See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/5426615

EMS and UV mutagenesis in yeast

Article in Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] · May 2008
DOI: 10.1002/0471142727.mb1303bs82 · Source: PubMed

CITATIONS READS
29 4,854

1 author:

Fred Winston
Harvard Medical School
178 PUBLICATIONS 20,655 CITATIONS

SEE PROFILE

All content following this page was uploaded by Fred Winston on 22 March 2018.

The user has requested enhancement of the downloaded file.

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.
Biol. 82:13.3B.1-13.3B.5.

C 2008 by John Wiley & Sons, Inc.

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) BASIC

PROTOCOL
Materials
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 13.3B.1

Published online April 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142727.mb1303bs82 Supplement 82
Copyright
C 2008 John Wiley & Sons, Inc.
 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 inactiva-
tion of EMS. If cells are to be stored before plating, pellet in a tabletop cen-
trifuge 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 concen-
tration 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).
For identification of other types of mutants, plates can be incubated at other temper-
atures. 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,
EMS and UV
Mutagenesis in depending upon the frequency of the desired mutant class. Up to 20,000 colonies (l00
Yeast plates, each with 200 colonies) can be screened if necessary.

13.3B.2
Supplement 82 Current Protocols in Molecular Biology
 11. Choose at least ten YPD plates that contain 100 to 200 colonies per plate. Replica-
plate (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 temperature-
sensitive, place into permanent storage (UNIT 13.1).

MUTAGENESIS USING UV IRRADIATION ALTERNATE

PROTOCOL
Additional Materials (also see Basic 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

13.3B.3
Current Protocols in Molecular Biology Supplement 82
 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 auxotrophic mutation present in the parental
Mutagenesis of yeast cells is an important strain.
genetic technique to increase the frequency
and spectrum of mutants obtained. Even when Critical Parameters
mutants arise spontaneously through genetic The degree of mutagenesis is a critical fac-
selection, treatment with a chemical mutagen tor. Too little mutagenesis will not sufficiently
or irradiation can alter the types of mutants increase the frequency of mutants in a popula-
obtained. EMS is known to cause primarily tion of cells; too much mutagenesis will result
GC to AT transversions. UV light has been re- in multiple mutations. Since some mutagens,
ported to cause a wide spectrum of mutational such as nitrosoguanidine, create multiple mu-
changes, including transitions and transver- tations in a nonrandom fashion (Botstein and
sions (Prakash and Sherman, 1973; Coulondre Jones, 1969), double mutants may not be eas-
and Miller, 1977). ily discovered by standard genetic analysis.
Mutagens will generally alter only one Following isolation, any mutants of inter-
DNA strand at any mutagenized site. This est should be subjected to basic genetic anal-
should create a double-stranded molecule that ysis (Rose et al., 1990). The first step in this
is heterozygous, possibly resulting in a yeast analysis should be a genetic cross (UNIT 13.2)
colony of mixed genotype. In practice, this to demonstrate that the mutant phenotype is
is rarely observed. Evidence exists that mis- caused by a mutation in a single gene (i.e.,
match repair may play a role in the degree and that the mutant phenotype segregates 2:2 in
spectrum of mutagenesis (Muster-Nassal and tetrads). If a large number of mutants are iso-
Kolodner, 1986; Siede and Eckardt-Schupp, lated, the number of genes identified can be
1986). determined by complementation and linkage
The degree of mutagenesis can be moni- analysis (Rose et al., 1990).
tored by measuring any easily detectable mu-
tational event. Instead of using canavanine re- Anticipated Results
EMS and UV
Mutagenesis in sistance as in the Basic Protocol, one could, Under optimal conditions of mutagenesis
Yeast for example, measure α-aminoadipate utiliza- as described in this unit, the frequency of
tion (UNIT 13.1) or the reversion of a revertable canavanine-resistant mutants should be at least
13.3B.4
Supplement 82 Current Protocols in Molecular Biology
 l00-fold greater after mutagenesis. This result Literature Cited
is important in order to know that the mu- Botstein, D. and Jones, E.W. 1969. Nonrandom
tagenesis has worked properly, since the fre- mutagenesis of the Escherichia coli genome
by nitrosoguanidine. J. Bacteriol. 98:847-
quency of obtaining the desired class of mu- 848.
tant is likely to be unknown. The frequency
Coulondre, C. and Miller, J.H. 1977. Genetic stud-
of finding a particular class of mutant will de- ies of the lac repressor. IV. Mutagenic specificity
pend entirely upon the nature of the mutation in the lacI gene of Escherichia coli. J. Mol. Biol.
sought. For example, random temperature- 117:577-606.
sensitive mutants will be found at a higher fre- Muster-Nassal, C. and Kolodner, R. 1986. Mis-
quency than temperature-sensitive mutations match correction catalyzed by cell-free extracts
that cause cell-cycle arrest in G2. of Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. U.S.A. 83:7618-7622.
Time Considerations Prakash, L. and Sherman, F. 1973. Mutagenic speci-
Mutagenizing cells with EMS takes ∼2 hr. ficity: Reversion of iso-1-cytochrome c mutants
Plating the cells requires ∼30 min, with colony of yeast. J. Mol. Biol. 79:65-82.
formation in 3 to 4 days. Mutagenizing cells Rose, M.D., Winston, F., and Hieter, P. 1990.
by UV irradiation requires ∼1 hr to prepare Laboratory Course Manual for Methods in Yeast
Genetics. Cold Spring Harbor Laboratory, Cold
serial dilutions and plate cells. Once the plates Spring Harbor, New York.
have been irradiated, colony formation takes 2
Siede, W. and Eckardt-Schupp, F. 1986. A mis-
to 4 days. For both methods, identification of match repair-based model can explain some fea-
temperature-sensitive mutants will require an tures of UV mutagenesis in yeast. Mutagenesis
additional 4 to 8 days. 1:471-474.

Yeast

13.3B.5
Current Protocols in Molecular Biology Supplement 82

View publication stats