EMS and UV Mutagenesis in Yeast
UNIT 13.3B
1
Fred Winston
1
Harvard Medical School, Boston, Massachusetts
ABSTRACT
Many fundamental biological processes have been elucidated by the isolation and analysis
of mutants that are defective in such processes. Therefore, the methods to generate
mutants are of great importance in model organisms. This unit describes two protocols
for mutagenesis of yeast—using ethyl methanesulfate (EMS) and ultraviolet (UV) light.
Each of these methods has been used successfully for many years. Curr. Protoc. Mol.
C 2008 by John Wiley & Sons, Inc.
Biol. 82:13.3B.1-13.3B.5. Keywords: yeast r mutagenesis r EMS r UV
INTRODUCTION
Because spontaneous mutations occur at a low rate, yeast cells are often treated with
mutagens to increase the frequency of mutants. Two common mutagens of yeast cells are
ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Mutagenesis can increase the
frequency of mutation up to 100-fold per gene without excessive killing of the cells and
without a significant frequency of double mutants. EMS and UV may produce different
spectra of mutants, but generally only one type of mutagenesis is necessary to generate
a sufficient number of mutants to study.
This unit presents protocols for both EMS and UV mutagenesis of yeast cells. Cultures of
the desired yeast strain are treated with the mutagen, and the effectiveness of the mutagen
is measured by determining the frequency of an event for which there exists a genetic
selection (e.g., canavanine resistance). Mutagenized cells can then be screened for any
phenotype of interest, including auxotrophies, cold sensitivity, and radiation sensitivity.
As an example, the Basic Protocol includes steps for screening for mutants that are
temperature-sensitive for growth. Once a gene of interest has been identified by such
procedures, it can be cloned, mutagenized, and manipulated in other ways to study its
function in greater detail (UNITS 13.8 & 13.10).
NOTE: All solutions, plasticware, glassware, and velveteens coming into contact with
yeast cells must be sterile.
MUTAGENESIS USING ETHYL METHANESULFONATE (EMS)
Materials
BASIC
PROTOCOL
Desired yeast strain
YPD medium and plates (UNIT 13.1)
Sterile water
0.1 M sodium phosphate buffer, pH 7.0 (see recipe)
Ethyl methanesulfonate (EMS; Kodak)
5% (w/v) sodium thiosulfate (Sigma), autoclaved for sterility
13 × 100–mm culture tube
Vortex
30◦ C incubator with rotating platform
Canavanine plates (UNIT 13.1)
Yeast
Current Protocols in Molecular Biology 13.3B.1-13.3B.5, April 2008
Published online April 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142727.mb1303bs82
C 2008 John Wiley & Sons, Inc.
Copyright 13.3B.1
Supplement 82
Additional reagents and equipment for growing cells, determining cell density, and
replica plating (UNIT 13.2) and for storage of strains (UNIT 13.1)
CAUTION: EMS is a dangerous mutagen. All solutions, plasticware, and glassware that
come into contact with EMS should be rinsed with 5% sodium thiosulfate to inactivate
the EMS.
Grow and mutagenize cells
1. Grow an overnight culture of the desired yeast strain (UNIT 13.1) in 5 ml YPD medium
at 30◦ C.
2. Determine the density of cells (UNIT 13.2) in the culture and record this number.
Adjust concentration to ∼2 × 108 cells/ml if necessary. Transfer 1 ml of the culture
to a sterile microcentrifuge tube.
3. Pellet cells in a microcentrifuge ∼5 to 10 sec at maximum speed, room temperature.
Discard supernatant and resuspend in 1 ml sterile water. Repeat wash. After the
second wash, resuspend cells in 1.5 ml sterile 0.1 M sodium phosphate buffer,
pH 7.0.
4. Add 0.7 ml cell suspension to 1 ml buffer in a 13 × 100–mm culture tube. Save
remaining cells on ice for a control.
5. Add 50 µl EMS to the cells and disperse by vortexing. Place on a rotating platform
and incubate 1 hr at 30◦ C.
EMS treatment should cause ∼40% of the cells to be killed.
6. Transfer 0.2 ml of the treated cell suspension to a culture tube containing 8
ml sterile 5% sodium thiosulfate, which will stop the mutagenesis by inactivation of EMS. If cells are to be stored before plating, pellet in a tabletop centrifuge 5 min at 3000×g, 4◦ C), resuspend in an equal volume of sterile water,
and store at 4◦ C.
Determine effectiveness of mutagenesis
7. Plate 0.1 ml mutagenized cells directly on each of two canavanine plates. As
controls, plate 0.1 ml nonmutagenized cells (from step 4) on duplicate canavanine
plates. Incubate at 30◦ C until colonies form (∼2 to 4 days).
