Part D:Mutation Experiments

Hunting for Red Mutants

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

Figure

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.

Diluting Cells:


1. Pipette 0.9 ml of sterile water into each of 9 sterile test tubes. Clearly label and arrange these tubes in a rack.
2. With a sterile toothpick remove a small sample of yeast from one of the subculture plates streaked in step 1 and suspend enough cells in the first tube to make the liquid just barely cloudy. A just cloudy (turbid) suspension has approximately one million (106) cells/ml.
3. Resuspend the yeast by gently tapping the end of the tube containing 106 cells/ml. Pipette 0.1 ml (100 l) of the yeast solution from the 106 cells/ml tube into the next tube (Figure 2).
4. From the 105 cells/ml dilution, pipette 0.1 ml into each of 6 different 104 cells/ml tubes.
5. From one of the 104 cells/ml tubes pipette 0.1 ml (100 l) into a separate 103 cells/ml tube to serve as a control and to determine the actual number of yeast cells in the dilution. (See step 11.)
6. Distribute a 104 cells/ml dilution tube to each student lab team.

Teacher Tips

CAUTION: The UV light used in this box is hazardous--particularly to your eyes. Do not circumvent the safety features of the box.

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)

Collecting Data and Isolating Mutants:

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

Figure 1

Figure 2

Figure 3

Figure 4

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.
2. Incubate the plates for 1-2 days.

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.
4. Incubate the plates for 1-2 days. ( Teacher Tips)

Test for adenine requirement: 1. Make a replica of these plates on MV and on MV plus adenine.
2. Incubate all the plates for 1-3 days.

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 -.
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.

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Last updated Friday July 11 1997