Mutagenesis
Encyclopedia
Mutagenesis is a process by which the genetic information of an organism
is changed in a stable manner, resulting in a mutation
. It may occur spontaneously in nature, or as a result of exposure to mutagen
s. It can also be achieved experimentally using laboratory procedures. In nature mutagenesis can lead to cancer and various heritable diseases, but it is also the driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller
, Charlotte Auerbach
and J. M. Robson
in the first half of the 20th century.
s. In 1927, Hermann Muller
first demonstrated mutation with observable changes in the chromosomes can be caused by irradiating fruit flies
with X-ray
, and lent support to the idea of mutation as the cause of cancer. His contemporary Lewis Stadler
also showed the mutational effect of X-ray on barley in 1928, and ultraviolet
(UV) radiation on maize in 1936. In 1940s, Charlotte Auerbach
and J. M. Robson
, found that mustard gas can also cause mutation
s in fruit flies.
While changes to the chromosome caused by X-ray and mustard gas were readily observable to the early researchers, other changes to the DNA induced by other mutagens were not so easily observable, and the mechanism may be complex and takes longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775, and coal tar was demonstrated to cause cancer in 1915. The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbon
s (PAH). PAHs by themselves are not carcinogenic, and it was proposed in 1950 that the carcinogenic forms of PAHs are the oxides produced as metabolites from cellular processes. The metabolic process was identified in 1960s as catalysis by cytochrome P450 which produces reactive species that can interact with the DNA to form adducts, the mechanism by which the PAH adducts give rise to mutation however is still under investigation.
DNA may sustain more than 50,000 damages per cell per day, and some estimates put the number of oxidative adducts per cell generated through reactive reactive oxidative species at 150,000. Left uncorrected these adducts can give rise to mutation. In nature, the mutations that arise may be beneficial or deleterious - it is the driving force of evolution, an organism may acquire new traits through genetic mutation, but mutation may also result in impaired function of the genes, and in severe cases, causing the death of the organism. In the laboratory, however, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel function, as well as mutant strains with useful properties. Initially the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, later techniques were developed to introduce specific mutations.
and DNA adducts, or through error in replication and repair. Mutagenesis may also arise as a result of the presence of environmental mutagens that induces changes to the DNA. The mechanism by which mutation arises varies according to the causative agent, the mutagen
, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on the DNA producing lesions. Some however may affect the replication or chromosomal partition mechanism, and other cellular processes.
Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450. Other enzymes that may also produce mutagenic metabolites include glutathione S-transferase
and microsomal epoxide hydrolase
. Mutagens that are not mutagenic by themselves but require biological activation are called promutagens.
Many mutations arise as a result of problems caused by the DNA lesions during replication. In bacteria, extensive damage to the DNA due to mutagens produces single-stranded DNA gaps during replication which induces the SOS response
, an emergency repair process that is also error-prone, thereby generating mutations. In mammalian cells, stalling of replication at a damaged sites induces a number of rescue mechanisms that help bypass DNA lesions but which also may result in errors. The Y family of DNA polymerase
s specialisze in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymrase, transits the lesion and extend the DNA until the lesion has been passed so that normal replication can resume.
may be hydrolyzed spontaneously and 10,000 purine
sites in DNA are estimated to be depurinated
each day in a cell.. Numerous DNA repair pathway exist for the DNA, however, if the apurinic site failed to be repaired, misincorporation of nucleotide may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site. Cytidine may also become deaminated to uridine at one five-hundredth of the rate of depurination and can result in G to A transition. Eukaryotic cells also contains 5-methylcytosine
, thought to be involved in the control of gene transcription, which can become deaminated into thymine.
s.
Many compounds, such as PAHs, aromatic amines, aflatoxin
and pyrrolizidine alkaloids may form reactive oxygen species
catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication, and the bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination
of the DNA, it is however uncertain how significant the depurination as caused by the adducts is in generating mutation.
Some alkylating agents such as N-Nitrosamine
s may also require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. Alkylation
and arylation of bases can cause errors in replication. N7 and O6 of guanine and the N3 and N7 of adenine are most susceptible to attack; while N7-guanine adducts, which form the bulk of DNA adducts, appear to be non-mutagenic, alkylation at O6 of guanine is harmful because excision repair
of O6-adduct of guanine may be poor in some tissues. The O6 methylation of guanine can result in G to A transition
, while O4-methylthymine can be mispaired with guanine. The type of the mutation generated however may be dependent on the size and type of the adduct as well as the DNA sequence.
