Question

The Ames Test In a nutrient medium that lacks histidine, a thin layer of agar containing \(\sim 10^{9}\) Salmonella typhimurium histidine auxotrophs (mutant cells that require histidine to survive) produces \(\sim 13\) colonies over a two-day incubation period at \(37^{\circ} \mathrm{C}\) (see Eig \(25-19\) ). How do these colonies arise in the absence of histidine? When investigators repeat the experiment in the presence of \(0.4 \mu \mathrm{g}\) of 2 -aminoanthracene, the number of colonies produced over two days exceeds 10,000 . What does this indicate about 2-aminoanthracene? What can you surmise about its carcinogenicity?

Short answer

The colonies arise from spontaneous mutations in absence of histidine. 2-aminoanthracene increases mutation rate, suggesting carcinogenicity.

Step-by-step solution

Understanding Histidine Auxotrophs

Salmonella typhimurium histidine auxotrophs are mutant cells that cannot synthesize histidine on their own. When introduced to a nutrient medium that lacks histidine, these cells cannot grow unless they undergo a mutation that allows them to synthesize histidine again.

Observation of Spontaneous Mutation

In the absence of histidine, approximately 13 colonies arise over two days. This indicates that a small number of cells have mutated 'reverted' back to non-mutant forms that can synthesize histidine; these spontaneous mutations are rare occurrences.

Effect of 2-aminoanthracene

When the experiment is conducted with 0.4 µg of 2-aminoanthracene, the resultant colony count exceeds 10,000. This indicates that 2-aminoanthracene significantly increases the mutation rate, causing many cells to revert to histidine independence.

Inferring Carcinogenicity of 2-aminoanthracene

Since a high mutation rate is typically associated with carcinogenic compounds, the large number of colonies suggests that 2-aminoanthracene may promote changes in DNA that could potentially lead to cancer. Therefore, it's likely carcinogenic.

Key concepts

Histidine Auxotrophs

Histidine auxotrophs are a specialized class of mutant organisms. In this context, we're discussing a specific strain of bacteria called _Salmonella typhimurium_. These mutant bacteria are unable to synthesize an essential amino acid, histidine, a vital building block for protein synthesis.
Without histidine, these cells cannot grow or reproduce unless they acquire a mutation allowing them to synthesize histidine again. In an environment lacking this amino acid, histidine auxotrophs serve as critical indicators of genetic mutations. When a mutation occurs that corrects their genetic defect, these bacteria regain the ability to produce histidine and subsequently can form colonies.

Spontaneous Mutation

Spontaneous mutations refer to random genetic changes that occur naturally without any external influence. In the Ames test scenario, where no histidine is present, only a few bacterial colonies arise due to spontaneous mutation.
These small number of mutations change the bacteria back to non-mutant forms, enabling histidine synthesis. In a laboratory setting, observing these rare changes highlights the inherent randomness of genetic mutation. Even in a seemingly constant environment, natural processes can induce these genetic modifications.
This background rate of spontaneous mutation is crucial for studying the effects of external agents, as it establishes a benchmark for mutation rates in a controlled environment.

2-Aminoanthracene

2-Aminoanthracene is a chemical compound used in the Ames test to study mutagenicity—a compound’s ability to induce genetic mutations. In the original exercise, when _Salmonella typhimurium_ histidine auxotrophs are exposed to 2-aminoanthracene, thousands more colonies appear compared to the control.
This dramatic increase suggests that 2-aminoanthracene induces mutations at a much higher rate than what occurs naturally. By reverting auxotrophs to histidine-producing forms, it helps scientists evaluate the compound's mutagenic potential.

• It serves as a potent example in genetic research for assessing mutation risks.
• Laboratory experiments with this compound reveal how potentially harmful mutations could be triggered in a living organism.

Carcinogenicity

Carcinogenicity refers to the potential of a substance to cause cancer. The Ames test provides insights into whether a compound could be carcinogenic by measuring how much it increases the rate of genetic mutation.
In the case of 2-aminoanthracene, its ability to significantly boost the mutation rate suggests its potential as a carcinogenic agent. Substances that elevate genetic mutation rates often correlate with carcinogenic properties because cancer typically involves genetic changes. A high mutation rate can lead to faulty DNA replication, uncontrolled cell division, and tumor formation.
Thus, the presence of over 10,000 colonies in the test indicates that 2-aminoanthracene might cause similar mutations in more complex organisms, raising concerns regarding its safety and potential health risks.