Part B: Basic Genetics Experiments

A Simple Cross

You may have heard the saying "What you see is what you get." Wouldn't it be great if that was always true? Often we can't see what is happening within organisms or inside individual cells even when we use a microscope. However, yeast is an exception. One of the most appealing features of Baker's yeast, Saccharomyces cerevisiae, is that in its life cycle "What you see is what you get." In yeast, the complete sexual life cycle can be seen, which makes it ideal for learning about the basic concepts of cell biology and genetics. Yeast cells have unique shapes at key points in the life cycle so you can learn to recognize each step. An outstanding feature of the yeast life cycle is the ease with which you can manipulate it. You can have yeast cells do whatever you want to study at a convenient time. Control of the growth and reproduction of yeast cell cultures by temperature changes or by type of food source is a most useful tool for doing experiments.

Experiment:

The entire experiment can be completed in less than two weeks. First you will mix two haploid yeast cultures that have grown overnight on a rich growth medium. Day-by-day you will change their growth conditions (environment) and observe the cell mixtures as they appear on the agar, and through the microscope. As you go along, you will be able to build a diagram of the yeast life cycle. You will see the main events of the sexual reproduction cycle and also examine the inheritance pattern for a color trait. After completing the life cycle experiment, you will be able to use your new skills to set up your own experiments to study influence of the environment patterns of inheritance.

Time Line:

If you incubate your cultures at 30øC, you will be able to follow this schedule. At room temperature, you may have to double the incubation times suggested in each procedure.

1st Day : 5 min Getting Ready
2nd Day : 50 min Mating Two Haploid Strains and Observing Zygotes
3rd Day: 15 min Selecting the Diploids
4th Day: 15 min Presporulation
5th Day: 15 min Sporulating the Diploids
6th Day: 50 min Observation of Asci and Germination of Spores
10th Day: 15 min Looking for the Missing Color

Figure 1

Materials:

Getting Ready:

(Your teacher may have already done this for you.)

 Time Line: First Day: 5 min

 1. Touch a sterile toothpick to a colony of HA2 yeast. Make a short (1/2 inch ) streak at the top of a YED agar plate. Discard the toothpick.
2. Touch another toothpick to a colony of HBT yeast.
Make a short (1/2 inch ) streak on the left side of the YED agar plate. Discard the toothpick.
3. Your plate should look like the plate diagramed in Figure 1.
4. Turn the plate over and label the bottom of the plate with "Getting Ready", HA2, HBT, YED, your name and the date.
( Teacher Tips )

Technical Tip :

When you mix haploid cells of opposite mating types a and  together on a nutrient agar medium, they become pear-shaped (we call them "shmoos"). They then mate (conjugate) to form diploid zygotes. The zygotes can be distinguished by their characteristic shapes. Before they start budding they are peanut-shaped and after they bud, they look more like clover leaves.

Figure 2 & 3
 

Mating Two Haploid Strains and Observing Zygotes:

Time Line: 2nd Day: 50 min

Materials:

1. Touch a sterile toothpick to the HBT streak on the YED plate and then touch it to the agar at the center of the plate as illustrated in Figure 2. Discard the toothpick.
2. Touch another sterile toothpick to the HA2 streak and touch it to a spot close to the spot you just made at the center of the plate. Discard the toothpick.
3. Then use a third sterile toothpick to mix the two spots of yeast together as illustrated in Figure 3. Be gentle, don't tear up the surface of the agar. 4. Draw a sketch of the yeast cultures in the first circle on the Data Record Sheet or in your lab journal. Label the yeast strains, the streak color, the kind of medium, and the date of the procedure.

(Teacher Tips )

Technical Tip:

Time Shifting: Yeast cells follow a clock that depends on temperature, so you can easily control how fast they grow and develop. If you can't look at the mating mixture after three or four hours, you or a friend (ask your teacher) can put the plate in the refrigerator until you have time. Another way of slowing the growth of the yeast, is to put the plate in the refrigerator as soon as you mix the cells and then taking it out four or five hours before looking at the cells. If you leave the plate in the refrigerator for one day, then you need to add another day to your experiment.

Figure 4

Figure 5

5. Make a wet-mount slide from either one of the parent strains and look at it through the microscope.
6. Draw a sketch of about 10 of these cells in the first square of your Data Record Sheet or in your lab journal. Label the square "Haploids".
7. After 3 hours, make a wet-mount slide of the mating mixture and look at it through the microscope. See if you can find some zygotes (peanut or clover-leaf shaped.) These zygotes are diploid. They contain the chromosomes from both of their haploid parents. You may also see some shmoos that look much like pear-shaped cells. Shmoos are haploid cells getting ready to mate. Some cell shapes are shown in Figure 4.
8. Draw a sketch of about 10 cells in the second square of your Data Record Sheet. Label the square "Mixture". Label unbudded zygotes with "Z," budded zygotes with "BZ," and shmoos with "S."
9. Put the plate back in the incubator with the agar side up for another day or two to let the diploid cells grow.
(Teacher Tips)

Technical Tip:

Geneticists use the word trait to mean any feature of an organism that can be inherited such as color or the ability to grow on a certain kind of food. Traits can come in different forms which are called variations. For example, in the two haploid yeast strains you are studying, the trait is the colony color while red and cream are the variations. The color of the yeast is part of the phenotype of the strain.
 