Ionizing radiations and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.
, mitomycin C and cisplatin
are used as anticancer chemotherapeutic
because their high degree of toxicity to proliferating cells.
. In human skin cells, thousands of dimers may be formed in a day due to normal exposure to sunlight. DNA polymerase η
may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as sufferers of Xeroderma pigmentosum
, are sensitive to sunlight and may be prone to skin cancer.
and proflavine
allows them to insert between bases in DNA, and cause frameshift mutation
. The intercalation into DNA of anthracycline
s such as daunorubicin
and doxorubicin
interferes with the functioning of the enzyme topoisomerase II, blocking replication as well as causing mitotic homologous recombination.
s may produce highly reactive free radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation
and deletion of part of a chromosomes. Alkylating agents like mustard gas may also cause breakages in the DNA backbone. Oxidative stress
may also generate highly reactive oxygen species
that can damage the DNA. Incorrect repair of other damages induced by the highly reactive species can also lead to mutations.
and virus
may insert DNA sequence into coding region or functional elements of a gene and result in inactivation of the gene.
may substitute for thymine in replication. Some metals such as cadmium, chromium, and nickel may alter the fidelity of DNA replication.
may be exposed to UV radition, then plated onto agar medium. The colonies formed are then replica-plated
, one in rich medium
, another in minimal medium, and mutants that have specific nutritional requirement can then be identified by its inability to grow in minimal medium and isolated.
A number of methods for generating random mutation in specific protein were later developed to screen for mutants with interesting or improved properties. This may be done by using doped nucleotides in oligonucleotides synthesis, conducting a PCR reaction in conditions that enhance misincorporation of nucleotide thereby generating mutants.
, while nitrosoguanidine,, bisulfite,, and N4-hydroxycytidine may induce GC to AT transition. These technique allows specific mutations to be engineered into a protein, however, they are not flexible in the kinds of mutants generated.
Current techniques for site-specific mutation commonly involve using mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation
, or deletion or insertion
of small stretches of DNA to be introduced at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.
The site-directed approach may be done systematically in such technique as alanine scanning
mutagenesis whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein.
. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.
More recently, techniques such as phage display
has been used to discover short peptide sequences of up to 12 residues with binding affinity for other proteins.
and murine leukemia virus
may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. Various insertional mutagenesis techniques may also be used to study the function of particular gene.
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
is changed in a stable manner, resulting in a mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
. It may occur spontaneously in nature, or as a result of exposure to mutagen
Mutagen
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are therefore also likely to be carcinogens...
s. It can also be achieved experimentally using laboratory procedures. In nature mutagenesis can lead to cancer and various heritable diseases, but it is also the driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller
Hermann Joseph Muller
Hermann Joseph Muller was an American geneticist, educator, and Nobel laureate best known for his work on the physiological and genetic effects of radiation as well as his outspoken political beliefs...
, Charlotte Auerbach
Charlotte Auerbach
Charlotte Auerbach FRSE FRS was a German zoologist and geneticist.Born in Germany, she fled to Scotland because of anti-Semitism. She became well known after 1942 when she, with A. J. Clark and J. M. Robson, discovered that mustard gas could cause mutations in fruit flies...
and J. M. Robson
J. M. Robson
John Michael 'Rab' Rabinovich, later known as J. M. Robson was a geneticist and physicist who co-founded the science of mutagenesis by mutations in fruit flies exposed to mustard gas, and who first observed neutron beta decay.-Biography:...
in the first half of the 20th century.
Background
DNA may be modified, either naturally or artificially, by a number of physical, chemical and biological agents, resulting in mutationMutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s. In 1927, Hermann Muller
Hermann Joseph Muller
Hermann Joseph Muller was an American geneticist, educator, and Nobel laureate best known for his work on the physiological and genetic effects of radiation as well as his outspoken political beliefs...
first demonstrated mutation with observable changes in the chromosomes can be caused by irradiating fruit flies
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
with X-ray
X-ray
X-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...
