Part B : Yeast Experiments

The Yeast Mating Pheromone

When a substance produced by one organism influences the behavior of another individual of the same species we call it a phermone. In multicellular organisms, when one cell produces a substance that influences the behavior of another, we call it a hormone. In a unicellular organism, such as yeast, it becomes a fine distinction. The yeast mating factors are a good example. Technically, microbiolial physiologists call them pheromones, but they share many characteristics with hormones. We think they make an excellent model for studying how many hormones work. Haploid yeast cells secrete small peptides mating pheromones that have strong similarities to peptide hormones in mammals. The yeast pheromones stimulate cells of the opposite mating type to differentiate into gametes, which have a distinctive pear shape that makes them easy to identify under the microscope. You may have seen them in your mating mixture.

Because they resemble the cartoon characters drawn by Al Capp, they are often called shmoos, and have become the unofficial mascot of many teachers, students, and other yeast workers. While shmoos are commonly found in mixtures of cells of the two yeast mating types, that does not prove that a pheromone is involved. That requires showing that a diffusible molecule from cells of one mating type has the same effect on cells of the other mating, without direct cell-to-cell contact.

Experiment:


In this experiment you will produce shmoos without the benefit of direct cell-to-cell contact. The mating-type cells secrete the mating pheromone, -factor, into the medium where it diffuses through the agar and stimulates the mating-type a cells to form shmoos.

This simple procedure -- the confrontation test -- is known as a bioassay, a test that uses a biological response to detect or measure some agent in the environment. This particular bioassay was used to demonstrate the existence of yeast mating pheromones and to isolate and purify the peptides that are secreted into the medium. This led to the identity of its structure (amino acid sequence). Biochemical companies now produce it synthetically and sell it as a reagent for studying yeast. Knowledge of its amino acid sequence also aided molecular geneticists to isolate the gene that codes for it. Studies of these genes shed light on how cells regulate the production of such molecules.

Time Line:

1st Day: 5 min Subculturing strain
2nd Day: 30 min Plating a cells and observing the shmoos

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-type a cells after three or four hours, you or a friend (ask your teacher) can put the plate in the refrigerator until you have time.
You can also put the plate in the refrigerator as soon as you transfer the a cells and then take the plate out four or five hours before looking at the cells.

Materials:

For each student or group:

Common materials:

Accumulating the pheromone in the agar: 1. 1st Day: With the flat end of a toothpick take some mating-type strain cells and make a streak, about an inch long, near the edge of a YED plate.
Incubate the plate overnight.

Detecting the pheromone with the bioassay: 2. 2nd Day: Prepare a suspension approximately 1 x 107 cells/ml of mating-type a cells. Use a toothpick to spread the suspension of a cells on the agar near the grown streak of cells. The a cells should be put within one microscope field of the cells.
Incubate the plate for 3 to 5 hours.

Examine the preparation directly on the plate under the microscope. The shmoo-shaped mating-type a cells should be apparent near the streak of cells.( Teacher Tips)

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