Technical Tip:

After your mating mixture has grown up, you can see its color but you need some way to tell the diploids from the cream-colored haploid parent. To make this easy, we have used yeast that need different food (growth medium). Both of the haploid strains can grow on the rich YED medium, but neither can grow on the nutritionally poor MV medium. But, the diploid cells formed from these two haploid strains can grow on MV. By putting the cells on MV you can be confident that you have a purified diploid colony.

Figure 6

Selecting the Diploids:

Materials: Time Line: 3rd Day: 15 min

 1. First record the appearance of your plate by making a drawing of it in the 2nd blank circle on your Data Record Sheet. Label the parents and the mixture and describe their colors. Be sure to write down what kind of medium (YED or MV) cells are growing on and today's date.
2. Make a copy (replica) of the YED plate by transfering the mating mixture onto an MV plate. Make the streaks the same size and shape as those on the original plate. With a sterile toothpick pick up some HBT cells streak from your YED plate. Then make a streak on the left side of the MV plate (See Figure 5). Discard the toothpick. With another sterile toothpick pick up some HA2 cells from YED plate and make a streak of HA2 cells at the top of the MV plate. Discard the toothpick. With a third sterile toothpick pick up some of the mixture in the middle of the YED and then make a dot of cells in the center of the MV plate. Discard the toothpick. Label this MV plate with your name, the date and "Selecting the Diploids". Incubate the plate overnight.
(Teacher Tips)

Technical Tip

Before inducing the yeast to sporulate, it is better to transfer the diploid cells back to a YED plate. Cells on YED medium will grow faster than on MV medium and rapidly growing cells will sporulate better.

Figure 7

Presporulation:

Materials: Time Line: 4th Day: 15 min

 1. Make a sketch of the MV plate you made in the last procedure in the 2nd empty circle on your Data Record Sheet. Record the color of the diploid cells growing on the plate. Does one color phenotype (pink or cream-colored) seem to hide the other phenotype?
2. Make a wet-mount slide from the diploid cells growing on MV and look at them through the microscope.
3. Draw a sketch of about 10 of these cells in the third square of your Data Record Sheet. Label the square "Diploids".
4. Use a sterile toothpick to transfer some of the mating mixture (from the middle of the MV plate) to a plate of YED medium (see Figure 6). Discard the toothpick.
5. Incubate the plate overnight.

Teacher Tips

 Technical Tip:
By this point you have seen half of the yeast life cycle: mating between two haploids to form a stable diploid cell. The diploid cells will divide and can be cultured just as you have done with the haploid parents. You can now see the other half of the life cycle by sporulating the diploid, to obtain four haploid spores. To do this you need to transfer the diploid cells to sporulation medium (YEKAC). YEKAC contains no nitrogen source and only a nonfermentable carbon source (acetate). When diploid cells try to grow on YEKAC, they sporulate and go through meiosis. Meiosis produces two important results:
1) The chromosome number is reduced from diploid to haploid, and
2) the resulting haploid cells have all possible combinations of the adenine, tryptophan and mating-type genes.
Through the microscope, you can see the products of sporulation as four spores enclosed in a sack called an ascus.

Figure 8

Sporulating the Diploids:

Time Line: 5th Day: 15 min

 Materials:

1. Draw a sketch of the YED plate you prepared in the previous procedure on your Data Record Sheet and record the color of the diploid colony growing on YED.
2. Use a sterile toothpick to pick up some of the freshly grown diploid cells from the YED plate and make three streaks on the YEKAC plate (See Figure7).
3. Incubate the plate for at least 3 days.

Technical Tip:

When you examine the sporulation culture through the microscope, the asci will be easy to find. They look like lumpy cells. These are actually sacks containing four spores. After you observe the asci you will grow the spores into colonies to see if any of them have the original color phenotypes of the parent strains.

Figure 9

Observation of Asci and Germination of Spores:

Materials: Time Line: 8th Day: 50 min

Draw a sketch of the YEKAC sporulation plate on your Data Record Sheet.

 Make a wet-mount slide of a sample from the YEKAC plate and examine it with a microscope. Look for lumpy- cells that appear to have two, three, or four round spores inside a membrane. These are the asci containing ascospores. They should all have four spores, but sometimes some of the spores don't develop. If most of the cells do not have spores, incubate the plate for another day or two.

 Draw a sketch of about 10 of the asci in the third square of your Data Record Sheet. Label the square "Sporulation".

 Touch a sterile toothpick to one of the streaks of the YEKAC plate. Make a streak on a new YED plate (See Figure 8). Then use a new sterile toothpick to make another zigzag streak across the first one on your YED plate. Continue using fresh sterile toothpicks to make 4 or 5 more zigzag streaks in this manner. The last streaks should give you some single colonies. Each colony will grow from a single ascus or from parts of broken asci that may contain single spores.

Teacher Tips

Technical Tip:

When you put spores back onto YED growth medium, they germinate, begin budding, and grow into colonies. Since some will be mating type a and some mating type , they may also mate. Therefore, the colonies that grow may be either haploid or diploid cells and either pink or cream-colored.

Looking for the Missing Color:

Time Line: 10thDay: 15 min

1. Look for different colors among the colonies. Can you find both of the colony color phenotypes expressed by the original haploid parent strains?
2. Draw and label a sketch of this plate on your Data Record Sheet.

Return to Yeast Experiments
 

Last updated Wednesday, 04-Dec-02 14:51:34