, and lent support to the idea of mutation as the cause of cancer. His contemporary Lewis Stadler
Lewis Stadler
Lewis John Stadler was an American geneticist. His research focused on the mutagenic effects of different forms of radiation on economically important plants like maize and barley.- Background :...
also showed the mutational effect of X-ray on barley in 1928, and ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
(UV) radiation on maize in 1936. In 1940s, Charlotte Auerbach
Charlotte Auerbach
Charlotte Auerbach FRSE FRS was a German zoologist and geneticist.Born in Germany, she fled to Scotland because of anti-Semitism. She became well known after 1942 when she, with A. J. Clark and J. M. Robson, discovered that mustard gas could cause mutations in fruit flies...
and J. M. Robson
J. M. Robson
John Michael 'Rab' Rabinovich, later known as J. M. Robson was a geneticist and physicist who co-founded the science of mutagenesis by mutations in fruit flies exposed to mustard gas, and who first observed neutron beta decay.-Biography:...
, found that mustard gas can also cause mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s in fruit flies.
While changes to the chromosome caused by X-ray and mustard gas were readily observable to the early researchers, other changes to the DNA induced by other mutagens were not so easily observable, and the mechanism may be complex and takes longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775, and coal tar was demonstrated to cause cancer in 1915. The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbon
Polycyclic aromatic hydrocarbon
Polycyclic aromatic hydrocarbons , also known as poly-aromatic hydrocarbons or polynuclear aromatic hydrocarbons, are potent atmospheric pollutants that consist of fused aromatic rings and do not contain heteroatoms or carry substituents. Naphthalene is the simplest example of a PAH...
s (PAH). PAHs by themselves are not carcinogenic, and it was proposed in 1950 that the carcinogenic forms of PAHs are the oxides produced as metabolites from cellular processes. The metabolic process was identified in 1960s as catalysis by cytochrome P450 which produces reactive species that can interact with the DNA to form adducts, the mechanism by which the PAH adducts give rise to mutation however is still under investigation.
DNA may sustain more than 50,000 damages per cell per day, and some estimates put the number of oxidative adducts per cell generated through reactive reactive oxidative species at 150,000. Left uncorrected these adducts can give rise to mutation. In nature, the mutations that arise may be beneficial or deleterious - it is the driving force of evolution, an organism may acquire new traits through genetic mutation, but mutation may also result in impaired function of the genes, and in severe cases, causing the death of the organism. In the laboratory, however, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel function, as well as mutant strains with useful properties. Initially the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, later techniques were developed to introduce specific mutations.
Mechanisms
Mutagenesis may occur endogenously, for example through spontaneous hydrolysis, or through normal cellular processes that can generate reactive oxygen speciesReactive oxygen species
Reactive oxygen species are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons....
and DNA adducts, or through error in replication and repair. Mutagenesis may also arise as a result of the presence of environmental mutagens that induces changes to the DNA. The mechanism by which mutation arises varies according to the causative agent, the mutagen
Mutagen
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are therefore also likely to be carcinogens...
, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on the DNA producing lesions. Some however may affect the replication or chromosomal partition mechanism, and other cellular processes.
Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450. Other enzymes that may also produce mutagenic metabolites include glutathione S-transferase
Glutathione S-transferase
Enzymes of the glutathione S-transferase family are composed of many cytosolic, mitochondrial, and microsomal proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.GSTs can constitute up to 10% of...
and microsomal epoxide hydrolase
Epoxide hydrolase
Epoxide hydrolase functions in detoxication during drug metabolism. It converts epoxides to trans-dihydrodiols, which can be conjugated and excreted from the body. Epoxides result from the degradation of aromatic compounds...
. Mutagens that are not mutagenic by themselves but require biological activation are called promutagens.
Many mutations arise as a result of problems caused by the DNA lesions during replication. In bacteria, extensive damage to the DNA due to mutagens produces single-stranded DNA gaps during replication which induces the SOS response
SOS response
The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced. The SOS uses the RecA protein . The RecA protein, stimulated by single-stranded DNA, is involved in the inactivation of the LexA repressor thereby inducing the response...
