Objectives and Applications:

This experiment is designed to be used in conjunction with the traditional textbook treatment of a simple dominant/recessive two-factor cross. It gives graphical realism to the ever-popular Punnett's Square. (See A Traditional Dihyrid Cross segment in video tape I).

Objectives:
1. To see first-hand how the genotypes of the haploid gametes combine to form the phenotypes of the diploid offspring for dominant and recessive alleles.

2. To see that there is no uncertainty in the outcome (phenotypes of the offspring) when there is no uncertainty in the genotypes of the gametes. In the diploid dihybrid cross the uncertainty is the result of random sampling of the pool of gametes from the parental cross.

Yeast strains usually come from the supplier growing on agar slants. Contamination may be a problem when students use the master set of yeast strain slants as the source of their strains. You may wish to subculture the strains on YED plates. One subculture plate containing all eight haploid strains will supply enough yeast for a class. If several groups need access to the yeast at the same time, you may want to make several subculture plates (See Subculturing Yeast segment in video tape III). Alternatively you may wish to prepare the Day 1 plates for the students. A quick method for preparing these plates is for the teacher to make a master plate, incubate it overnight, and then use the replica plating method to make copies for the students (see Replica Plating segment in video tape III).

See the Laboratory Methods section for agar media recipes. You will need to make the plates several days in advance.

Each student or team will require at least 50 sterile toothpicks for this experiment. Toothpicks are sterile from the box and there are 750 toothpicks in each box. (See Laboratory Methods F: Using Toothpicks and Inoculating Loops)

Each student or team will require at least 1 mating grid. A copy master of six grids is included with these materials.

1st Day:
Have the students write the expected genotypes and phenotypes on the mating grid and then tape the grid to the bottom of the YED plate as a pattern for plating the yeast.
Only the eight haploid strains are plated on Day 1.

2nd Day:
Stress that fresh toothpicks should be used for each different strain or mating mixture. The best results are obtained when only small amounts of yeast are used to make each mating mixture.

3rd Day:
Once again the best results are obtained when only small amounts of yeast are transferred during replica plating. If you use replica plating equipment you will need a fresh sterile velvet for each plate that is copied.

4th Day:
If the mixtures were not mixed thoroughly there may be pink areas in otherwise cream-colored mixtures. These mixtures should be scored as cream-colored.

Comments on genetic notation: We have chosen to use traditional notation for the genotypes in this experiment so that it will be more consistent with usual textbook treatments of the dihybrid cross and Punnett's Square. In later experiments we will introduce the more sophisticated notation of modern yeast genetics. Even so, you have some options for how much you want to simplify things.

Case 1

We can represent the hypothetical dihybrid cross between two diploids as follows:

But in yeast, we don't actually cross diploids. We first sporulate these diploids, obtaining two types of spores, each in both mating types:

These represent the gametes in the parental cross. We then mate either ARt x BrT or BRt x ArT to obtain the F1 diploids, which would be:

If we sporulate this diploid, we get the following eight haploid genotypes (spores or gametes) shown on the previous page.

In this experiment the students start here to obtain the F2 generation, without the complication of statistical sampling, by setting up the following grid of crosses:

F2:

	ART ARt ArT Art
BRT	c+ c+ c+ c+
BRt	c+ c- c+ c-
BrT	c+ c+ r+ r+
Brt	c+ c- r+ r-

where c = cream, r = red, + = grows on MVA, and - = doesn't grow on MVA Case 2 In yeast, mating type is controlled by the mating type genes that segregate as alternative Mendelian alleles, so it seems natural to include them in the formal description of the genotypes, as we have done above. However, it may simplify the discussion to treat mating type separately. The A's and B's could, therefore, be omitted in all the above genotypes. If, for example, you describe a cross as Rt x rT, it is implicit that they are different mating types. If this approach is taken, then the whole scheme simplifies to the following:

P1: RRtt x rrTT

gametes: Rt and rT

F1/P2: RrTt

F2:

