie Bartel and Alexander Varshavsky Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary

Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D20. Using chemical mutagen- esis we obtained a number of strains that grow on HzO-containing media but not on otherwise identical media containing 90% D20. The frequency of these &O-sensitive (ds) mutants is comparable to the fre- quency of conventional temperature-sensitive (ts) mu- tants in the same mutagenized sample, and the ds mutations are distributed over a large number of com- plementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle fs mutants tested, only 2 are also ds. Thus, the ds technique should be useful for pro- ducing conditional mutations in genes that are not amenable to the fs and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the ad- vantages of conditional phenotypes to mammalian and avian genetics.

Introduction

Much of the advance in the understanding of life pro- cesses that has occurred in this century was made possi- ble by the application of genetic methods. Mutations in some genes are compatible with the organism’s viabil- ity, and therefore such mutants can be studied directly. However, mutational inactivation of an essential gene product results in a lethal phenotype, which imposes se- vere limitations on the repertoire of genes and their func- tions amenable to genetic analysis. Horowitz (1948, 1950) addressed these limitations by introducing the use of con- ditional mutants, which display a wild-type phenotype at a certain (permissive) temperature but are either inviable or otherwise defective at another (nonpermissive) temper- ature (Horowitz and Leupold, 1951). Such conditional (high temperature-sensitive, ts, or cold-sensitive, cs) mu- tants can be propagated at a permissive temperature and then shifted to a nonpermissive temperature where spe- clfic effects of inactivating a gene product can be ob- served and analyzed. Moreover, by allowing controlled functional inactivation of a mutant gene product at the nonpermissive temperature, this method allows temporal analysis of ensuing changes in the affected cell or organ- ism, thereby increasing the range and power of the

genetic approach (reviewed by Pringle, 1975; Botstein and Maurer, 1982).

The WCS method for generating conditional mutants has several limitations. First, some genes, in spite of at- tempts to mutate them to a ts or cs form, have not yielded the desired mutants. For instance, Kaback et al. (1984) found only three complementation groups in a screen for fs lethal mutations mapping to the Saccharomyces cerevisiae chromosome I, which is estimated to contain about 100 genes. Second, homeothermic animals, in con- trast to other organisms, cannot withstand the consider- able deviations from their normal internal temperature that are required for a fs or cs approach. Indeed, the rela- tively unadvanced state of mammalian and avian genet- ics (as compared with the genetics of poikilothermic metazoans such as flies and nematodes) may be due not only to the increased sizes, generation times, and genetic complexity of these animals, but also to the virtual nonex- istence of conditional mutants in homeotherms. Aside from limitations of the ts/cs approach, it is desirable to have a greater diversity of generally applicable condi- tional phenotypes. For example, pseudorevertants of a conditional mutant can be used to identify other genes whose products interact with the product of the mutant gene (Jarvik and Botstein, 1975; Botstein and Maurer, 1982). This approach is greatly facilitated if the pseu- dorevertants have a conditional phenotype distinct from that of the original mutant. However, if the original mutant is fs, there are at present few alternative conditional phenotypes available. The same difficulty is relevant in other genetic approaches that involve the use of two dis- tinct conditional phenotypes (Moir and Botstein, 1982).

General physical and chemical considerations suggest that it should be possible to generate mutants hypersensi- tive to heavy water (D,O), and that the resulting condi- tional mutants should not be necessarily ts or cs. If, in ad- dition, a significant proportion of genes in an organism could be mutated to the D2 O-sensitive (ds) phenotype, this approach might alleviate the limitations of the cur- rently available methods discussed above. As shown be- low, the ds approach is feasible, and should be generally applicable.

Results

Figure 1A shows the plating efficiencies of a wild-type yeast strain (DBY1705) on media containing various con- centrations of l&O at several growth temperatures. Plat- ing efficiencies are reduced at high i&O concentrations, especially at temperatures above 30°C (Figure 1A). Growth rates are also reduced at high concentrations of D20. For example, DBY1705 grown in SD-97% D20 at 30% has a doubling time of ~8 hr, compared with -4 hr in SD-80% D20 and ~3 hr in SD-30% D20. At the same time, DBY1705 and other wild-type strains grow well enough at 30°C on minimal (SD) medium containing 90%

Cell 936

16 20 24 28 32 36

Growth temperature (“C)

m 0

Percent D,O

Figure 1. Plating Efflclences of Wild-Type and ds Yeast Strains on DzO-Contalnlng Media