, an emergency repair process that is also error-prone, thereby generating mutations. In mammalian cells, stalling of replication at a damaged sites induces a number of rescue mechanisms that help bypass DNA lesions but which also may result in errors. The Y family of DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
s specialisze in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymrase, transits the lesion and extend the DNA until the lesion has been passed so that normal replication can resume.
Spontaneous hydrolysis
DNA is not entirely stable in aqueous solution. Under physiological conditions the glycosidic bondGlycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....
may be hydrolyzed spontaneously and 10,000 purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
sites in DNA are estimated to be depurinated
Depurination
In molecular genetics, depurination is an alteration of DNA in which the purine base is removed from the deoxyribose sugar by hydrolysis of the beta-N-glycosidic link between them. After depurination, an apurinic site is formed where the sugar phosphate backbone remains and the sugar ring has a...
each day in a cell.. Numerous DNA repair pathway exist for the DNA, however, if the apurinic site failed to be repaired, misincorporation of nucleotide may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site. Cytidine may also become deaminated to uridine at one five-hundredth of the rate of depurination and can result in G to A transition. Eukaryotic cells also contains 5-methylcytosine
5-Methylcytosine
5-Methylcytosine is a methylated form of the DNA base cytosine that may be involved in the regulation of gene transcription. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered .In the figure on the right, a methyl group, is...
, thought to be involved in the control of gene transcription, which can become deaminated into thymine.
Modification of bases
Bases may be modified endogenously by normal cellular molecules. For example DNA may be methylated by S-adenosylmethionine, and glycosylated by reducing sugarReducing sugar
A reducing sugar is any sugar that either has an aldehyde group or is capable of forming one in solution through isomerisation. This functional group allows the sugar to act as a reducing agent, for example in the Tollens' test or Benedict's test.-Chemistry:...
s.
Many compounds, such as PAHs, aromatic amines, aflatoxin
Aflatoxin
Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus, the most notable ones being Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are toxic and among the most carcinogenic substances known...
and pyrrolizidine alkaloids may form reactive oxygen species
Reactive oxygen species
Reactive oxygen species are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons....
catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication, and the bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination
Depurination
In molecular genetics, depurination is an alteration of DNA in which the purine base is removed from the deoxyribose sugar by hydrolysis of the beta-N-glycosidic link between them. After depurination, an apurinic site is formed where the sugar phosphate backbone remains and the sugar ring has a...
of the DNA, it is however uncertain how significant the depurination as caused by the adducts is in generating mutation.
Some alkylating agents such as N-Nitrosamine
Nitrosamine
Nitrosamines are chemical compounds of the chemical structure R1N-N=O, some of which are carcinogenic.-Usages:Nitrosamines are used in manufacture of some cosmetics, pesticides, and in most rubber products. -Occurrences:...
s may also require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. Alkylation
Alkylation
Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene . Alkylating agents are widely used in chemistry because the alkyl group is probably the most common group encountered in...
and arylation of bases can cause errors in replication. N7 and O6 of guanine and the N3 and N7 of adenine are most susceptible to attack; while N7-guanine adducts, which form the bulk of DNA adducts, appear to be non-mutagenic, alkylation at O6 of guanine is harmful because excision repair
Excision repair
Excision repair is a term applied to several DNA repair mechanisms. They remove the damaged nucleotides and are able to determine the correct sequence from the complementary strand of DNA.Specific mechanisms include:...
of O6-adduct of guanine may be poor in some tissues. The O6 methylation of guanine can result in G to A transition
Transition (genetics)
In genetics, a transition is a point mutation that changes a purine nucleotide to another purine or a pyrimidine nucleotide to another pyrimidine . Approximately two out of three single nucleotide polymorphisms are transitions....
, while O4-methylthymine can be mispaired with guanine. The type of the mutation generated however may be dependent on the size and type of the adduct as well as the DNA sequence.
Ionizing radiations and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.
Crosslinking
Some alkylating agents may produce crosslinking of DNA. Some natural occurring chemicals may also promotes crosslinking, such as psoralens after activation by UV radiation, and nitrous acid. Interstrand cross-linking blocks replication and transcription and can cause chromosomal breakages and rearrangements. Some crosslinkers such as cyclophosphamideCyclophosphamide
Cyclophosphamide , also known as cytophosphane, is a nitrogen mustard alkylating agent, from the oxazophorines group....