8. Calculate the relative levels of canavanine-resistant mutants in the mutagenized and
nonmutagenized cultures.
The percentage survival can be calculated by comparing the number of cells in the initial
culture (step 2) and the number of viable cells following EMS treatment (calculated from
the number of colonies obtained in step 7).
Identify temperature-sensitive mutants
9. Dilute the EMS-treated cells from step 6 with sterile water to obtain 100 to 200
viable cells per plate.
A dilution factor of 1:1000 may be sufficient, but this will depend on the initial concentration of cells used.
10. Plate 0.1 and 0.2 ml of the diluted cells on separate sets of YPD plates, using ten
plates in each set. Incubate all plates 3 to 4 days at room temperature (23◦ C).
EMS and UV
Mutagenesis in
Yeast
For identification of other types of mutants, plates can be incubated at other temperatures. For example, if screening for auxotrophs, incubate plates at 30◦ C, the optimal
temperature for growth of yeast cells. In addition, the number of plates used may vary,
depending upon the frequency of the desired mutant class. Up to 20,000 colonies (l00
plates, each with 200 colonies) can be screened if necessary.
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11. Choose at least ten YPD plates that contain 100 to 200 colonies per plate. Replicaplate (UNIT 13.2) each to two fresh YPD plates. For temperature-sensitive mutants,
incubate one YPD plate per set at 37◦ C and one at room temperature (23◦ C),
overnight.
The 23◦ C plate serves as a positive control for growth, as some temperature-sensitive
mutants may grow poorly at 30◦ C.
Be certain that each plate is numbered and has an orientation symbol on the back. In
general, one of the two replica plates should be permissive for mutant growth (typically
a YPD plate). The other plate should represent conditions that do not permit growth of
the desired class of mutants.
12. Compare the 37◦ C plate with the 23◦ C and 30◦ C plates. Any colonies that failed
to grow at 37◦ C are candidates for temperature-sensitive mutants. To recheck, pick
corresponding colonies from YPD plates incubated at 23◦ C and streak for single
colonies on fresh YPD plates. On each plate, also streak the parental control strain
for single colonies. Incubate the plates at 23◦ C until colonies form (2 to 4 days).
Six to eight strains can be purified on each YPD plate.
13. After single colonies form, replica-plate (UNIT 13.2) the YPD plates with the
temperature-sensitive candidates to two YPD plates as before. Incubate one plate
at 37◦ C and the other at room temperature for 1 day.
14. Record the growth response. For each candidate that is reproducibly temperaturesensitive, place into permanent storage (UNIT 13.1).
MUTAGENESIS USING UV IRRADIATION
Additional Materials (also see Basic Protocol)
ALTERNATE
PROTOCOL
UV germicidal light bulb (Sylvania G15T8; 254 nm wavelength) or Stratagene UV
Crosslinker
UV dosimeter (optional)
23◦ C incubator
CAUTION: Wear safety glasses to protect eyes from UV light.
1. Grow an overnight culture of the desired yeast strain (UNIT 13.2) in 5 ml YPD medium
at 30◦ C.
2. Pellet 1 ml of cells in a microcentrifuge ∼5 to 10 sec at maximum speed, room
temperature and discard supernatant. Resuspend in 1 ml sterile water and repeat
wash. After the second wash, resuspend in 1 ml sterile water.
3. Determine the cell density and record this number. Adjust to 2 × 108 cells/ml if
necessary.
4. Make serial dilutions of the culture in sterile water so that each plate has 200 to
300 viable cells. Plate 0.1 and 0.2 ml on YPD plates as described in step 10 of the
Basic Protocol.
5. Irradiate all but two plates from each set with UV light using a dosage of
300 ergs/mm2 . There should be ∼40% to 70% survival. The nonirradiated plates
will serve as controls to determine the degree of killing by the UV light.
Light from the UV germicidal bulb can be measured using a UV dosimeter. From this
measurement, the proper length of time can be calculated for irradiation to attain 300
ergs/mm2 . Alternatively, the proper time of irradiation can be empirically determined
by measuring the time that results in 40% to 70% survival.
Yeast
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Most UV lights should be warmed up for ∼20 min before use. In addition, petri plate lids
block transmission of UV light; therefore, be sure to remove them prior to irradiation.
Alternatively, a Stratagene UV Crosslinker, commonly used for cross-linking DNA or
RNA to membranes, can be used. As for a germicidal lamp, the proper time should be
determined empirically.
6. As a control to determine the degree of UV mutagenesis, plate 0.1 ml of the
original culture on duplicate canavanine plates and irradiate one plate for the time
determined in step 5. From the number of canavanine-resistant colonies, calculate
the frequency of mutations with and without UV irradiation.
