Ames Test
Developed by Bruce Ames and his colleagues in the 1970s. Tests the mutagenicity of different compounds Is used by FDA to test many chemical rapidly and inexpensively Uses special bacteria that are very sensitive to many mutagenic agents
Ames Test
The picture (courtesy of Bruce Ames) shows a qualitative version of the Ames test. A suspension of a histidinerequiring (His) strain of Salmonella typhimurium has been plated with a mixture of rat liver enzymes on agar lacking histidine. The disk of filter paper has been impregnated with 10g of 2-aminofluorene, a known carcinogen. The mutagenic effect of the chemical has caused many bacteria to regain the ability to grow without histidine, forming the colonies seen around the disk. The scattered colonies near the margin of the disk represent spontaneous revertants.
Characteristics of mutants strains of S.typhimurium used for Ames Test cannot synthesize histidine. are very susceptible to additional mutations because they lack the normal repair mechanisms found in bacteria. more permeable than wild-type bacteria to external chemicals, including potential mutagens.
Different mutant strains of S.typhimurium with different mutations in their DNA
TA 1535 has a base substitution that produces a missense mutation in the gene coding for the first enzyme of histidine synthesis. The mutant enzyme has a proline where a leucine is in the wild-type enzyme. TA 100 is very similar to 1535, but is also supposed to detect a different range of mutagens. TA 1537 has a frameshift mutation (deletion of one nucleotide) in a different gene than is mutated in 1535. TA 1538 has a different frameshift mutation (insertion of one nucleotide) in the same gene that is mutated in TA 1537. TA 98 is similar to 1538 but is supposed to detect more mutagens than 1538 does. TA 102 is significantly different from the others. It has an ochre mutation which means that it has a nonsense mutation. This mutation occurs in the same gene as is mutated in the strain TA 1535.
His Operon
An operon is a group of key nucleotide sequences including an operator, a common promoter, and one or more structural genes that are controlled as a unit to produce messenger RNA (mRNA). Operons occur primarily in prokaryotes and nematodes. They were first described by Francois Jacob and Jacques Monod in 1961. The his operon encodes the enzymes for the biosynthesis of the amino acid histidine. His- mutants are histidine auxotrophs (that is, they require histidine for growth). The hisD gene encodes the enzyme which catalyzes the last step of histidine biosynthesis, the conversion of histidinol to histidine.
Mutations
A mutation is any change in a DNA sequence from the original sequence of nucleic acids Spontaneous mutation are just errors that occur during DNA replication when cells divide, there is no obvious cause on which to blame the mutation. - An average of nearly one mutation (error) in DNA every time one cell divides. - Cells have ways to repair the mutated DNA, and they usually do, but if the mistake is overlooked, the change in the DNA is carried on in future replications in the cell. Induced Mutations - New mutations created by treating an organism with a mutagenizing agent. - Mutations may be induced by exposure to ultraviolet rays and alpha, beta, gamma, and X radiation, by extreme changes in temperature, and by certain mutagenic chemicals such as nitrous acid, nitrogen mustard, and chemical substitutes for portions of the nucleotide subunits of genes. - H. J. Muller, an American geneticist, pioneered in inducing mutations by Xray radiation (using the fruit fly, Drosophila).
Types of point mutations
Missense mutations: transitions, transversions. Nonsense mutations: amber, ochre, opal. Frameshift mutations: deletion, insertion. .
Mutagenic Agents
These are substances, conditions and forms of energy that significantly increase the frequency of mutations. Examples of a forms of energy are ultraviolet light, x-rays, cosmic energy, gamma radiation, alpha particles, beta particles and neutrons. Examples of substances that are mutagens are nitrous acid, hydroxylamine, ethyl methanesulfonate, 5-bromouracil, celery, benzo (a) pyrene, acridine dyes, fungally contaminated peanuts or peanut butter, and many, many more.
Mode of action of chemical mutagenic agents
Alkylating agents are chemicals that donate alkly groups to other molecules. Ethyl methanesulfonate (EMS) is an example.
Base Analogs
Base analogs are similar to the actual correct base that get incorporated into the DNA as would its natural counterpart. The problem is that, if they are more prone to tautomeric shifts than the natural base, the frequency for mutation goes up, substantially. E.g., 5-bromouracil is an analog to thymine. It undergoes a tautomeric shift to base pair with guanine instead of adenine, causing a transition.
Deaminating agents
Cause the loss of the amino group. We covered spontaneous deamination of cytosine above. Deaminating agents would increase the frequency of cytosine deamination, greatly. Nitrous acid is a deaminating agent.
Intercalation agents
Compounds that can slide between the nitrogenous bases in a DNA molecule. This tends to cause a greater likelihood for slippage during replication, resulting in an increase in frameshift mutations.
Mutagens
Hydroxylating agents add an OH group to a position on the DNA base cytosine, causing a G:C to A:T transition. Viruses that can integrate into the genome are also mutagenic agents.
The Genetic Code
Ala - Alanine Arg - Arginine Asn - Asparagine Asp - Aspartic Acid Cys - Cysteine Gln - Glutamine Glu - Glutamic Acid Gly - Glycine His - Histidine Ile - Isoleucine Leu - Leucine Lys - Lysine Met Methionine Phe - Phenylalanine Pro - Proline Ser - Serine Thr - Threonine Trp - Tryptophan Tyr - Tyrosine Val - Valine