(A) $0 sensltlwty of the wild-type strain DBY1705 as a function of growth temperature An exponential culture of DBY1705 grown in SD-Hz0 was diluted in HTO. and 50 PI aliquots were spread onto SD plates of different D20 content. The plates were Incubated at the Indl- cated temperatures until colonies >i mm I” dl- ameter formed. All experiments were per- formed III duplicate or trlpllcate, mean values are plotted Circles, SD-H20: crosses, SD- 80% D@ triangles, SD-99% D20 The con- trol value of relative plating efflclency (100%) corresponded to platmg on SD-H,0 at 30% yielding approximately 200 colomes per plate (6) Hypersensttlwty of ds mutants to D20. Shown are the relative plating efflclenc;es at 30% and at different DZO concentrations, de- termined as m (A), of the haplold segregants dsla-dsld of a single meiotlc tetrad from the cross dsl x DBY1500. Open triangles, dsla (wild type); open circles, dslb (wild type): closed triangles, dslc (ds), closed circles, dsld

(ds). Plating efflclency curves of the wld-type segregants dsla and dslb were similar to that of the wild-type parent DBY1500 (data not shown) SD plates contalnlng O%, 30%, 50% 80%, 90% and 99% D20 were Incubated at 30% for 3. 4, 5. 10, 19. and 30 days. respectively. These times were suiflclent for colonies >1 mm II- diameter to form, except on SD-99% D20 plates, where no colonies formed

D20 to meet the goals of the ds approach. These parameters were therefore chosen as the nonpermissive condition In a screen for ds mutants.

Individual yeast colonies derived from an ethyl meth- anesulfonate (EMS)-mutagenized culture were tested for their ds, ts, and cs phenotypes by stamping onto the appropriate plates as described in Experimental Proce- dures. The results of a typical screen of this type are shown in Table 1 (see also Figures 1B and 2). It is clear that mutations conferring a ds phenotype can arise at least as often as those resulting in a ts phenotype (16% versus 7% of all colonies screened in this experiment) (Ta- ble 1). In addition, the overlap between the screened mu-

Table 1. Relative Frequency of ds Mutants

Percent of All Phenotype Number of Isolates Colonies Screened

ds 77 16 ts 33 7 cs 4 0.8

ds and ts 11 2.3 ds and cs 1 0.2 ts and cs 1 0.2

Strain DBY1705 was mutagenired wth EMS to ~30% survival, 480 colomes from the mutagemzed sample were screened for their ds, ts, and cs phenotypes as described II? Experimental Procedures. Mutants with a double phenotype were also scored as having the correspond- mg sngle phenotypes. Although the mdlwdual mutant isolates from a single batch of mutagenlzed cells are not expected InvarIably to represent Independent mutants, the complementatlon test of Table 3 showed that at least a substantial proportlon of the above ds isolates carried mutations in different genes.

tant classes (ds, ts, and cs) is small, with only 11 of the 77 ds strains isolated also showing a tight ts phenotype. Plating efficiency tests showed that ds strains are hyper- sensitive to D,O not only in the presence of 90% D20 but also at all D20 concentrations tested (Figure 18). To con- firm that mutations conferring the ds phenotype represent a set that is largely nonoverlapping with the set of ts muta- tions, we tested several cell division cycle (cdc) mutants, originally isolated as ts mutants (Hartwell et al., 1973), for their sensitivity to D20. Of the 17 ts cdc mutants tested, only two display a ds phenotype (Table 2).

To characterize the ds strains of Table 1 further, we de- termined their phenotypes on media of high osmolarity. Three of the 47 ds strains tested are hyperosmotic- remedial, i.e., they do not grow on SD-90% D20 plates, but can grow on SD-90% D20 supplemented with either 1.2 M KCI or 1 M sorbitol (data not shown). Two other ds strains are hyperosmotic-sensitive, i.e., they do not grow either on SD-90% D20 or on SD-H20 supplemented with 1.2 M KCI (data not shown). We conclude that most ds mutants do not display other known conditional pheno- types.

We also tested whether the conditional lethal phenotype of any of our ds strains (Table 1) on SD-90% D20 was due to a conditional auxotrophy for specific amino acids. Two strains, ds30, which is both ds and ts, and ds36, which is ds but not ts, have their ds phenotype (and in the case of ds30, the ts phenotype as well) complemented by the addition of either aspartic acid or asparagine to the SD-90% D20 medium (data not shown). In addition, al- though ds30 is an adenine prototroph, its colonies turn pink on SD-90% D20 supplemented with either Asp or Asn. These properties of ds30 are consistent with it aris-

D?O-Sensitive Mutants 937

Table 2. D,O Senswty of Strains Originally Iso!ated As ts

0% D20 0% D,O 90% D20 Strain (23%) (36°C) (23OC)