	RT Rt rT rt
RT	c+ c+ c+ c+
Rt	c+ c- c+ c-
rT	c+ c+ r+ r+
rt	c+ c- r+ r-

Comments:
The main difference between this experiment and the analogous case in higher organisms, is that in yeast we have control of the life cycle so that we can deal directly with the haploid stages. We can isolate, as pure strains, the haploid parents of the F2 generation, which correspond to the gametes from the P2 parents. By being able to identify these, isolate them as pure strains, and make all the possible crosses, we remove all the chance factors. In higher organisms, we hypothesize the segregation of alleles in a hybrid when it goes through meiosis. If we make two assumptions, 1) that the alternative alleles segregate independently into the gametes, and 2) that there is dominant/recessive expression of the respective alleles, then we can predict the 9:3:3:1 ratios among the F2 offspring. Accordingly, this experiment provides a direct demonstration of how the diploid phenotypes are derived from the genotypes of the parents.

This does not demonstrate independent segregation, which requires showing that the haploid phenotypes did, in fact, occur in equal numbers among the spores from the F1 diploids.

In the context of animal breeding, or even human genetics, what we do with yeast when we isolate two haploids and mate them has a parallel in in vitro fertilization. However, we do not yet have the ability to score many genetic traits in gametes of higher forms, nor can we propagate them asexually.

Environmental effects on Colony Color

• The interaction between genetic and environmental determinants of the color phenotype
• The genetic control of the biosynthetic pathway for adenine
• The pathway control mechanism of feedback inhibition
• The diffusion of nutrients through agar

Numbers refer to steps in the student procedure.
Materials: 1. If HA2 or HB2 is not available, you can substitute any of the following adenine mutant strains: HA1, HB1, HA12, HB12, HAR, HBR. The yeast should be subcultured overnight on a YED plate. (See Subculturing Yeast segment in video tape III.)

2. YED agar plates may be substituted for MV agar plates in this experiment. However, since YED agar provides a small amount of adenine, the pink lawn will grow all the way to the edge of the plate without a section of no growth. It may also take longer for the pink color to develop.

The procedure for this experiment is illustrated in the Effect of Adenine on Red Mutant Yeast segment in video tape II.

1. Any sterile container will work for mixing the yeast cell suspension such as baby food or other types of jars. It is possible to recycle them for this purpose by washing and sterilizing them in an autoclave or a pressure cooker. You can also purchase presterilized plastic specimen containers from a science supply vendor.
The amount of water in relation to the yeast cells is not critical as long as the suspension is visibly turbid. It takes about 1 milliliter of suspension for each plate, so multiply 1mL times the desired number of plates. The best results are obtained by using a fresh yeast suspension, but yeast cells are relatively stable in water so you might store the suspension in the refrigerator and use it for several days if necessary.

4. The surface water must be absorbed by the agar before the adenine disk is placed on the surface. If there is surface moisture, the adenine will diffuse in an uneven pattern across the surface instead of moving uniformly through the agar. The best results are obtained when freshly poured plates are allowed to sit at room temperature for 3 or more days before use.

5. Sterile forceps may be used to handle the adenine disk. The forceps may be sterilized by wiping them with an alcohol wipe instead of using the traditional flame sterilization method.

6. Note that the plates are incubated with the agar side down in order to keep the adenine disk in place. Usually plates are incubated with the agar side up in order to prevent any condensation from forming on the lid and dripping onto the yeast colonies.

Observe and Respond:

Development of growth and color rings:

1. How can you explain the white growth near the center where the adenine concentration is excessive? The answer to this question will vary based on the background of the students and the context in which you are using this experiment. What seems to be happening here is that the adenine concentration is high enough to support growth and also high enough to turn off the adenine synthesis pathway by feedback inhibition. Red pigment is only produced when the adenine synthesis pathway is turned on but it is blocked by an ade1 or ade2 mutation.
Do these white cells require adenine? Yes, this is an environmental difference, not a genetic change.
How can you test your answer experimentally? Move some of the white cells to YED agar without an adenine disk. Cells that do not require adenine will grow as cream colored colonies on YED and cells that still carry the red mutation will grow as pink colonies on YED. (See the next section "Retest the white yeast".)

