A haploid strain of yeast is treated with a mutagen called EMS.

The different steps in the treatment and the analysis are the following: Step 1:

Two tubes, labeled A and B contain 0.2 ml each of same yeast liquid culture. The concentration of yeast cells

in the liquid culture was determined by a measure of the turbidity of the culture. This concentration is 4.4 x 109 cells/ml.

Step 2:

In each tube (A and B) 1.5 ml of phosphate buffer was added. A volume of 0.05 ml of EMS was then added to tube A alone. Therefore, culture A contains treated yeast cells while culture B is a control. In your calculations you will ignore the volumetric contribution of EMS in culture A (A and B contains 1.7 ml of diluted yeast culture).

Step 3:

After 1 hour, 0.1 ml aliquots of culture are drawn from each tube and each aliquot is mixed with 3.9 ml of thiosulfate to neutralize EMS. The neutralized samples are then centrifuged to pellet the cells and each cell pellet (A and B) is resuspended into 4 ml of complete culture medium. A complete culture medium contains all the compounds (amino acids, nucleotides, carbon source, vitamins...) needed for cell growth. The new cultures are now called A1 (treated cells) and B1 (control).

Step 4:

The culture A1 and B1 are grown long enough to allow for 1 cycle of cell division.

A 0.1 ml aliquot of culture A1 is diluted with 9.9 ml of complete culture medium. Then a 0.1 ml aliquot of this diluted culture is diluted again with 9.9 ml of complete culture medium. The culture produced by the last dilution is called A2. Similarly, the same two rounds of dilutions described above are applied to the B1 culture to obtain the B2 culture.

Step 5:

Forty 0.1 ml aliquots of culture A2 and four 0.1 ml aliquots of culture B2 are plated on separate dishes with solid complete medium (40 plates for A2 and 4 plates for B2).

After several days of culture at 28°C, colonies were formed on the 44 plates.

The number of colonies determined on 4 representative plates (2 for A2 and 2 for B2) is shown on the following table.

Culture A2

Culture B2

Plate A2-­?1

Plate A2-­?2

Plate B2-­?1

Plate B2-­?2

# of colonies

44

48

238

274

Step 6:

Colonies were then transferred to plates containing a solid minimum media. To grow on minimum medium, yeast cells must be wild type and able to produced their own amino acids, and nucleotides.

The results are the following: out of 1800 colonies derived from plates A2, 52 could not grow on minimum

medium. In contrast, out of the 1500 colonies derived from B2 only 1 does not grow on minimum medium.

1) Is the determination of the cell concentration in the original yeast culture based on cell culture turbidity 4.4 x 109cells/ml accurate? To answer this question you need to compare the average number of colonies growing on plate B2 (see Table above) with the expected number of colonies growing on B2. This expected number is calculated using the concentration of cells based on turbidity and the series of dilutions (steps 1 to 5).

1)The treated (A) and control (B) samples give a different number of colonies on complete medium plates.

a) By comparing the average number of colonies on plates A2 and the expected number of colonies on B2 (see part 1), calculate the proportion of colonies derived from treated cells that grow on complete medium. (3 pts)

b)Based on this proportion, what is the main consequence of EMS treatment? (2 pts)

2) The treated (A) and control (B) samples give a proportion of colonies that grow on complete medium but cannot grow on minimum medium.

a)Calculate for both samples, the proportion of colonies that cannot grow on minimum media.(3 pts)

b)Based on these proportions, what is the second consequence of the EMS treatment? Briefly explain. (2 pts)