Teacher Tip

This experiment is a mutant hunt. The goal is to obtain a set of mutant strains in each mating-type. You can then extend your work by characterizing the strains further using complementation and mating tests. Most of the "white" mutants will still require adenine and will be double mutants, containing the original "red" adenine mutation plus a mutation earlier in the AMP synthesis pathway. Petite colony mutations are also likely. Occasionally an adenine-independent revertant will occur as the result of another mutation that reversed the effect of the original one. Spontaneous mutations occur with a small, constant probability at each cell division (or more precisely, at each round of DNA replication.) Consequently, the mutants occur in clones, with most of the clones occurring late in the growth of a colony when there are many cells. (See Ultraviolet Lethality and Mutations in Yeast for a way to induce these mutations.)


1st Day: Streak the strains for single colonies on YED plates. (See Laboratory Methods: Isolating Single Colonies) Be sure to have the students label the bottom of each plate with the name of the strain and the date.
It isn't necessary to keep the plates in the incubator for the whole two weeks. They won't dry out as much if you simply leave them on the lab bench out of direct sunlight.

10th Day:Using the pointed end of a toothpick pick white spots from colonies, try to get as few red cells as possible. Use a fresh sterile toothpick for each different sample.
You will need a separate YED plate for each mutant that the students pick. Once again be sure to label each plate with the mating type and date. Ideally the students should pick and streak out at least 10 mutants of each mating type. You will have the best results if you incubate these plates at 30 C for two to three days. If you incubate them at room temperature you will need to extend the incubation time. 12th Day:Pick several white colonies from each sample plate and make short streaks on a YED plate. Use a different sterile toothpick for each colony. Incubate the plate overnight.

15th Day: Replica plate the samples from the YED plate to YED, MV, and PETITE media. If you use toothpicks to replica plate you may omit the replica on YED. If you use replica plating equipment you need to include a replica on YED since your master plate may become contaminated while you are using the replica plating equipment. 16th Day: Record and analyze your results.
Continued "white" growth on YED indicates that the mutation is stable. Sometimes the mutants start to show some red pigment indicating that the mutation is unstable.

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Teacher Tip

Numbers refer to steps in the student procedure.

  • Presumed early AMP pathway mutants collected from HA2
  • Tester strains, HB1 and HB2
    You may wish to subculture these strains overnight on YED. One plate will supply enough cells for a whole class but if several students or groups need access to the strains you may wish to make several plates. To avoid contamination of the master set of strains it's usually best to limit student access. If the master slant vials are kept tightly closed and refrigerated they will remain viable for up to a year.
  • YED plates, MV plates, YEKAC plates
    See Laboratory Methods for media recipes and sterilization instructions
  • Sterile toothpicks
    toothpicks are sterile from the box, see Laboratory Methods:Using Toothpicks and Inoculating Loops

    (You may use mutants collected from HB2 if you use HA1 and HA2 as the tester strains)
    1st Day: Be sure to write some sort of identification code on the bottom of the plates under each haploid strain.

    2nd Day: Stress the use of a fresh sterile toothpick for each different strain and mixture. You could incubate the plates overnight and then streak out each mixture on YED, but that isn't necessary in this case. You can just incubate the mating mixture plate until the cells have had time to turn red.

    4th Day: What does it mean if the cross with HB1 is cream-colored and the same mutant crossed with HB2 is red?

    The cream-colored, adenine-requiring mutant probably had the ade2 allele in addition to an early pathway mutation. The red mating mixture is probably made of diploid cells that are homozygous for ade2/ade2 and heterozygous for ADEX/adeX. This diploid has the phenotype of the original red haploid strain (HA2), red and adenine-requiring.

    If you use replica plating equipment instead of toothpicks you will need to have a sterile velvet for each master plate that you replicate. See Laboratory Methods: Replica Plating

    8th Day: Use a microscope to examine each mating mixture for asci.
    See Laboratory Methods:Microscopic examination of Yeast

    Which mixtures contained diploid cells?
    If all of your cream-colored mutants were in fact mating-type a haploids all of the mating mixtures should contain diploid cells and produce asci.

    Another method to demonstrate that the cells are diploid uses the following steps:
    -- Streak the mixture for single colonies
    -- Pick several of the single red colonies and mix them with ade1 tester strains of both mating types
    Diploid cells will not mate. If neither mating mixture produces cream-colored cells by complementation with the testers the samples were probably diploids.