As described in the Basic Protocol, the degree of mutagenesis can be determined by
measuring traits other than canavanine resistance.
7. Incubate plates from step 4 at 23◦ C until colonies form (∼2 to 4 days).
8. Screen colonies for temperature-sensitive mutants as described in steps 9 to 14 of
the Basic Protocol.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.
Sodium phosphate buffer, 0.1 M, pH 7.0
Prepare 1 M solutions of Na2 HPO4 and NaH2 PO4 . Mix 5.77 ml Na2 HPO4 with
4.23 ml NaH2 PO4 and add water to 100 ml (the pH will be 7.0). Autoclave for
sterility. Store up to 1 year at room temperature.
COMMENTARY
Background Information
EMS and UV
Mutagenesis in
Yeast
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Mutagenesis of yeast cells is an important
genetic technique to increase the frequency
and spectrum of mutants obtained. Even when
mutants arise spontaneously through genetic
selection, treatment with a chemical mutagen
or irradiation can alter the types of mutants
obtained. EMS is known to cause primarily
GC to AT transversions. UV light has been reported to cause a wide spectrum of mutational
changes, including transitions and transversions (Prakash and Sherman, 1973; Coulondre
and Miller, 1977).
Mutagens will generally alter only one
DNA strand at any mutagenized site. This
should create a double-stranded molecule that
is heterozygous, possibly resulting in a yeast
colony of mixed genotype. In practice, this
is rarely observed. Evidence exists that mismatch repair may play a role in the degree and
spectrum of mutagenesis (Muster-Nassal and
Kolodner, 1986; Siede and Eckardt-Schupp,
1986).
The degree of mutagenesis can be monitored by measuring any easily detectable mutational event. Instead of using canavanine resistance as in the Basic Protocol, one could,
for example, measure α-aminoadipate utilization (UNIT 13.1) or the reversion of a revertable
auxotrophic mutation present in the parental
strain.
Critical Parameters
The degree of mutagenesis is a critical factor. Too little mutagenesis will not sufficiently
increase the frequency of mutants in a population of cells; too much mutagenesis will result
in multiple mutations. Since some mutagens,
such as nitrosoguanidine, create multiple mutations in a nonrandom fashion (Botstein and
Jones, 1969), double mutants may not be easily discovered by standard genetic analysis.
Following isolation, any mutants of interest should be subjected to basic genetic analysis (Rose et al., 1990). The first step in this
analysis should be a genetic cross (UNIT 13.2)
to demonstrate that the mutant phenotype is
caused by a mutation in a single gene (i.e.,
that the mutant phenotype segregates 2:2 in
tetrads). If a large number of mutants are isolated, the number of genes identified can be
determined by complementation and linkage
analysis (Rose et al., 1990).
Anticipated Results
Under optimal conditions of mutagenesis
as described in this unit, the frequency of
canavanine-resistant mutants should be at least
Current Protocols in Molecular Biology
l00-fold greater after mutagenesis. This result
is important in order to know that the mutagenesis has worked properly, since the frequency of obtaining the desired class of mutant is likely to be unknown. The frequency
of finding a particular class of mutant will depend entirely upon the nature of the mutation
sought. For example, random temperaturesensitive mutants will be found at a higher frequency than temperature-sensitive mutations
that cause cell-cycle arrest in G2.
Time Considerations
Mutagenizing cells with EMS takes ∼2 hr.
Plating the cells requires ∼30 min, with colony
formation in 3 to 4 days. Mutagenizing cells
by UV irradiation requires ∼1 hr to prepare
serial dilutions and plate cells. Once the plates
have been irradiated, colony formation takes 2
to 4 days. For both methods, identification of
temperature-sensitive mutants will require an
additional 4 to 8 days.
Literature Cited
Botstein, D. and Jones, E.W. 1969. Nonrandom
mutagenesis of the Escherichia coli genome
by nitrosoguanidine. J. Bacteriol. 98:847848.
Coulondre, C. and Miller, J.H. 1977. Genetic studies of the lac repressor. IV. Mutagenic specificity
in the lacI gene of Escherichia coli. J. Mol. Biol.
117:577-606.
Muster-Nassal, C. and Kolodner, R. 1986. Mismatch correction catalyzed by cell-free extracts
of Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. U.S.A. 83:7618-7622.
Prakash, L. and Sherman, F. 1973. Mutagenic specificity: Reversion of iso-1-cytochrome c mutants
of yeast. J. Mol. Biol. 79:65-82.
Rose, M.D., Winston, F., and Hieter, P. 1990.
Laboratory Course Manual for Methods in Yeast
Genetics. Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
Siede, W. and Eckardt-Schupp, F. 1986. A mismatch repair-based model can explain some features of UV mutagenesis in yeast. Mutagenesis
1:471-474.
Yeast
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