Part B: Basic Genetics Experiments

Two Genes/One Trait

The study of genetics in yeast and other microorganisms started getting very interesting with the discovery that different genes could determine the same, or at least very similar, traits. This immediately raised all kinds of questions about what a gene really is and, given two mutants, how you can tell if their mutations are in the same gene or in different genes that effect the same trait.

Let's start by trying to make sense out of all the different words geneticists use to talk about genes. We know that a gene is a sequence of DNA that codes for a protein or sometimes an RNA molecule. The molecule the gene codes for is often called the gene product. We also know that a mutation is a change in a gene. Does that make it a different gene? Well, yes and no. It is certainly different, but it is a different form of the same gene. It is different because it is changed, but it is still the same gene because it codes for the same gene product (or a modified form of that product.)

Now, suppose you did an experiment to find red mutants of Baker's yeast and you found several different mutants after exposing a few million cells to ultraviolet radiation. How could you tell if these were different mutations in the same gene (alleles) or if there is more than one gene controlling this color phenotype?

Experiment: In this experiment you will make crosses with four red strains, two of each mating type. HA1 and HA2 are both mating type a and HB1 and HB2 are both mating type , so you can only make four crosses: HA1 HB1, HA1 HB2, HA2 HB1, and HA2 HB2. By making these crosses and observing the color phenotypes of the diploids, you should be able to figure out how many genes are involved and how these strains are related.

Time Line:

1st Day: 10 min Subculture Parent Strains
2nd Day: 15 min Cross all four strains
4th Day: 15 min Record the results of the color test
15 min The Growth Requirement
5th Day: 15 min Record the results of the growth testFigure 1

Materials:

Subculture Parent Strains:

Time Line: 1st Day: 10 min You will need to grow these strains overnight on YED before mating them. Your teacher may have done this for you.

1. Touch a sterile toothpick to the stock sample of yeast and then make a streak of the cells on a YED agar plate.
Use a different sterile toothpick for each strain to make a plate like in Figure 1.
2. Label each strain by writing its' name on the bottom of the plate.

3. Incubate the plate for 1-2 days. (Teacher Tips)

Figure 1

Cross all four strains:

Time Line: 2nd Day: 15 min

1. Make crosses of the four strains on the plate you made on the first day. Use fresh sterile toothpicks to transfer a small amount of yeast from each of the freshly grown overnight haploid cultures to the position shown in figure 2. Place each pair of strains close together, but not touching. Discard the toothpick used for each strain. (See Figure 2)
2. Use fresh sterile toothpicks to mix each pair of strains together, as shown in the diagram below, be careful not to tear up the surface of the agar. (See Figure 3)
3. Incubate the plate overnight.

A. Record the results of the color tests:

Time Line: 4th Day: 15 min

1. Sketch a diagram of the plate after it has grown enough that the parent haploids have turned pink. Label the colors of the mating mixtures in the center of the plate.

2. Summarize your observations.

3. What can you conclude about the "two-gene hypothesis"?(Teacher Tips)

Figure 2 & 3

  • In the life cycle experiment you found that the mutant haploid parent strains would not grow on MV medium, but that the diploid formed from them would. Now that you have tested four different combinations of red mutants give a hypothetical answer to the following questions:

  • Is there a relationship between the color and the ability to grow on MV?

  • Do the same genes control the growth requirements and the color?

  • You can test your hypothesis by testing your haploid and diploid strains on MV.

    The Growth Requirement:

    Time Line: 4th Day: 15 min

    1. Make a replica of your YED plate on MV medium.
      Use a fresh sterile toothpick to transfer a small amount of yeast from each growing spot on the YED plate to the corresponding position on the MV plate.
    2. Label the plate.
    3. Incubate the plate overnight.

    (Teacher Tips)

    Figure 4

    Figure 5

    Record the results of the growth tests:

    Time Line: 5th Day: 15 min
    1. Sketch a diagram of the MV plate after the diploid from HA2 and HB1 has clearly grown. On your diagram label those that have grown with a "+" and those that have not grown with a "-".

    2. Do your observations support the hypothesis that there are two genes that can produce the red color?

    3. Describe a model for how these genes are related to each other and how they are involved in the metabolism of the cell.

    (Teacher Tips)

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    Last updated Wednesday, 04-Dec-2002 20:58:28 UTC