Module 5: Ames Test & types of mutations

How is the Ames Test described?

As a fast and sensitive assay of the ability of a chemical compound or mixture to induce mutations in DNA.

What are some of the benefits of using the Ames Test?

Because it doesn't use a live animal model, it is inexpensive, easy, and fast. (ALSO sensitive)

Because there are different his-‐ mutations in these strains what happens?

Because there are different his-‐ mutations in these strains, reversion to the his+ phenotype will require different molecular lesions (mutations). Since different mutagens can exert their effect on DNA through different mechanisms, using strains containing different mutations allows us to identify mutagens that have differing effects on DNA

How to calculate reversion frequency

Calculate reversion frequency as the average number of revertant colonies per half for each strain, based on your negative control.

How to determine the colonies in each experimental compound

Determine the colonies in each experimental compound, subtract the # of spontaneous reversion colonies then divide by each positive control and multiply by 100%.

What does a positive result in the Ames test indicate?

It is mutagenic, but not necessarily carcinogenic to humans. that would require further testing in mammalian model systems

What does the Ames Test test for?

Mutagenicity of certain compounds

Does the Ames Test use a live animal model?

NO

What types of mutations were in our mutated strains of bacteria? (2 different)

TA1535 contains a T to C missense mutation in hisG gene, leading to a leucine to proline amino acid substitution. TA1538 has a deletion of 1 base pair (C) in the hisD gene, causing a -1 frameshift mutation, changing two amino acids and brings a stop codon into the reading frame 133 amino acids prematurely

What are the results of the assay?

The # of colonies growing on the plate indicates the # of revertants large amounts of growth = mutagen (positive result) little or no growth = not mutagen (negative result)

How to calculate relative mutagenicity

To calculate the relative mutagenicity, you will determine the average colonies in each positive control and subtract the average spontaneous reversion number.

Is the bacteria we test susceptible to contamination by other microbes?

Yep, that is why sterile technique is super important

Why can bacteria be used to test for mutagens?

because DNA is the same in all organisms, any living organism can be used to test for mutagens so bacteria can be used as a 1st step in identifying potential human carcinogens w/out waiting for long-lived mammals to develop cancer

Why are the secondary mutants called revertants?

because they have reverted to the his+ genotype and phenotype and can now grow just fine in media lacking histidine

What are some additional mutations in the bacterial strains?

both have a defect in the lipopolysaccharide cell wall causing them to be more vulnerable to exogenous mutagens. both have a defective DNA excision-repair mechanisms which would normally correct mutations arising during DNA replication or form exogenous mutagens.

How can the rate of mutagenicity differ in different organisms?

due to the rate of chemical absorption by cells and differential metabolism of compounds (like in the mammalian liver)

Are S. tymphimurium haploid or diploid bacteria?

haploid

What was Dr. Ames work critical in?

linking mutations in DNA to carcinogenesis like with DDT, AF-2, tris-BP, and hair dye mutagenic coumpounds

What bacteria did we use?

mutant strains of salmonella tymphimurium

What mutations do the S. Tymphimurium contain already?

mutations in the gene encoding an enzyme used to synthesize the amino acid histidine (His- genotype) since the bacteria require histidine to make many of their proteins, these mutant bacteria will die unless the media in which they are grown contains histidine

What do we do in the assay?

plate his- bacteria onto media with trace amounts of histidine and add chemicals to be tested for mutagenicity

What were the positive controls of the Ames Test?

sodium azide 4-nitro-o-phenylenediame

What are secondary mutations and how often do they occur?

they typically occur at a low spontaneous rate; these mutants are called revertants

In the original Ames Test a liver extract is added to the plates as well, why?

to simulate how mammalian liver enzymes can modify compounds in some cases the liver detoxifies the compounds but some are actually rendered more toxic after modification.