Part C: UV Experiments

Ultraviolet Induction of Mitotic Segregation

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:

Getting Ready:

Time Line:Day before: 10 min

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.
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.)

Teacher tip

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.
Irradiate the plates with the covers removed.
Incubate some control plates that are not irradiated.

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.

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Last updated Wednesday, 04-Dec-2002 21:00:06 UTC