1. Why are we using more dilute cultures for the killing curve while we are using the arg selection culture at full strength?

2.How would you find the arg+ mutants on the killing curve plates?

3. when designing a protocol to produce auxotrophs for biochemical pathways, how long should you irradiate yeast in this system to maximize the number of auxotrophic mutants? Explain your reasoning.

4. If the mutation in the Saccharomyces cerevisiae is an ochre mutation (see introduction), what amino acids may be substituted into the polypeptide by a single base mutation? Show the codon created and the amino acid for which it codes.

5.Why do we have to incubate the plates in the dark?

6. How does our experimental design allow us to determine that the UV caused the reversion from arg- to arg+ and not simple spontaneous suppressor mutations?

Exposure to UV light causes pyrimidine dimer formation. The pyrimidine dimer can be repaired by direct reversal and NER. It can also be bypassed by activation of the SOS response.

a. Which of these three pathways maintains the arginine auxotrophy? Why?

b. Which of these three pathways changes the arginine auxotrophy to prototrophy? Why?

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  1. We use more dilute cultures because

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  2. 1. Full strength cultures of the arg selection culture were used because arg was placed on a medium that did not contain arginine therefore, once exposed to ultraviolet light only those with a specific mutation will be able to grow on these plates. Since we are looking for growth and only a specific mutation (arg- to arg+) will grow, it is better to have more of the culture so growth will be more numerous. More dilute cultures were used for the killing curve because they were placed on a medium enriched with arginine and will grow numerously with small amounts.

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