    Your original hypothesis stated that the early AMP pathway mutants also contained the ade2 allele. Does your data support this hypothesis?

    Remember the production of a red adenine-requiring diploid indicates that the ade2 mutation was present in the beginning cream-colored mutants. The cross with the "other" red adenine mutation (ade1) serves as a control in this experiment. If the white mutant was isolated from the ade2 haploid, then the diploids formed in the control cross would be ade1/ADE1 ADE2/ade2 ADEX/adeX. With all the mutant genes being heterozygous, this diploid should be white and adenine-independent. This experiment proves that the original ade2 mutation is still in the white mutant.

    To demonstrate directly that the white mutant contains a mutation in another gene for adenine biosynthesis requires separating the two mutations by genetic segregation. (See Separating the Early AMP Pathway Mutants from the Red Mutants) Additional evidence can also be obtained by crossing several mutants against each other. (See Testing Early AMP Pathway Mutants for Allelism )

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    Teacher Tip

    1st Day: Be sure to write some sort of identification code on the bottom of the plates under each haploid strain.

    2nd Day: Stress the use of a fresh sterile toothpick for each different strain and mixture.

    You could incubate the plates overnight and then streak out each mixture on YED, but that isn't necessary in this case. Both of the parent mutants are cream-colored in these crosses, so only diploids in which the two early mutations are in different genes will form red cells. You can just incubate the mating mixture plate until the cells have had time to turn red. If you subculture one of the red tester strains on each plate, you will be able to tell when the mating mixtures should have turned red if they are going to produce the red color.

    The number of complementation groups is often indicative of the number of steps in the associated metabolic pathway (in this case AMP synthesis).

    Once the unknowns are placed into complementation groups one strain from each group may be crossed to a known set of tester strains (ade4 through ade8) for another round of complementation testing. The results will determine which specific gene is mutant in each group.

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    Teacher Tip

    1st Day: Be sure to write some sort of identification code on the bottom of the plates under each haploid strain. You may wish to subculture the strains in a grid pattern. See Mutant Hunt and The Two Gene Hypothesis for ideas about grids.

    2nd Day: Stress the use of a fresh sterile toothpick for each different strain and mixture.

    3rd Day: See Laboratory Methods: Isolating Single Colonies.

    8th Day: Use a fresh sterile toothpick for each colony you pick.

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    15th Day: Be sure to label the samples in some way on the bottom of the plate. Note: the red colonies are from spores that are ade2.

    16th Day: See Laboratory Methods: Replica Plating. Be sure to label the samples the same way you did on Day 15.

    17th Day: Note: the samples that grow on MV are ADEX ADE2 or they are unsporulated diploids. The samples that don't grow on MV are adeX ade2 or adeX.

    Which of the four expected spore types have you been able to identify so far?
    ade2 on Day 15
    ADE2 ADEX on Day 17

    adeX ade2 and adeX are still not distinguishable. We need to make a test cross to tell them apart.

    19th Day: ade2 adeX crossed with ade2 will be red
    adeX crossed with ade2 will be cream-colored

    Once the unknowns are isolated as single mutants they may be crossed to a known set of tester strains (ade4 through ade8) for another round of complementation testing. The results will determine which specific gene is mutant in each strain.

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    Teacher Tip

    Numbers refer to items in the student steps.

    Inducing Mutations:
    The procedure, below, outlines one possible strategy for a class of 15 to 25 students to induce mutations in yeast. It can be done easily in a normal 50 minute class and can probably be completed in 45 minutes. In this procedure, the new mutants are crossed against known red mutants to identify the affected gene. The experiment can be expanded by crossing the new mutants among each other. For this purpose, the procedure may be repeated in another class with the other yeast mating type. Also one class can be divided into two groups with one group mutating HA0 mating type and the other group mutating HB0 mating type. Each class or group will need to expose 40-50 plates.

    (Steps 1 through 6 of the procedure are done for the entire class. The teacher may wish to do these steps as a demonstration, otherwise a student will need to be selected to do this part. This procedure ensures that all cells to be mutated are from the same strain and dilution.)

