Question: You are the chief scien............. Edit
Answer: The following schematic represents the only pathway for the synthesis of the amino acid lysine in yeast cells. Each step in this pathway is catalyzed by a specific enzyme, which is encoded by a specific gene. This pathway begins with a hypothetical compound A and proceeds via three intermediates B, C and D to produce lysine. A yeast cell that is missing any of genes encoding these four enzymes cannot synthesize lysine. A cell that cannot synthesize lysine cannot grow and form a colony on minimal media (media that does not have any added supplements) but can grow and form a colony on media that contains lysine. You do a mutant hunt to identify the enzymes that are involved in lysine biosynthesis. You start with a population of wild type yeast cells, mutagenize them using UV and allow the UV irradiated cells to grow and divide on a master plate containing rich medium (i.e. medium containing all essential compounds and added supplements). You then replica plate the cells from each colony on the master plate to the following plates containing minimal media or minimal media and the specified nutrients.
a) From these plates…
i) Identify the colony or colonies of wild-type cells. Explain why you selected these colonies.
Colonies #1 and 3 are composed of the wild-type cells since they can grow on minimal medium plates.
ii) Assume that each colony is composed of cells that carry a single mutation. Identify the colony or colonies of cells that cannot synthesize lysine. Explain why you selected these colonies. Colonies #2, #4, #5, #6, #7 and #9 are the colonies of the cells that do not grow on minimal media, but can grow on minimal media with added lysine.
iii) Identify the colony or colonies of cells that carry a mutation in a gene that codes for a protein involved in biosynthetic pathway different from the lysine biosynthetic pathway. Explain why you selected this colony or colonies. Colony #8. These cells are auxotrophs since they cannot grow on minimal. Since they also cannot grow on minimal with lysine, they must be missing a gene important for some other pathway,
iv) Identify the colony or colonies of cells that carry a mutation in either the gene that encodes the enzyme that converts A to B or the gene that encodes the enzyme that converts B to C. Explain why you selected this colony or colonies. Colonies #6 and #9. These cells cannot make lysine (compare plate 1 and 2), so they are clearly carrying a mutation in one of the enzymes needed to make lysine. Because they can grow on plates with minimal + intermediate C, we know that these cells can take intermediate C and make lysine. This indicates that the final two steps in the pathway are intact, which means that either the enzyme that converts A to B or the enzyme that converts B to C is missing. By repeating the mutagenesis experiment you isolate 10 mutants that cannot synthesize lysine. You then perform a complementation test on these mutants.
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