A364A + + + cdcl-1 + + cdc2.1 + - + cdc4-1 + + cd&1 + + cd&-1 + -

cdc7-1 + - + cd&l + - + cdc9.1 + - + cdclO-1 + - + cdcl3-1 + +

cdcl4-1 + - + cdcl5-1 + cdcl5-2 + - + cdcl7-1 + + cdcle-1 + + cdcl9-1 + - + cd@1 -1 + - +

lndlwdual colonies of strains grown on SD-H,0 plates at 23OC were dispersed in HP0 and stamped onto YPD plates contalmng the Indl- cated amounts of DrO Growth was compared after 3 days at the ap- propriate temperature Symbols + , wild-type (strain A364A) or near wild-type growth. - no growth

ing by a ds mutation In the ASP5 gene (de Robichon- Szulmajster et al., 1966; Jones and Fink, 1982). To test this possibility, the strains ds30 and ds36 were crossed to an asp5 strain, X1049-9C. The ds30/asp5 diploid is both ds and ts in the absence of aspartic acid but neither ds nor ts in its presence, while the ds36/asp5 and the DBY17051 asp5 diploids are neither ds nor fs regardless of the pres- ence of aspartic acid in the growth medium (data not shown). In addition, when a ds30/asp5 diploid was sporu- lated and tetrads dissected, no segregants (out of 12 tetrads analyzed) were Asp+ on both D*O and HZ0 plates (data not shown), indicating that the corresponding alleles are tightly linked. This linkage, along with the lack of com- plementation between the X1049-9C (as@) and ds30 strains, strongly suggests that the ds phenotype of ds30 is due to a mutation in the ASP5 gene.

Several of the ds strains were crossed to a wild-type

B C

strain (DBY1500). Tetrad analysis of the resulting diploids confirmed that the ds phenotype segregates in all cases tested as a Mendelian trait (Figure 2 and data not shown). In addition, for several of the ds strains that are also ts (e.g., ds30, ds34, and ds46), the ds and ts phenotypes of the origtnal strains cosegregated in all of the tetrads ana- lyzed (usually eight for each diploid strain), indicating that the mutations responsible for the ds and ts phenotypes of the original strain are tightly linked and therefore most likely identical.

It is known that a significant fraction of genes can be mutated to yreld a ts phenotype (reviewed by Pringle, 1975; Kaback et al., 1984). The high frequency of ds mu- tants after EMS mutagenesis (Table 1) is consistent with the ,possibrlity that the fraction of genes able to yreld ds alleles is comparably large. In a test of this conjecture, we assessed the ability of several of the recessive ds mutants to complement other recessive ds mutants in a doubly het- erozygous diploid (Table 3). All of these ds mutants, while unable, as expected, to complement themselves, were able to complement all of the other 38 ds mutants tested (Table 3). These results Indicate that the set of yeast genes which can mutate to yield a ds phenotype IS very much larger than the set of ds isolates in this Initial screen, and may be comparable In srze to the set of genes yrelding ts mutants.

Discussion

This work Introduces a new conditional phenotype, hyper- sensrtrvrty to D20. Using the yeast S. cerevisiae as a model, we have shown that mutatrons resulting In the DzO-sensitive (ds) phenotype occur frequently upon con- ventional mutagenesis, that these mutations Involve a large number of genes, and that most of the resulting ds mutants do not display other known condrtronal pheno- types. Thus the ds method may prove useful for producing conditronal mutations in genes that are not amenable to the ts and cs approaches, and also for generating alterna- tive conditional (ds) alleles in many other genes. Several other potential applicatrons of the ds approach are drs- cussed below

SD-O% D20 SD-O% D20

30” c 36” C

SD-go% D20

30” c

Figure 2 Growth of ds, ts. and Wild-Type S cerevislae Strains I” the Presence and in the Absence of D?O

lndudual colomes of strains grown on SD- H,O plates at 30°C were dispersed in HP0 and stamped, using a 4&p1n Inoculator. onto two SD-HI0 plates (A and B) and one SD- 90% D?O plate (C) The SD-H,0 plates were Incubated for 3 days at either 30°C (A) or 36°C (B), and the SD-SO% D1O plate was ~ncu- bated at 30°C for 4 days(C) Rows 1, 2, 3. and 4 contain the haplold segregants from single melotlc tetrads produced by crosses dsl x DBY1500. ds15 x DBY1500, dsZ0 x DBY1500 and ds30 x DBY1500, rsepectlvely Row 5 con- talns (from left to right) A364A. cdcl-1. cdc4-1 and DBY1500