2. Describe the nutritional conditions in the red zone. There is enough adenine to support growth but not enough to shut off the adenine synthesis pathway.

3. Why is there no growth at the edges of the plate? The adenine concentration is too low.

1. Streaking for single cells is illustrated in the experimental procedure. This method is useful for examining the range of cell types in a sample. An alternative method, the single streak subculture method, will be adequate for this experiment if you wish to use fewer plates and have several students test their yeast on the same YED plate. (See the Streaking For Single Cells and Subculturing Yeast segments in video tape III)

Analyze your observations:

1. Explain whether the change from red to white near the center of the plate was a genetic or an environmental effect? The white yeast from the center of the adenine disk plate should grow as pink colonies on YED. The change from pink to white is environmental.

2. Give an explanation for this change or suggest another experiment you might do to learn more about it. In the YED environment, there is enough adenine to support growth but not enough to shut off the adenine synthesis pathway, so the cells form pink colonies. In the MV+ adenine environment, there is enough adenine to support growth. The concentration is also high enough to shut off the adenine synthesis pathway, so the cells form cream-colored colonies. One possible experiment would be to plate the white yeast from the first MV+ adenine plate onto an MV plate. If the red to white change was genetic, the yeast should now be able to grow on a plate with just MV agar. If the change was environmental, the white yeast should not be able to grow on just MV.

1. If HA2 or HB2 is not available, you could substitute any of the following adenine mutant strains: HA1, HB1, HA12, HB12, HAR, HBR. The yeast should be subcultured overnight on YED agar. (See Subculturing Yeast segment in video tape III)

Growing the red strain anaerobically:

The procedure for this experiment is illustrated in the Anaerobic Growth of Red Mutant Yeast segment in video tape II.

1. See the previous section Effect of Adenine of Red Mutant Yeast for a discussion of making suspensions of yeast cells.

4. Most of the surface water should be absorbed by the agar before the agar is cut and folded. The best results are obtained when 1 mL or less of yeast cell suspension is placed on each plate and the freshly poured plates are allowed to set out at room temperature for 3 or more days before use.

7. Note that in order to keep the agar "sandwich" together, the plate is incubated with the agar side down. Normally, plates are incubated with the agar side up in order to reduce condensation problems.

Exposing the cells to air(O2):

1. The cells outside the "sandwich" should be pink and the cells inside the layers should be cream-colored. It is likely that bubbles of carbon dioxide gas will form between agar layers. 5. After about one hour the cream colored cells that have been exposed to the air will start to develop a pink color. The cells remaining between the agar layers should still be cream-colored. The responses to the following questions will vary according to the background information that has been given to the students. The following responses are examples.

Analyze your observations:

1. Is there any part in the sandwich where the yeast has not turned pink or red? Cream-colored inside, pink outside. 2. Is there a reason to think that the yeast growing there might be anaerobic? It is common for colored compounds to be formed by oxidation. If oxygen is not present (anaerobic conditions), the color does not develop. The lack of pink pigment in the cells between the agar layers supports the idea that anaerobic conditions were present between the agar layers.

3. Did you see any gas bubbles between the layers of agar? Bubbles of gas are formed between the agar layers. If so, can you think of an explanation for them? The yeast cells produce CO2 gas as a waste product during their normal growth. This helps keep the environment between the agar layers anaerobic.

4. What conclusion, if any, can you draw from this experiment about the role of oxygen in the formation of the red pigment? Oxygen is necessary for the formation of the red pigment.

5. Explain whether the change from white to red was a genetic or environmental effect? The effect was probably environmental. The yeast that stayed between the agar layers remained cream-colored, but still formed the pigment when grown aerobically. If the effect was genetic, the change would not be reversed for all cells.

6. Give an explanation for this change or suggest another experiment you might do to learn more about it. This was an environmental effect produced by the pigments formed in the cells by a reaction involving the oxygen in the air. The yeast that turned pink could be replated between agar layers to see if the change was genetic. If it turned once again cream-colored between the layers and then turned pink when exposed to air, the idea of an environmental effect would be supported.

