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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We use more dilute cultures because
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.
1. The reason we use more dilute cultures for the killing curve is to ensure that the effect of the antibiotic on the bacterial cells can be clearly observed. By starting with a larger number of cells, the antibiotic can have a greater impact, leading to a more accurate measurement of its effectiveness. On the other hand, arg selection culture is used at full strength because the goal is to select for arg+ mutants specifically, and using a more concentrated culture allows for better detection and isolation of these mutants.
2. To find the arg+ mutants on the killing curve plates, we need to look for colonies that are able to grow in the presence of the antibiotic. This can be done by visually inspecting the plates and identifying colonies that have a larger size or exhibit better growth compared to the surrounding arg- colonies. These colonies are likely to be arg+ mutants.
3. The duration of irradiating yeast to maximize the number of auxotrophic mutants depends on several factors, including the sensitivity of the yeast strain to radiation and the desired mutation rate. It is generally advisable to perform a time course experiment where yeast cells are irradiated for varying lengths of time and then plated to determine the optimal duration. The reasoning behind this is that longer exposure to radiation can increase the likelihood of inducing mutations, but it also increases the risk of damaging cells beyond repair. Therefore, finding the balance between mutation induction and cell survival is crucial.
4. If the mutation in Saccharomyces cerevisiae is ochre mutation, it means that a single base mutation has occurred, resulting in the formation of a premature stop codon. Possible amino acid substitutions caused by a single base mutation would depend on the specific codon affected. For example, if the base mutation occurs in the UAA codon, it would create UAG, UGA, or UAA itself, which are stop codons and do not code for any amino acid.
5. Incubating the plates in the dark is necessary to prevent the formation of unwanted UV-induced mutations during the incubation period. UV light can continue to cause DNA damage even after the initial exposure, especially if exposed to light afterwards. By incubating the plates in the dark, we can minimize the possibility of these secondary mutations and ensure that any observed changes in the arginine auxotrophy are directly attributed to the initial UV exposure.
6a. The pathway that maintains the arginine auxotrophy is NER (Nucleotide Excision Repair). NER is responsible for repairing DNA lesions, including pyrimidine dimers caused by UV exposure. If the arginine auxotrophy is dependent on the presence of these lesions, then NER would play a role in maintaining the auxotrophy state.
6b. The pathway that changes the arginine auxotrophy to prototrophy is SOS response. The SOS response is a DNA damage response mechanism that is activated in the presence of significant DNA damage, such as pyrimidine dimers. It can induce the expression of multiple genes, including those involved in repair and bypass mechanisms. Activation of the SOS response can lead to the bypass of the DNA lesion, allowing for the synthesis of arginine and converting the cells from arg- to arg+.