Cdl 938

Table 3 Complementatron Analysis of ds Mutants

MA Ta

MATa dsl ds20 ds26 ds27 ds29 ds30 ds46 DBY1500

dsl ds + + + + -+ + + ds20 + ds + + + + + + ds26 + + ds + + + + + ds27 + + + nt + + nt + ds29 + + + + ds + + + ds30 + + + + + ds, ts 7 + ds46 + + + + + + ds. ts + ds2 + + + + + + + + ds3 + + + + + + + + ds4 + + + + + + + + ds5 + + + + + + + + ds7 + + + + + + + + dsl0 + + + + + + + + dsll + + + + + + + + ds12 + + + + + + + + ds13 + + + + + + + + ds14 + + + + + + + + ds15 + + + + + + + + ds16 nt + + nt + + nt + ds18 + + + + + + + + ds19 + + + + + + + + ds21 + + + + + + + + ds22 + + + + + + + + ds24 + + + + + + + + ds25 + + + + + + + + ds28 + + + + + + + + ds32 + + + + + + + + ds33 + + + + + + + + ds34 + + + + + + + + ds35 + + + + + + + + ds36 + + + + + + + + ds37 + + + + + + + + ds38 + + + + + + + + ds39 + + + + + + + + ds41 + + + + + + + + ds42 + + + + + + + + ds44 + + + + + + + + ds45 + + + + + + + + ds47 + + + + + + + + Seven ds Isolates (see Table 1 and Experimental Procedures) were crossed by DBY1500, and tetrad drssectrons were carried out to ob- tain ds strains of the opposrte matmg type These strains were then crossed to the ongrnal ds strarns. The ds. ts, and cs phenotypes of the resultant diploids were tested by stamping as described in Ex- perimental Procedures. ds isolates that were found to be erther dominant or partrally dommant were not included in the analysrs. Sym- bols: +, wild-type growth on SD-Ha0 (30% and 36OC) and SD-90% DsO (30°C); ds, wild-type or near wild-type growth on SD-H20 (30°C), and no growth on SD-90% D,O (3OOC); ts, weld-type or near wild-type growth on SD-HP0 at 30°C, and no growth on SD-Ha0 at 36°C; nt, not tested.

Mechanistic Aspects of the ds Phenotype, and Its Potential Utility for Studies of Biopolymers It is likely that most ds mutations will be found to reside in genes encoding specific proteins, with the mutant pro- teins being functionally inactivated by growing cells in the presence of D20. In fact, one of our randomly isolated ds mutants maps to a known gene, ASP5 (see Results). Since DZO is known to influence the conformational and func- tional properties of biopolymers (Scheraga, i960; Thom- son, 1963; Lemm and Wenzel, 1981; Nakagawa et al..

1982) the existence of ds mutants is, in hindsight, not un- expected. Furthermore, given the different nature of the effects of D20 and temperature on the conformations and chemical properties of biopolymers (Sheraga, 1960; Grechko et al., 1963; Tanford, 1968; Alber et al., 1987), it is also not unexpected that most ds mutants display nei- ther a ts nor a cs phenotype.

D20 is unique as a physiological perturbant in that the perturbing factor (deuterium) can be both an integral part of the bulk intracellular solvent and an integral part of a biopolymer, where deuterium resides as either exchange- able (N-D, O-D, S-D bonds) or nonexchangeable (C-D bonds) hydrogen atoms. (The only other analogous situa- tion would occur in the case of water containing isotopes of oxygen instead of deuterium. In this case, however, the relevant isotopic effects would be vanrshingly small com- pared with those of deuterium.) Thus, the origins of the ds phenotype may be more varied than is initially apparent. Figure 3 schematically depicts the possibilities to be con- sidered. When cells are grown in a minimal medium of high DzO content, most hydrogen-containing bonds in water and other intracellular compounds (including bio- polymers) will contain deuterium (D) instead of protium (H) (Figure 3DD). This configuration is approximated by the growth conditions used in the present work. However, a different isotopic distribution is also possible in which deuterium is a component of the bulk intracellular water and of the bonds in biopolymers and other metabolites that contain an exchangeable hydrogen, but is largely ab- sent from the bonds that contain a nonexchangeable hydrogen (Figure 3DH). This configuration would be ap- proximated when cells are grown in a medium of high D20 content that is supplemented with “light” (protium- containing) amino acids and other metabolites. An oppo- site bias in the distribution of intracellular deuterium could be created by growing the cells in a medium that lacks D20 but is supplemented by “heavy” (deuterated) amino acids and other metabolites (Figure 3HD).

