17. Mutation, Repair, and Recombination

Types of Mutations

17. Mutation, Repair, and Recombination

Types of Mutations

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Problem 33c

Textbook Question

In an experiment employing the methods of the Ames test, two strains of Salmonella are used. Strain A contains a base-substitution mutation, and Strain B contains a frameshift mutation. Four plates are prepared to test the mutagenicity of the compound ethyl methanesulfonate (EMS). Plate 1 is a control plate with Strain A and S9 extract but no EMS. Plate 2 is also a control plate and contains Strain B and S9 extract but no EMS. Plate 3 contains Strain A along with S9 extract and EMS, and Plate 4 contains Strain B, S9 extract, and EMS.
Why is the S9 extract added to each of the plates?

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The S9 extract is a mixture of enzymes derived from the liver of a rodent, typically a rat. It is used in the Ames test to mimic the metabolic processes that occur in a living organism.

Many compounds are not directly mutagenic but become mutagenic after being metabolized by enzymes in the liver. The S9 extract contains these metabolic enzymes, such as cytochrome P450, which can activate or detoxify potential mutagens.

By adding the S9 extract to the plates, the experiment simulates the metabolic activation of the compound (in this case, EMS) as it would occur in a living organism. This ensures that the test accounts for both direct and metabolically activated mutagens.

The inclusion of S9 extract in both control and experimental plates ensures that any observed effects are due to the compound being tested (EMS) and not the metabolic enzymes themselves.

In summary, the S9 extract is essential for assessing the mutagenicity of compounds that require metabolic activation, providing a more accurate representation of how the compound might behave in a biological system.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Ames Test

The Ames test is a widely used method for assessing the mutagenic potential of chemical compounds. It employs specific strains of bacteria, typically Salmonella, that are sensitive to mutations. By observing the growth of these bacteria in the presence of a potential mutagen, researchers can infer whether the compound causes genetic changes, which may indicate carcinogenic properties.

S9 Extract

S9 extract is a metabolic activation system derived from the liver of rodents, often used in mutagenicity testing. It contains enzymes that mimic the metabolic processes that occur in living organisms, allowing researchers to evaluate how a compound is metabolized and whether it becomes mutagenic after such transformation. The addition of S9 extract helps to simulate in vivo conditions, providing a more accurate assessment of a compound's mutagenic potential.

Types of Mutations

Mutations can be classified into various types, including base-substitution mutations and frameshift mutations. A base-substitution mutation involves the replacement of one nucleotide with another, potentially altering a single amino acid in a protein. In contrast, a frameshift mutation results from the insertion or deletion of nucleotides, shifting the reading frame of the genetic code and often leading to significant changes in protein structure and function. Understanding these mutations is crucial for interpreting the results of the Ames test.

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Textbook Question

Mutations in the IL2RG gene cause approximately 30 percent of severe combined immunodeficiency disorder (SCID) cases in humans. These mutations result in alterations to a protein component of cytokine receptors that are essential for proper development of the immune system. The IL2RG gene is composed of eight exons and contains upstream and downstream sequences that are necessary for proper transcription and translation. Below are some of the mutations observed. For each, explain its likely influence on the IL2RG gene product (assume its length to be 375 amino acids).

Nonsense mutation in a coding region

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Textbook Question

Alkaptonuria is a human autosomal recessive disorder caused by mutation of the HAO gene that encodes the enzyme homogentisic acid oxidase. A map of the HAO gene region reveals four BamHI restriction sites (B1 to B4) in the wild-type allele and three BamHI restriction sites in the mutant allele. BamHI utilizes the restriction sequence 5′-GGATCC-3′. The BamHI restriction sequence identified as B3 is altered to 5′-GGAACC-3′ in the mutant allele. The mutation results in a Ser-to-Thr missense mutation. Restriction maps of the two alleles are shown below, and the binding sites of two molecular probes (probe A and probe B) are identified.

DNA samples taken from a mother (M), father (F), and two children (C1 and C2) are analyzed by Southern blotting of BamHI-digested DNA. The gel electrophoresis results are illustrated.

Explain how the DNA sequence change results in a Ser-to-Thr missense mutation.

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Textbook Question

In an experiment employing the methods of the Ames test, two strains of Salmonella are used. Strain A contains a base-substitution mutation, and Strain B contains a frameshift mutation. Four plates are prepared to test the mutagenicity of the compound ethyl methanesulfonate (EMS). Plate 1 is a control plate with Strain A and S9 extract but no EMS. Plate 2 is also a control plate and contains Strain B and S9 extract but no EMS. Plate 3 contains Strain A along with S9 extract and EMS, and Plate 4 contains Strain B, S9 extract, and EMS.

Characterize the expected distribution of colony growth on the four plates. Defend your growth prediction for each plate.

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Textbook Question

What event is being detected by growth of a colony on any of the four plates?

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Textbook Question

Suppose the compound being tested was proflavin instead of EMS. Would this change the Ames test results? Explain why or why not.

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Textbook Question

Infantile cardiomyopathy is a devastating disorder that is fatal during the first year of life due to defects in the function of heart muscles resulting from mitochondrial dysfunction. A study, performed by Götz et al. [(2011). Am. J. Hum. Genet. 88:635–642), identified two different causative mutations in the gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS). One mutation changes a leucine residue at amino acid position 155 to arginine (p.Leu155Arg). The other mutation changes arginine at position 592 to tryptophan (p.Arg592Trp). The mtAlaRS enzyme has an N-terminal domain (amino acids 36–481) that catalyzes tRNA aminoacylation and an internal editing domain (amino acids 484–782) that catalyzes deacylation in the case that the tRNA is charged with the wrong amino acid.

Consider the position of the disease causing missense mutations in the mtAlaRS gene in the context of the known protein domains of this enzyme. What predictions can you make about how these mutations impair protein synthesis within mitochondria in different ways?

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Textbook Question

Which mutation would you predict has a more severe impairment of translation in mitochondria, and why?

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Textbook Question

Base-substitution mutations often change the amino acid specified by a codon. For each of the amino acid changes listed, determine which ones can result from a one–base-pair substitution. For those that can result from a one–base-pair substitution, give the possible wild-type and mutant codons, listing multiple possibilities if there is more than one option.

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