Unformatted text preview: BIS101-001Genes and Gene Expression Ames Test; Complementation Test Chapters 12 Lecture #13 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 1 Molecular Mechanism of Mutation The Ames Test March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 2 Key Terms: The Ames Test is a simple bacterial assay that allows researchers to quickly screen thousands of chemicals for their potential genotoxicity and carcinogenicity. The test is based on the principles of mutation analysis and bacterial genetics. Reversion analysis is the study of the frequency, cause and nature of back mutations. Wild type bacteria (prototrophs) are mutagenized with well characterized mutagens and mutant strains are isolated that are incapable of growth on minimial medium. These mutants are called auxotrophs. The risk of a real or potential mutagen can be assessed by calculating the HERP %: Human Exposure/Rodent Potency percentage. Bacteria have a means for the sexual exchange of genetic information. In addition to a circular chromosome, bacteria also contain extrachromosomal elements called plasmids or F factors. Complementation is the process in which heterozygosity for chromosomes bearing mutant recessive alleles for 2 different genes produces a normal phenotype. Complementation Test is a method of discovering whether 2 recessive mutations are in the same or separate complementation groups (i.e., genes). To visualize the placement of a large number of mutations in to several complementation groups, a complementation table is constructed.
BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 3 March 19, 2012 The Ames Test Since World War II, the chemical industry has created more than 150,000 new chemicals, many of which have been introduced into the environment. New chemical compounds are being created at a rate of 1,500 to 2,000/year. Many of these compounds are noxious and/or toxic to humans and other animals. Some of these compounds are genotoxic, or capable of damaging the DNA in our chromosomes. We are also now discovering that many compounds, which occur naturally in plant and animal tissues, can also be highly mutagenic. For these reasons researchers have developed methods to monitor the environment for potential genotoxic compounds using rapid, inexpensive, and reliable tests like the Ames test. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 4 Ames Test
Because there is a 90% correlation between mutagens and carcinogens (cancer-causing compounds) and teratogens (cause of birth defects), it is important to identify and characterize these mutagens in terms of their mode of action and potency. During the period between 1964 to 1974, Dr. Bruce Ames, Professor of Biochemistry at UC Berkeley, developed a rapid screen for potential mutagenic compounds using a series of defined hismutants of Salmonella typhimurium. With the "Ames Test", thousands of chemicals can be quickly tested for mutagenicity. Chemicals that test positive for mutagenicity could then be screened for potential carcinogenicity in small animal models. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 5 Ames Test The Ames Test is based on the principles of mutation analysis and bacterial genetics. The test uses a common bacterial system for reversion analysis which includes a series of welldefined histidine deficient (his-) Salmonella auxotrophic strains. These strains can grow on minimal medium only when histidine is added to the medium. Once the spontaneous reversion rate has been determined (e.g., 10-9 or 1 his+ revertant in 1 billion his- cells), one can examine the effects of compounds of unknown toxicity on the reversion rate.
March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 6 Ames Test: Basic Bacterial Genetics Bacterial mutants incapable of growing on minimal medium (glucose and salts) are called auxotrophs. These mutants have lost (by mutation) the ability to perform biochemical functions such as the ability to utilize the milk sugar, lactose, as a carbon source or to synthesize one or more amino acids. Wild type strains, capable of performing these functions, are called prototrophs and therefore they are capable of growing on minimal media. Some Common Bacterial Genotypic Symbols Symbol bioargmethisthi lacgalstrr strs
March 19, 2012 Character of phenotype Requires biotin for growth Requires arginine for growth Requires methionine for growth Requires histidine for growth Requires thiamin for growth Unable to use lactose as a carbon source Unable to use galactose as a carbon source Resistant to streptomycin Sensitive to streptomycin
BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 7 Ames Test: Reversion Analysis
Much of what we now know about the molecular mechanism of mutation was determined by reversion analysis of bacterial mutants (e.g., Salmonella typhimurium and E. coli). Reversion involves the reversal of a mutation back to wild type by a second spontaneous or induced mutation in the same position. Unlike intragenic or extragenic suppression, reversion involves a back mutation that restores the original base pair in the wild type strain or cell. Transversion Forward mutations can be induced with well-characterized mutagens at a frequency characteristic for that mutagen. Back mutations occur spontaneously or they can be induced by a mutagen. UAC
Forward Mutation UAC
Back Mutation (reversion) UAG UAG March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 8 Reversion Analysis If, for example, a his- mutant called strain "C" was generated with hydroxylamine (i.e., promotes GC to AT transitions), it is possible to deduce the nature of an unknown mutagen by exposing this mutant to it. If the unknown compound increases the reversion rate of mutant C from 10-9 to 10-6, then it must be a mutagen of the type that causes AT to GC transitions, which is the only way to reverse the GC to AT transition caused by hydroxylamine. 109 Strain "C" cells H2O X Minimal Medium without histidine
March 19, 2012 Minimal Medium without histidine
9 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Components of the Ames Test A collection of defined his- Salmonella mutants with known spontaneous reversion rates: q q q his- mutant A (GC to CG transversion, 10-8 ) his- mutant B (single bp deletion, 10-10 ) his- mutant C (GC to AT transition, 10-9 ) Salmonella cells made permeable to various compounds as a result of a "permeability" mutation in Salmonella. Introduction of a mutation that prevents repair of mutations into Salmonella cells. Growth medium that incorporates rat liver microsomes to help modify some compounds into their mutagenic forms. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 10 Ames Test Protocol
Spread each of six histidine minimal agar plates (lacking histidine) with 109 Salmonella cells of hismutants A, B and C, respectively. Make a sterile solution of unknown compound "X". Soak 3 small filters disks in "X" and place them in the center of the three histidine minimal agar plates. Soak three disks in sterile water and place them in the center of the other three plates as negative controls. Incubate plates at 37C for 20 hours and count the number of his+ colonies around each disk.