Given these possibilities, ads phenotype could in prin- ciple result from at least two mechanisms. In one, ads pro- tein is active in the presence of Hz0 (Figures 3HH and 3HD) irrespective of whether the nonexchangeable hydro- gen in a protein is predominantly deuterium or predomi- nantly protium, but is inactive when the bulk intracellular water has a high content of D20 (Figures 3DD and 3DH). A second type of ds protein would require for its activity the absence of deuterium from certain (nonexchangea- ble) carbon-hydrogen bonds. Such a ds protein would be inactive in the configurations of Figures 3DD and 3HD, whereas the preceding mutant protein would be active in the configuration of Figure 3HD. It is possible to distin- guish further between these and other possibilities, but those cited are sufficient to illustrate the potential muf- tiplicity of mechanisms underlying the ds phenotype.

If some ds mutants are indeed based on the second of the above mechanisms (Figure 3HD), how could such mu- tants be identified? Among auxotrophic ds mutants one might expect to have representatives of both of the above classes. These ds mutants could be distinguished by their ability to be complemented by the “heavy” (deuterated)

D,O-Sensrtrve Mutants 939

HH

N-H

Figure 3 On the Mechamsttc Aspects of the ds Phenotype: Deutertum As a Component of Both Water and Btopolymers

(DD) Cells growmg rn a mammal medium of high D20 content contain deutertum (D) not only as part of the bulk solvent but also In biopolymers and other compounds (DH) Cells growtng rn a medium of high D,O content supplemented wtth “ltght“ (protrum-contatmng) amino acids and other metabolic precursors Such cells could be expected to differ from those in (DD) by contaming mostly protrum (H) in those covalent bonds that are dertved directly from precursors in the medium and that contarn a nonexchangeable hydrogen (HD) Cells growrng rn a medium that lacks D,O but IS supplemented wtth “heavy” (deuterated) amino acids and other metabolrc precursors. Such cells would be expected to retatn deutertum (D) rn those covalent bonds that are derived dtrectly from precursors rn the medium and that contain a nonexchangeable hydrogen (HH) Cells growing rn a medium that lacks deuterrum.

Cartoons (DD) and (HH) approximate the configurations studted rn the present work. See also Drscusston.

form of an amino acid whose “light” (protium-containing) form was initially identified as complementing the ds phenotype. In other words, there may exist two classes of ds amino acid auxotrophs: a “true” ds auxotroph, where a mutant enzyme in an amino acid biosynthetic pathway is unable to function at a high concentration of D20 (Figures 3DD and 3DH); and a “false” ds auxotroph, which requires protium rather than deuterium in a particular nonexchangeable carbon-hydrogen bond in at least one of the amino acid residues of a mutant protein. Such a re- quirement would be satisfied by supplementing the D20- containing growth medium with specific “light” amino acids but not with their “heavy” counterparts. Thus second type of ds auxotroph could be due to a mutation in any es- sential gene whose function is not necessarily related to amino acid biosynthesis.

Most ds mutants of this work were ds In both minimal (SD-90% DsO) and complete media (SD-90% DPO sup- plemented with nonessential amino acids). The few ds amino acid (Asp) auxotrophs among these ds mutants (see Results) could be complemented by either “light” or “heavy” aspartic acid (data not shown). Given the small size of the initial set of ds mutants, these data do not pre- clude the possibility of mechanistically different ds pheno- types, including the “false” auxotrophic ds phenotypes discussed above. If such a class of ds mutants is identi- fied, the ds approach will acquire an additional dimension- the possibility of Isolating apparent auxotrophic ds alleles of a given gene whose ds phenotype is complementable by “light” but not “heavy” amino acids.

Irrespective of whether one or several of the above mechanisms (Figure 3) are relevant to the observed ds mutations, the ds technique not only provides a new tool for in vivo genetic analysis, but also allows a new ap- proach to studies of biopolymers. In this approach, anal- ogous to the established use of ts proteins in struc- ture-function analyses (Alber et al., 1987), the protein of

interest would be mutated to ads form, isolated with differ- ent intramolecular distributions of nonexchangeable deu- terium, and compared with the corresponding wild-type protein in the presence of varying concentrations of D20. Both general considerations (see above) and the fact that one of our randomly isolated ds mutants maps to a known gene, ASPS, suggest that it should be possible to obtain ds mutations in other known genes using either site- directed mutagenesis (Smith, 1985) or gene-specific screens (Shortle, 1983; Alber et al., 1987).

