Ames Test for Detecting Chemical Mutagens

What is the Ames test used for?

Ames test: uses bacteria to show whether or not a certain chemical substance is a mutagen -If the test is positive then the chemical can be further tested in animals to see if it is a possible human carcinogen

What would we need to do to determine if any of the chemicals we tested are human carcinogens?

Test on single-celled Eukaryotic cells and then scale it up to multicellular organisms such as rodents

Explain what the CM plates tell you about each of the chemicals (water, NaAz, 4-Nitro, and test) that we used for the toxicity test

*CM - H2O (histidine, bacteria, H2O) -Control to show viable culture and mutants grow with histidine supplied by the media; should see a lawn *CM - NaAz or Nitro (histidine, bacteria, mutagen) -Shows growth of culture with mutagen -If a zone of clearing is present, then we know the chemical on the disc is toxic *CM - Test (histidine, bacteria, test mutagen) -Shows growth of culture with unknown chemical -Looking for a zone of clearing for toxicity test

Explain the difference between insertion mutations and base substitutions mutations. Which of our positive control chemicals induces which type of mutation? Which strain of Salmonella was used to detect each type of mutation?

*Insertion/frameshift mutations: few nucleotides are inserted or deleted, changing the reading frame, or proper groupings of 3 nucleotides in mRNA codons -Nitro -Strain F *Base substitutions mutations: one nucleic base is switched for another nucleic base -Sodium Azide (NaAz) -Strain M

Explain what the MM plates tell you about each of the chemicals (water, NaAz, 4-Nitro, and test) that we used for the Ames test

*MM - H2O (bacteria + water) -Shows rate of spontaneous back mutations (mutations created during RNA replication) -Should have few colonies *MM -NaAz or Nitro (bacteria + mutagen) -Shows the rate of induced back mutations (mutations caused by mutagen above the rate of mutations) -Should see more single colonies than on MM H2O plate *MM - Test (bacteria + unknown mutagen) -This determines if the test chemical is a mutagen and if it causes back mutation

How are the terms mutagen and carcinogen related?

*Mutagen: chemical substance or physical agent that can induce changes in the base sequence of DNA -Many mutagens are also carcinogens as the mutations they induce can cause cancer in humans and animals over time

Explain the difference between a spontaneous mutation and an induced mutation. Which of your test plates contains colonies produced by spontaneous mutation? Which contains colonies produced by induced mutation?

*Spontaneous mutation: random mutations due to DNA replication errors -MM H2O *Induced mutation: mutations as a result of a mutagen -MM NaAz or Nitro; MM Test (potentially)

What is a mutagen?

A chemical or agent that is capable of altering nucleotide sequence of DNA

Explain how the Ames test works (what is a back mutation and how can these occur?)

Back mutation: a second mutation that reverts certain mutated genes in bacteria back to its wild type -Ames test relies on back mutations -A filter paper disc is saturated with a sample of the chemical to be tested and placed on MM and is spread with his - bacteria -Chemical diffuses out onto media and if it is mutagenic, it will induce back mutations in his - causing it to be his + ***Bacteria with back will grow on MM

Explain how the CM plates and MM plates differ from each other

CM = Complete Media -Contain histidine and should allow growth for all strains of his - bacteria (F and M strain) MM = Minimal Media -Does not contain histidine and therefore both strains of his - bacteria will be unable to grow unless a back mutation occurs

Did the test chemical cause mutations? If so, which type(s) of mutations (insertions or base substitution) did it cause? Explain how you know.

Yes -Strain M -Know this because there were single colonies on the MM test plate, indicating a mutation occurred -Since strain M was base substitution, then the mutation that must have occurred must have been base substitution