March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 11 Mammalian Metabolism of Benzo(a)pryrene Shown at the left is a sequence modifications of benzo(a)pryrene by liver enzymes. BP is a component of car exhaust. Note the product (3) is reactive with guanine residues in DNA. Product number (4) is not reactive. This demonstrates how potential genotoxic compounds can be created by liver metabolism and subsequently detoxified by other enzymes of metabolism.
BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 12 March 19, 2012 Ames Test
Enzymes in the rat liver are induced by Arochlor or PCB. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 13 Ames Test of Unknown Compound X
109 Cells of Strains "A", "B" and "C" added to each plate his- Mutant A CG GC, 10-8
X his- Mutant B deletion, 10-10
X his- Mutant C GC AT, 10-9
March 19, 2012 0
BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 2
14 Aflotoxin B1 Toxin produced by certain strains of the fungi Aspergillus flavus and A. parasiticus. A potent mutagen and carcinogen that commonly contaminates grains and peanuts. Promotes hepatocarcinogenesis in most mammalian species. Produces GC to TA transversions. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 15 Assessing Risk of Different Mutagens
(contaminant of peanuts) Nitrosamines AF-2
(BBQ meats cosmetics) # of his+ Revertants (food preservative) Tris-BP
(flame retardant) Lady Clairol
(balsam natural brown) Saccharine
(artificial sweetener) EDB
(fumigant) Microgram of Mutagen March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 16 Conclusion As shown on the previous slide, compound X is a potent mutagen that promotes CG to GC transversions 100-fold above the negative controls (i.e., above the normal reversion rate for His- mutant Strain A). This compound is a candidate for carcinogenicity testing. Some questions for further consideration include:
q q Are all genotoxic compounds equally hazardous to our health? What precautions should be taken to regulate these compounds in the environment? To help answer these questions, Dr. Ames developed the concept of Human Exposure/Rodent Potency Percentage or HERP%. Human exposure is defined as the annual exposure per 70kgs of body weight, while rodent potency is defined as the amount of mutagen needed to cause cancer in laboratory rats. For example, HERP% can be use to compare the potential dangers of aflatoxin, a fungal toxin found in peanut butter and ethylene dibromide, a common insect fumigant.
Aflatoxin 0.000064mg/70kg X 100 = 2.1% 0.003mg/kg 150mg/70kg X 100 = 10,000% 1.5mg/kg
17 Ethylene dibromide March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Complementation Test What is a complementation group?
(How many genes are needed to produce a phenotype?) March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 18 Complementation Test: 2 gene example In the example below, two genes are required for a phenotype. The complementation or Cis/Trans test got its name from genetic studies in which different recessive mutations were put in a cis (on the same chromosome in the same gene or "cistron") or in the trans configuration (on separate chromosomes in the same gene). When two different mutations (red and black boxes) are in the trans configuration in the same gene, they do not complement each other. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 19 Molecular Basis of Complementation (2 genes)
A a a A a X X X b X B B b B B b b' X Mutant b Mutant b' Mutant
20 Mutant a Mutant b Wild type X Mutant a Mutant b Wild type X a' A A Mutant a Mutant a' Mutant X March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Complementation Analysis Many phenotypes are the result of the action of several genes. Mutations in any one of the genes can produce the mutant phenotype. These requires that mutants be placed into complementation groups before mapping.
Lactose Operon I P O Z Y (A) Tryptophan Operon P O att E D C B A March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 21 Complementation Matrix for 9 Mutants
1 1 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Group Mutants A B C D E
+ - 1, 3, 7 2 4, 6 5, 9 8 P O
March 19, 2012 att E 8 D 5,9 C 4, 6 B 2 A 1,3,7
22 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Summary:
Molecular Basis of Mutation. Single base pair substitutions, insertions, or deletions can change the way the triple code of the correct open reading frame (zero reading frame) is read. These forward mutations can lead to loss of function or a gene or create a new function. Suppressor Mutations. Mutations can be reversed by a back mutation or by a suppressor. A suppressor is a second mutation within the gene (intragenic) or in another gene (extragenic) Spontaneous Mutations. These mutations are rare and occur as a result of errors in DNA replication, tautomeric shifts in certain bases, and permanent chemical changes in nucleotides. These changes can be caused by chemicals or by physical agents such as ionizing radiation (e.g, X-rays and ultraviolet light). Induced Mutations. Chemical and physical agents can be used to induce (i.e., increase the rate of) mutations by several orders of magnitude. Mutations can be caused by slippage in DNA replication. Ames Test. Using our knowledge of mutations to monitor mutagens in the environment. Complementation analysis: Determining the number of genes involved in producing a particular phenotype.
March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 23 ...
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This note was uploaded on 03/18/2012 for the course BIS 101 taught by Professor Simonchan during the Winter '08 term at UC Davis.
- Winter '08