Implications for Genetics of Organisms Other Than Yeast Most wild-type single-cell organisms, Including bacterra. fungr, algae, and protozoa, can grow at relatrvely high con- centrations of D20, the maxrmum concentration varying from ~50% to more than 90% D20 for drfferent species (reviewed by Thomson, 1963). Thus the application of the ds method to these organrsms should be technically straightforward.

Insects such as Drosophila are sterilized but not killed by growth on a medium containing 500/o DZO (reviewed by Thomson, 1963). Mammals such as mice and rats can be maintained for months In the presence of up to 30% D20 in their drinking water (Czajka and Finkel, 1960; Thomson. 1960, 1963), and for shorter periods of time on higher concentrations of D20 (Katz et al.. 1962; Thom- son, 1963). Specific organs in both mammals and insects appear to be differentially affected by long-term exposure to high levels of deuteration. the reproductive system be- ing one of the most vulnerable (Czajka and Finkel, 1960; Amarose and Czajka, 1962; Thomson, 1963). Although it remains to be determined whether the ds approach of the present work can be extended to metazoans, the relatively high levels of deuteration tolerated by these organisms justify the attempt. Note that while in the present work we limited the screen to ds lethals, a number of ds phenc-

Cell 940

types in addition to conditional lethality are expected to be of value In metazoans. For Instance, distinct neurological, muscular, reproductive, and developmental ds pheno- types may require only a partial inactivation of a relevant ds protein in the presence of D20.

While genetic studies of single-cell organisms and poi- kilothermic metazoans utilize conditional phenotypes pro- vided by the ts and cs approaches, conditional pheno- types have been virtually unavailable in genetic studies of homeothermlc animals such as mammals and birds. It is with these organisms that the ds method, If successful, may have its greatest impact outside of the area of appli- catlon discussed above.

Solvent-Hypersensitive Mutants Generalizing the idea of the ds approach, it should be pos- sible to produce conditional mutants hypersensitive to a variety of cell-penetrating solvents other than D20. Many solvents are known to have significant effects on the struc- ture of specific biopolymers and noncovalent assemblies such as membranes (Gordon and Jencks, 1963; Tanford, 1968). Thus the previously known osmotic-hypersensitive and pH-hypersensitive phenotypes (reviewed by Pringle, 1975) may represent a subset of a much larger, still unex- plored set of solvent-hypersensitive phenotypes. Two de- sirable characteristics of a solvent to be used are meta- bolic inertness and the ability to enter cells without the need for a specific carrier system. Many organic solvents, such as dioxane, dimethyl sulfoxide, and formamide, possess at least the latter property. The approach would consist of determining the maximal concentration of a sol- vent compatible with an acceptably high rate of growth of a wild-type organism, followed by the generation, via mutagenesis, of mutant strains that either cannot grow or have other distinct phenotypes under the same solvent conditions. Since some organic solvents, such as dimethyl sulfoxide, are particularly effective in destabilizing nucleic acid duplexes, it is possible that some of the correspond- ing solvent-hypersensttive mutants will map in regions of physiologically relevant DNA or RNA secondary struc- tures. Thus, just as the ds technique may tap genes and specific alleles that are beyond the reach of the ts and cs approaches, specific solvent-hypersensitive mutants may eventually define yet other genes and alleles that are missed by existing techniques, including the ds method.

Experimental Procedures

Yeast Strains and Media The genotypes and sources of S cerevwae strains used in this work are as follows: DBY1500, MATa hfs4-539 ad&107 /ys2-807 trpl-J 7 leu2-3,772 (D. Botstem); DBY1705, MATu /eu2-3.772 ura3 /ys2-807 ga/2 (D Botsteln); A364A. MATa adel ade2 ural h/s7 lys2 tyrl gall (Yeast GenetIc Stock Center, Umverslty of Callfornla. Berkeley); cdcl-cdc21. essentially lsogenic to A364A (Yeast Genetic Stock Center); X1049-9C. MATo asp5 trpl ura3 hfs3 arg8 (Yeast Genetlc Stock Center); dsl-ds47. obtalned by EMS mutagenesis of DBY1705 (this study); and dsla- dsld. haplold segregants of a single melotlc tetrad from a dsl x DBY1500 cross (this study, see Flgure IB)

