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10.2: Testing for Mutagenic Chemicals in Bacteria and Mice

  • Page ID
    4897
  • Ames test

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    Figure 10.2.2 Blue Mouse

    Big Blue mice are transgenic for a segment of DNA that contains the DNA of bacteriophage lambda, a virus that infects E. coli, and which serves here as the vector for 3 genetic elements from the lac operon of E. coli:

    • the lacI gene
    • the operator of the operon
    • the beta-galactosidase (lacZ) gene

    The Assay

    The transgenic mice are given repeated doses of the suspected carcinogen for a week or two. If the chemical is mutagenic, it will cause random mutations throughout the genome of each mouse cell. If a mutation occurs in either the lacI gene (which encodes the lac repressor) or the operator,

    the gene (lacZ) for beta-galactosidase will no longer be repressed. To detect this,

    • The DNA is extracted from the tissues of the treated mouse.
    • The vector is isolated and used to make functional bacteriophages.
    • E. coli cells are mixed with the bacteriophage and spread on a solid culture medium.
    • The bacteriophages infect and destroy ("lyze") the E. coli cells.
    • This causes clear circular zones, called plaques, to appear in a "lawn" of bacteria.
    • Before they die, cells that have been infected by bacteriophages carrying a mutated lacI or operator will produce beta-galactosidase.
    • This reacts with a substrate in the culture medium turning it blue.
    • Bacteriophages with unmutated genes produce colorless plaques because no beta-galactosidase is synthesized.
    • Count both colorless and blue plaques.
    • The number of blue plaques divided by the total number of plaques gives the mutation frequency.
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    Figure 10.2.4 Blue Plaque courtesy of Stratagene

    This photograph shows one mutant (blue) plaque on a lawn of E. coli containing many non-mutant (clear) plaques.

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