BIOA01H3 Lecture Notes - Lecture 31: Auxotrophy, Arginine, Citrulline

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12 Jun 2016

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Beadle and tatum grew nutritional mutants (auxotrophs) of neurospora on a minimal medium supplemented with a single amino acid such as arginine (arg). They are working on neurospora, some type of mold. They"re looking at mutations that are a problem in these cells. We"re looking at the phenotypic inability to grow. Some mutants can"t grow on certain food; they need something to be added to it. They need supplements, in this case it"s amino acids. They need a specific type of food added into their medium in order for them to grow. So, auxotrophas are ones that need something special added into their diet. They hypothesized that each auxotrophic strain had a defect in a gene that codes for an enzyme needed to synthesize a particular amino acid (one gene one enzyme hypothesis). The mutations occurring in those mutants are changes that resulted in a particular enzyme being functioning properly.

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You isolate 3 new yeast mutants (mutants 1, 2, and 3) that are not able to grow unless the amino acid glutamine is added to the growth medium. Genetic analysis tells you the mutants are defective for different genes. You first analyze the mutants for glutamine synthetase (GS) enzyme activity, and all three have undetectable levels of it. Mutant 1 makes normal levels of GS mRNA and protein and the protein is normal in size, while mutants 2 and 3 do not make GS mRNA or protein. You next analyze levels of histone acetylation at the glutamine synthetase gene promoter, and find that they are normal in mutant 1 and mutant 3, but low in mutant 2. Finally, you do a DNAseI hypersensitivity assay and find that mutant 1 looks the same as the wild type, but mutants 2 and 3 have lost a hypersensitive site that is present near the promoter of the GS gene in the wild type.

a. If the mutations causing the 3 GS gene defects are in a chromatin âwriterâ gene, a chromatin âreaderâ gene, and in the GS gene, which mutant is defective for which gene? Explain your answers.

Mutant 1:

Mutant 2:

Mutant 3 :

b. For the GS gene mutant, indicate where in the GS gene the mutation must be (promoter, enhancer, splice junction, coding region missense, coding region nonsense) and explain why.

c. For the chromatin writer mutant, name the enzymatic activity it must encode, and explain why. d. For the chromatin reader mutant, name the complex or complexes that could be affected, and explain how you know this.

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BIOA01H3 Lecture Notes - Lecture 31: Auxotrophy, Arginine, Citrulline

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