Mutants are useful tools for geneticists. By studying the characteristics of mutant genes, it is possible to get information about the corresponding normal, or wild type, genes. A mutant hunt is a very exciting event in a research laboratory since there is always the chance that a completely new mutant may be discovered.
Normal yeast is cream-colored and will grow on minimal medium (MV). The mutants that you will be hunting for are red and are not able to grow on MV.
It is possible to search for mutants that
arise spontaneously, but it is more efficient
to treat the cells with something that induces
mutation by damaging their DNA.
Ultraviolet (UV) radiation, damages cells in
a number of ways. Some are killed but the
survivors, those that can still reproduce to
form a colony, often have new errors or
mutations in their DNA.
As the UV exposure goes up, so does the
number of mutations. This is what you
want. However; as the number of mutations
increases, the survival rate decreases, therefore fewer cells are able to reproduce
and form colonies. Usually there is an
optimal exposure that produces many
mutations but doesn't kill off too many cells.
You canproduce this optimal exposure rate
by using UV-C irradiator containing a
germicidal lamp to expose HA0 and HB0 to
a dose that results in about 20 percent
survivors. For each plate containing 1000
cells before irradiation, about 200 survive to
form yeast colonies after incubation. Each
of the surviving colonies is a potential new
mutant strain. You need about 10,000
surviving yeast cells of each mating type to
be sure of finding several pink mutants. The
more looking you do, the more likely you
are to find a mutant.
Experiment:
Use a serial dilution technique (see
Laboratory Methods: Serial dilution) to
plate approximately one thousand (1 103)
cells per plate of normal cream-colored
strains. Expose the cells to UV-C radiation.
When the cells grow to form colonies, some
will have the mutant red or pink color.
Isolate these mutants and study their new
phenotypes.
Time Line:
1st Day: 5 min Getting ready
2nd Day: 50 min Diluting, Plating and Irradiating Cells
5th Day: 50 min Collecting
Data and Isolating Mutants
7th Day: time will Characterizing vary your mutants
Inducing mutations:
Getting ready:
1. Use a sterile toothpick to make 4 or 5
streaks of HA0 and/or HB0 onto
separate YED plates. (Figure 1)
2. Incubate plates at 30øC overnight.
Plating and Irradiating Cells:
1. Resuspend the cells in the 104 cells/ml
dilution and pipet 0.1 ml (100 l) of
onto a fresh YED plate.
Use a sterile spreader to distribute the cells evenly
over the surface of the agar. (see Yeast Culture:
Spreading cells).
Use the same techniques to make as many plates
as you plan to irradiate.
Avoid spreading cells too near the edge of the
plate.
2. Place your plates (up to 2 plates at a
time) in the irradiation box and remove
the lids before exposing the cells to UV.
The plastic in the petri dish lid will act as a filter
and block the UV from the yeast on the plate
surface.
If that happens there will be no mutants.
Exposure of 20 seconds should result in about
20% survival. Since you had about 1000 cells on
the plate before the UV exposure, you ahould now
have about 200 that have survived and will
produce colonies.
3. Don't forget the controls.
Label three YED plates on the bottom: Control,
Your Name, 102 cells, no UV and the date. From
the 103 cells/ml dilution tube, pipette 0.1 ml (100
l) onto each of the 3 YED control plates. This
should result in about 100 cells per plate.
Spread the cells over the surface of the agar with a
sterile spreader.
4. Incubate the plates overnight at 30øC.
( Teacher
Tips)
1. When surviving cells have grown to
visible colonies, examine your plates
for possible red mutant colonies. If
you find one, circle it and tell the
teacher.
2. Count the colonies on each plate.
Use a felt tip pen to mark on the bottom of each
colony as you count them.
3. Compile your data and calculate the
percent survival as illustrated for the
sample data shown below
4. Collect all of the red mutants.
Carefully pick a small sample from each suspected
mutant colony using the pointed end of a
toothpick.
Collect several samples on a single plate for
further testing. If, when they grow up they appear
to be contaminated with white cells, purify them
by streaking for single cells (See Yeast Culture,
Streaking for Single Cells and video segment)
5. Incubate the plates for 1 to 2 days.
( Teacher
Tips)
Sample Data:
Plate 1 Survival | Plate 2 Survival | Plate 3 Survival | Total Survival | Average Survival | Colonies Plated (10 X control ) | Percent Survival | Red Mutants |
---|---|---|---|---|---|---|---|
265 | 302 | 247 | 814 | 271.3 | 1240 | 0.22 | 2 |
Characterizing your mutants:
The red mutants ade1 or ade2 result from
mutations in different genes, but both are
unable to grow if the chemical adenine is
missing from the medium. What about your
red mutants? Do they also require adenine
for growth? Have they mutated in the same
genes as the known ade1 or ade2?
You can answer both of these questions at
the same time.
1. Streak your mutants and the known
strains on a YED plate as shown in the
top diagram at the left.
Complementation Test:
In a complementation test you cross
your unknown mutant with a strain that has
a mutation in the ade1 gene and with a strain
that has a mutation in the ade2 gene. When
the unknown mutant is crossed to a mutation
in the same gene, the mating mixture will
still be red and require adenine. When it is
crossed to a strain with a mutation in a
different gene, the mating mixture will
contain cream colored yeast that does not
require adenine.
3. Make mating mixtures on the test plate
that you set up yesterday. The sequence
of steps is illustrated in the last three
diagrams.
Test for adenine requirement:
1. Make a replica of these plates on MV
and on MV plus adenine.
A Final Look:
1. Examine the MV and the MV plus
adenine replica plates. Sketch the
results. Do your red mutants require
adenine for growth? Mark the ones that
grow with a + in you sketch and the
ones that don't grow with a -.
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2. Incubate the plates for 1-2 days.
4. Incubate the plates for 1-2 days.
( Teacher
Tips)
2. Incubate all the plates for 1-3 days.
2. Examine your complementation plate
results. You have actually done two
tests for complementation. On the YED
plates you will see whether your
mutants complement the known ade1
or ade2 mutants for their color, and on
the MV plates you will see whether they
complement for adenine requirement.
After analyzing these results, go back
and label all red mutants as ade1 or
ade2 or unknown.
Last updated Wednesday, 04-Dec-2002 14:56:44 CST