H20-contalnlng culture media were prepared essentially as de- scribed by Sherman et al (1981) YPD contalned 1% Bacto-Yeast Ex- tract. 2% Bacto-Peptone, and 2% glucose YPD supplemented with 40 llgirnl tryptophan was used for spore germmatlon. SD-HP0 contamed 067% Difco yeast nitrogen base without ammo acids, 2% glucose, and

nutrients essential for auxotrophlc strains at concentrattons specified by Sherman et al. (1981). SD-D20 was ldentlcal to SD-H20 except for the substltutlon of D20 (99.8% Aldrlch Chemical Company) for Hz0 to fmal D20 concentrations mdlcated I” the text, usually 90% (vollvol) SD-H10 and SD-D20 plates contained the lndlcated medium. pre- pared wth 2% Bacto-Agar To conserve D20, which is costly, -15 ml of the medium was used per 10 cm plate. To prevent the dllutlon of D20 or- D20-containing plates through the exchange with atmospheric H20. plates were kept m sealed polyethylene bags both in storage and in the course of experiments Deuterated L-aspartic acid was from MSD Isotopes (Polnte Claire-Dorval, Quebec)

Mutagenesis Mutagenesis with EMS was cawed out essentially as described by Sherman et al (1981) One mllllllter of a culture of DBY1705 grown II- YPD to an A,,,, of approximately 5 was harvested by centrifugation ans resuspended in 1 ml of 0.1 M potassium phosphate buffer (pH 7.0). Thirty mIcrolIters of EMS (Sigma) was added, and the susperwon was Incubated at 30% for 45 min. with vortexlng every 5-10 mln The cells were washed extenwely with sterile water, resuspended in 1 ml of YPD, and Incubated on a roller drum for 3 hr at 3O”C, followed by dllu- tlon and plating on SD-H1O. This protocol resulted in approximately 30% viable cells relative to an unmutagenized control

Isolation of ds Mutants lndlvldual colomes from a mutagenized culture were resuspended III sterile water contained in wells of a 96.well mIcrotIter plate, and trans- ferred by stamping with a 48.pm Inoculator (Dan-Kar Corp Readmg, Mass.) onto three SD-H20 plates (Incubated at 16% 30°C, and 36% respectively) and one SD-90% D20 plate (incubated at 30%). After either 3 days (for SD-H20 plates at 30°C and 36%) or 6 days (for SD-H20 plates at 16% and for SD-90% D20 plates), the plates were compared for dlfferentlal growth patterns of Individual colonies (see Ta- ble 1) Colonies (ds mutants) that grew on SD-HP0 but failed to grow on SD-90% D,O were picked, purlfled by streaking on SD-HPO, and retested as above for their ds, ts, and cs phenotypes. All of the ds mu- tants retested in this manner had clear growth defects on SD-90% D,O when compared with the nonmutagemzed parental strain.

Characterization of ds Mutants Mutant strains dsl-ds47 were mated to the wld-type stram DBY1500. The phenotypes of the resultant diploids were assayed as above to check for dominance of the ds alleles (see legend to Table 3). Several of the recesswe dlplold strains were Induced to sporulate, and ascl were subjected to tetrad dissection as described by Sherman et al. (1981) The phenotypes of the haplold segregants in each tetrad were assayed, and the ds phenotype was found to segregate as a Mendellan trait (2:2) in all cases tested. Mutant (ds) segregants with a mating type opposite to that of the orlginal mutagenwed strain were used for com- plementatlon analysis (see Table 3).

To test for conditional auxotrophs among the ds strains, cells from mdwdual ds colomes were stamped as descrtbed above onto SD- 90% D20 and SD-Hz0 plates supplemented with various overlapping pools of L-ammo acids as described by Sherman et al (1981). Those strams that showed complementation of their ds and/or ts defect by two pools were retested on plates supplemented with the implicated amino acid alone.

SD-Hz0 and SD-SO% D20 plates supplemented with 1.2 M KCI or 1 M sorbltol were used in tests of osmotic sensltlwty

Acknowledgments

We thank Daniel Fmley and John McGrath for comments on the manu- script, and Barbara Doran for secretarial assistance One of us (A. V.) IS grateful to Alexandra Hawtger for helpful dIscussIons at the begin- ning of this work. This work was supported by a grant to A. V. from the National Cancer lnstltute (CA43309). B. B. was supported by a predoc- toral fellowship from the National Science Foundation.

The costs of publlcatlon of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘adverbsement” in accordance wth 18 U.S.C. Section 1734 solely to Indicate this fact

Received January 21, 1988; rewed February 24. 1988

D,O-Sensttrve Mutants 941

References Tanford, C (1968) Protean denaturatlon Adv Protean Chem 23, 121-282.

Alber. T, Dao-pin, S Nye, J A., Muchmore, D. C and Mathews, B W Thomson, J F. (1960) Physlologlcal effects of D20 tn mammals Ann (1987). Temperature-sensittve mutations of bacteriophage T4 lysozyme N.Y. Acad Sci. 84. 736-744 occur at sites with low mobility and low solvent accesstbtltty II- the folded protean. Btochemtstry 26, 3754-3758.