Yeast Mating Pheromone

Objectives and Applications:

Materials:
To reduce contamination it is usually best to limit student use of the master set of yeast strains. You may wish to make one YED subculture plate for each mating-type on the previous day before the students start work. Students can then use those plates when they do their work on 1st and 2nd days. One plate of each mating-type will provide enough yeast for a whole class.

2nd Day:
A suspension containing cells at approximately 1 x 10e7 cells/ml will appear very cloudy.

Before you do this lab, you should test your microscopes' ability to view yeast cells directly on the plate. You will most likely need to remove the Petri dish lid and get the objective lens very close to the agar surface. At a short working distance, some lenses fog up and obstruct the view.

To overcome this problem you can drop a coverslip onto the area you wish to observe. Naturally this will most likely contaminate the plate.

It is possible to buy synthetic alpha factor. (-Mating Factor, Sigma Chemical Co.)
If your microscopes can't view cells directly on the agar plates, you may wish to add the synthetic alpha factor to mating-type a cells which are growing on agar or in liquid culture, and then use those cells to make wet mount slides for viewing.

Yeast Transformation

Objectives and applications:

Materials notes:
- Plasmid DNA/carrier mixture: prepared as described in Schiestl and Gietz, 1989; this is enough for one transformation.
- MV medium: 1 plate for each experimental condition.
- Sterile pipettes: types for student use will depend on what materials you premeasure for the students.
- Spreaders: The use of alcohol to flame sterilize spreaders is dangerous and requires close supervision. Presterilized paper clip spreaders are much safer.
- Strain HA1L (ade1): one subculture plate will supply enough yeast for 4-5 students or teams. Plates can be grown several days in advance and stored in the refrigerator. Or, streak to isolate single colonies with sterile toothpicks and incubate 3-4 days until colonies are well grown. Use 3-4 colonies to make the yeast/lithium acetate suspension.

- Sterile Solutions: prepare working solutions from stock solutions just prior to use. Use distilled water and autoclave stock solutions and working solutions.

Working solutions:
• LiAc/TE solution: for 100 mL, mix 10 mL 1M LiAc stock solution + 1 mL 1M Tris-HCl stock solution + 1 mL 0.1M EDTA stock solution + 88 mL distilled water.
• TE solution: for 100 mL, mix 1 mL 1M Tris-HCl stock solution + 1 mL 0.1M EDTA stock solution + 98 mL distilled water.
• 40% PLT solution: for 200 mL, mix 160 mL 50% PEG stock solution + 20 mL 1M LiAc stock solution + 2 mL 1M Tris-HCl stock solution + 2 mL EDTA stock solution + 16 mL distilled water.

Stock solutions:

• 1M LiAc stock solution: 10.2 g LiAc.2 H2O + 80 mL distilled water; adjust pH to 7.5 with acetic acid; adjust volume to 100 mL with distilled water.
• 1M Tris-HCl stock solution: 12.1 g Trizma base + 80 mL distilled water; adjust pH to 8.0 with HCl; adjust volume to 100 mL with distilled water.
• 0.1M EDTA stock solution: 3.72 g ethylenediamine tetraacetic acid, disodium salt + 80 mL distilled water; adjust pH to 7.0 with sodium hydroxide; adjust volume to 100 mL.
• 50% PEG stock solution: 100 g polyethylene glycol 4000;to adjust to 200 mL with distilled water.

Yeast Transformation--Plating the cells

Compare your plasmid DNA plate with others in the class. Did all the plates have the same number of colonies? Were the colonies all the same color? Were the YCpADE1 plates and the YEpADE1 plates the same? Write an explanation for any differences observed in your class.
Colony numbers will probably vary. The colonies will probably all be cream colored. If you plate cells from MV to YED the YEpADE1 transformants will start to show red sectored colonies as the plasmid is lost. (See plasmid loss experiment)

3. Did any of the control plates have colonies? If they did offer a possible explanation.
There should not be any colonies on the negative control plate (no plasmid DNA).

Plasmid Loss