Mitosis is usually thought of as the process in which the chromosomes replicate and divide to produce two identical nuclei. These nuclei are then packaged in two identical cells. Meiosis is thought of as the process that introduces recombination and variation. This distinction between the two cell division processes is not clear in the real world. We sometimes say that a single yeast cell can give rise to a colony of cells that are all clones of each other. Is that always true? Can mitosis produce cells that are not exactly alike? Yes it can.
Spontaneous mitotic recombination, like mutation, occurs with a small constant probability at the time of cell division. Mitotic recombinants, therefore, will occur in clones during the development of a colony. This can be demonstrated using diploid yeast cells that are heterozygous for one of the red adenine mutations. If the cells are irradiated with UV, the small probability of spontaneous mitotic recombination can be increased toas high as one to two percent. This is detected by the appearance of red sectored colonies or occasionally of entirely red colonies. The occurrence of red sectors or completely red colonies can be understood as the result of either of two processes: mitotic crossing over or mitotic gene conversion which are described in the Background section on Genetics of Bakers Yeast. In addition to the red sectored colonies, one will observe small, truly white "petite" colonies. These petite colonies are usually not the result of mitotic recombination but rather result from UV damage that leads to the loss of respiratory metabolism in the mitochondria.
Experiment:
The procedure this experiment is
identical to the Ultraviolet lethality and
Mutations in Yeast experiment, except that
you will plate and irradiate a diploid strain
of yeast that is heterozygous for an adenine
mutation (ade2/ADE2). Then you will
examine the surviving colonies for red
sectors.
As an extension of the experiment you
may wish to test the genotype of the white
portions of the sectored colonies.
Time Line:
Day before:10 min Getting Ready
45 min Discussion of the
strategy and
objectives
Day 1: 50 min Dilution, Plating and
Irradiation of Cells
Day 3 or 4: 50 min Counting Colonies
and Analyzing
Results
Day 3 or 4: 30 min (optional) Plate and Irradiate White sectors Day 7: 30 min Record and Analyze Results of optional experiment Materials:
For each student or team:
Common Materials:
Optional Materials:
1. Make a clean sterile work space by
wiping the table or bench with an
alcohol wipe. Because most
contamination is airborne select a
place free from drafts.
Technical Tip:
White sectors that are homozygous
ADE2/ADE2 demonstrate that the red
sector was the result of a reciprocal
recombination event. The homozygosity
of the white cells can be confirmed by
sporulating them and demonstrating the
absence of red, adenine-requiring
spores. There are several explanations
that could account for the white sectors
containing heterozygous cells. The
colony could contain a mixture of
homozygous and heterozygous white
cells if the segregation did not occur at
the first division or if the colony formed
from two cells (a mother-daughter pair
still attached). Another type of genetic
event, closely related to reciprocal
recombination, but nonreciprocal, is
called gene conversion, and yields one
homozygous and one heterozygous
daughter cell.
Optional Analysis:
If a red sector (ade2/ade2) is the
result of reciprocal crossing over at
mitosis, then the white portion of the
colony should contain cells that are
homozygous ADE2/ADE2, in contrast to
the starting strain that was ade2/ADE2.
These white homozygous segregants
can be detected by their inability to
segregate red sectors. There is a
problem in this, however. One must test
a large sample of irradiated cells without
observing any red sectors to establish
the negative result as being statistically
significant
Procedure:
Time Line: Day 1
1. Streak out the overnight culture on
YED medium (or use the dilution-plating method) to obtain single
colonies.
Refer to the Laboratory Methods section for
different ways of obtaining single colonies.
Since sectored colonies occur at a frequency of
about one percent or less, you will need several
hundred colonies.
2. Irradiate the plates in the UV-C
chamber.
Time Line: Day 3 or 4
3. Examine the plates for the presence of
red sectors, either in the isolated
colonies or in the more dense areas of
growth.
Compare the irradiated plates with the
unirradiated control plates.
Optional:
4.Identify an isolated colony containing a
red sector.
Pick samples of white cells from several places
around the white portion of the colony and streak
out each on YED.
Irradiate these plates, and incubate.
Time Line: Day 7
5. Examine the irradiated plates for red
sectors.
Return to Contents
2. Open the yeast storage vial.
3. Using the broad end of a sterile
toothpick, pick up a small amount of
yeast from the agar slant in the vial.
4. Replace the lid. Tighten. ( Store in a
refrigerator to keep the cells viable for
up to nine months.)
5. Open the YED Petri dish just enough
so that you can reach into it with the
toothpick full of cells.
6. Gently make several streaks of the
culture on the surface of the agar.
(Remember that you need not be able
to see the streaks to have enough to
grow into a visible culture overnight.)
7. Close the lid and incubate the culture
overnight at 30oC, or 2 days at room
temperature. (Most microbial cultures
should be incubated with the agar side
up to prevent condensation from
dropping on the colonies.)
Irradiate the plates with the covers removed.
Incubate some control plates that are not
irradiated.
Last updated Friday July 11 1997