    Getting Ready 1. One subculture plate of each mating type will supply enough yeast for all the students. If contamination is likely or several groups need access to the yeast at the same time, you may want to make several subculture plates. (See Yeast culture "Subculture".)

    2. All incubation times will double if the plates are incubated at room temperature.

    Diluting Cells 1. A 106 cells/ml suspension is just barely turbid. The video segment serial dilution and viable cell counts illustrates this technique. It is important to stress the idea that one cell produces one colony. If an initial single cell is mutated it will pass that mutation to its offspring. That mutation will be carried by all the cells that are in the resulting colony. All the cells in the colony are clones of the original mutant cell.

    2. The sample should be about the size of the period at the end of this sentence. Now carefully transfer the yeast to the first tube by wiping it on the inside of the tube. Suspend the yeast by tipping the tube and thumping the end. Take care not to spill any over the top of the tube. Visually examine the turbidity (cloudiness) of the yeast suspension. You should just barely see that the yeast in water is slightly cloudy compared to tubes that have only water in them. If the solution is too turbid, then add more sterile water. If the yeast plus water is clear then add a very small amount of yeast and resuspend. This is a critical step, so be careful. (see. Yeast culture, and videotape segment: Serial dilutions and viable cell counts.) This starting tube has a concentration of approximately one million (1 106) cells per ml of water.

    3. This is a 1/10 dilution so the new tube now has about 100,000 (105) cells per ml. The suspension will be clear since yeast cells are not visible in suspensions less than 106 cells.

    4. Be sure to suspend the yeast before each transfer using the tapping technique.

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    Plating and Irradiating cells: While it is hypothetically possible to plate 10 plates from each tube, 9 plates per tube is more realistic.

    Label the bottom of the plate, near the edge, with a marking pen. Include in your label, your name, the date, 103 cells, 20 seconds UV exposure. Each group should have pipettors and spreading equipment. (See Yeast culture "Pipeting and Spreading Cells", Ref. Video Tape.) Don't forget to suspend the yeast with the thumping technique each time. This is again a 1/10 dilution so you are plating about 1000 (103) cells onto each plate.

    One group can be responsible for the controls. DO NOT expose these plates to UV. The actual number of colonies that grow on the plate after incubation will provide your basis for determining mutation Click here to return

    Collecting Data:
    1. Statement for teacher to make to students:

    "Look closely! It is amazing how easy it is to overlook one of these red mutants when they are surrounded by hundreds of white colonies. Don't be too disappointed if you do not find one on your plate but don't be too surprised either. Whether you find visible mutants or not, it is almost certain that some of the surviving colonies on your plates are mutants that now require the addition of nutrients to their media for survival. Later, you can work on designing an experiment to discover these mutants."

    3. Remember that the UV exposed plates were plated originally with approximately 1000 cells. The controls were plated with about 100 cells each. They are plated with a different dilution so that the colony numbers would not be too great to count on a single plate. Since all of the dilutions started from the same source, they should be diluted by factors of 10. For this reason multiply the average number of colonies on the control plates by 10 to estimate the actual number of colonies that were initially on the UV exposed plates. Each student should enter their data in a clear table. As a group exercise, compile the data. Total the number of surviving yeast colonies on all of the plates from your class. Sum the number of red mutants found in your class. Divide the number of red mutants by the number of surviving yeast to calculate the mutants per survivor. Compare class results to other classes for example, mating type A to mating type B, etc.

    4. Someone who has a gentle touch with toothpicks can do this for the entire class. Label the bottom of the plate with the class, the mating type of the mutants (important), and a number for each mutant. Touch the sharp end of a sterile toothpick to the suspected red mutant colony. Gently drag the end of the toothpick on the fresh YED to streak the red mutant yeast.. Keep the streaks on this plate orderly and separate. If your class has more than 8 or 9 mutants, then use two plates. If the mutant colony is growing next to a white colony, it may be necessary to streak for single cells. (See Yeast culture Streaking for single cells.)

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    Preparation of test plates:

    Use the master plates from the original mutant isolations that your class prepared earlier. Use separate, sterile toothpicks to set up the complementation plates. Put no more than 6 or 8 unknowns of each mating type on a single plate. (See replica plating video)

    Mating Mutants:
    Be careful not to tear the surface of the agar. Mix thoroughly but don't overlap into other mating mixes.

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    Last updated Friday July 11 1997