Thomson. J. F (1963). Brologrcal Effects of Deuterrum (New York. Per- gamon Press)

Amarose, A P, and Cza)ka, D M. (1962) Cytopathic effects of deu- tertum oxide on the male gonads of the mouse and dog Exp. Cell Res. 26, 43-61

Botsteln, D.. and Maurer, R (1982). Genetic approaches to the analysrs of mtcrobial development. Annu. Rev. Genet. 76, 61-83.

Czajka, D. M., and Finkel, A. J. (1960). Effect of deutertum oxtde on the reproducttve potentlal of mace Ann. N. Y, Acad. Sci. 84, 770-779

de Robichon-Szulmatster, H., Surdtn, Y., and Morttmer, R. K. (1966) Genettc and btochemical studtes of genes controlling the synthesis of threonme and methtontne tn Saccharomyces. Genetics 53, 609-619.

Gordon, J. A., and Jencks. W. P (1963). The relattonshtp of structure to effectrveness of denaturtng agents for proteins, Biochemtstry 2, 47-57.

Grechko. V,, Maslova, R., and Varshavsky, Y M. (1963). Influence of heavy water on the properttes of nucletc actds and proteins. Proc. Acad. SCI USSR 152, 740-744

Hartwell, L. H.. Mortimer, Ft. K, Culotti. J., and Culottt, M (1973) Genetic control of the cell dtvtston cycle in yeast: V. Genetic analysis of cdc mutants. Genetics 74, 267-286.

Horowitz, N. H (1948). The one gene-one enzyme hypotheses Genetccs 33, 612-613.

Horowttz, N. H (1950). Btochemtcal genetics of Neurospora Adv. Ge- net. 3, 33-68.

Horowrtr, N. H., and Leupold, U. (1951). Some recent studtes bearrng on the one gene-one enzyme hypotheses. Cold Spring Harbor Symp. Quant. Btol. 16, 65-74.

Jarvik. J., and Botstetn. D. (1975). CondItional-lethal mutattons that suppress genetic defects tn morphogenesis by altering structural pro- terns. Proc. Natl Acad. SCI. USA 72, 2738-2742.

Jones, E W.. and Fink, G R. (1982) Regulatton of amino acid and nucleottde biosynthests rn yeast. In The Molecular Biology of the Yeast Saccharomyces: Metaboltsm and Gene Expresston, J. N. Strathern, E. W. Jones, and J. R Broach, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 181-300

Kaback, D. B., Oeller, I? W., Steensma, H. Y., Hirschman, J., Rueztnsky, D., Coleman, K. G., and Prtngle, J. R (1984). Temperature- senstttve lethal mutations on yeast chromosome I appear to define only a small number of genes Genettcs 708, 67-90.

Katz., J. J., Crespt, H. L , Czajka, D M., and Ftnkel, A J (1962) Course of deuteriation and some phystologtcal effects of deuterium tn mace. Am. J Phystol. 203, 907-913

Lemm, U., and Wenzel, M. (1981). Stabtltzatton of enzymes and an- tisera by heavy water Eur. J Biochem. 176, 441-446.

Motr, D., and Botstetn. D. (1982). Determtnatton of the order of gene function tn the yeast nuclear dtvtsron pathway using cs and IS mutants. Genettcs 700, 565-577.

Nakagawa, Y,, Ghotb-Shartf, J , and Douglas, K T. (1982). Carboxy- pephdase Y from Saccharomyces cerevrsiae. conforma:tonal drffer- ences reflected in ktnetic behavtor in water and deutertum oxtde. BIO- chum. Biophys Acta 706, 141-143.

Pringle, J R. (1975). Inductron, selectton and expertmental uses of temperature-sensttive and other condtttonal mutants of yeast. In Methods tn Cell Btology, Vol 12. D M Prescott, ed. (New York. Aca- demtc Press), pp. 233-272.

Scheraga. H. A (1960). Heltxxandom co11 transformations tn deuter- ated macromolecules. Ann. N. Y. Acad SCI. 84, 608616

Sherman, F., Funk, G R., and Hicks, J. B. (1981). Methods tn Yeast Genettcs (Cold Spring Harbor, New York, Cold Sprmg Harbor Labo- ratory).

Shortle, D. (1983). A genetrc system for analysts of staphylococcal nuclease. Gene 22, 181-189.

Smfth. M. (1985). In vitro mutagenests Annu. Rev Genet. 79, 423-462.