, mitomycin C and cisplatin
Cisplatin
Cisplatin, cisplatinum, or cis-diamminedichloroplatinum is a chemotherapy drug. It is used to treat various types of cancers, including sarcomas, some carcinomas , lymphomas, and germ cell tumors...
are used as anticancer chemotherapeutic
Chemotherapy
Chemotherapy is the treatment of cancer with an antineoplastic drug or with a combination of such drugs into a standardized treatment regimen....
because their high degree of toxicity to proliferating cells.
Dimerization
UV radiation promotes the formation of a cyclobutyl ring between adjacent thymines, resulting in the formation of pyrimidine dimersPyrimidine dimers
Pyrimidine dimers are molecular lesions formed from thymine or cytosine bases in DNA via photochemical reactions. Ultraviolet light induces the formation of covalent linkages by reactions localized on the C=C double bonds. In dsRNA, uracil dimers may also accumulate as a result of UV radiation...
. In human skin cells, thousands of dimers may be formed in a day due to normal exposure to sunlight. DNA polymerase η
DNA polymerase eta
DNA polymerase eta is a eukaryotic DNA polymerase involved in the DNA repair by translesion synthesis. The gene encoding DNA polymerase eta is POLH, also known as XPV, because loss of this gene results in the disease Xeroderma Pigmentosum Variant...
may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as sufferers of Xeroderma pigmentosum
Xeroderma pigmentosum
Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet light is deficient. In extreme cases, all exposure to sunlight must be forbidden, no matter how small. Multiple basal cell carcinomas and other skin...
, are sensitive to sunlight and may be prone to skin cancer.
Intercalation between bases
The planar structure of chemicals such as ethidium bromideEthidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
and proflavine
Proflavine
Proflavine , also called proflavin and diaminoacridine, is an acriflavine derivative, a disinfectant bacteriostatic against many gram-positive bacteria...
allows them to insert between bases in DNA, and cause frameshift mutation
Frameshift mutation
A frameshift mutation is a genetic mutation caused by indels of a number of nucleotides that is not evenly divisible by three from a DNA sequence...
. The intercalation into DNA of anthracycline
Anthracycline
Anthracyclines are a class of drugs used in cancer chemotherapy derived from Streptomyces bacterium Streptomyces peucetius var...
s such as daunorubicin
Daunorubicin
Daunorubicin or daunomycin is chemotherapeutic of the anthracycline family that is given as a treatment for some types of cancer. It is most commonly used to treat specific types of leukaemia...
and doxorubicin
Doxorubicin
Doxorubicin INN is a drug used in cancer chemotherapy. It is an anthracycline antibiotic, closely related to the natural product daunomycin, and like all anthracyclines, it works by intercalating DNA....
interferes with the functioning of the enzyme topoisomerase II, blocking replication as well as causing mitotic homologous recombination.
Backbone damage
Ionizing radiationIonizing radiation
Ionizing radiation is radiation composed of particles that individually have sufficient energy to remove an electron from an atom or molecule. This ionization produces free radicals, which are atoms or molecules containing unpaired electrons...
s may produce highly reactive free radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
and deletion of part of a chromosomes. Alkylating agents like mustard gas may also cause breakages in the DNA backbone. Oxidative stress
Oxidative stress
Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage...
may also generate highly reactive oxygen species
Reactive oxygen species
Reactive oxygen species are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons....
that can damage the DNA. Incorrect repair of other damages induced by the highly reactive species can also lead to mutations.
Insertional mutagenesis
TransposonTransposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
and virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
may insert DNA sequence into coding region or functional elements of a gene and result in inactivation of the gene.
Error in replication
While most mutagens produce effects that ultimately result in error in replication, some mutagens may affect directly the replication process. Base analog such as 5-bromouracil5-Bromouracil
5-Bromouracil is a brominated derivative of uracil that acts as an antimetabolite or base analog, substituting for thymine in DNA, and can induce DNA mutation in the same way as 2-aminopurine...
may substitute for thymine in replication. Some metals such as cadmium, chromium, and nickel may alter the fidelity of DNA replication.
Mutagenesis as a laboratory technique
Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organism. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutants strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.Random mutagenesis
Early approaches to mutagenesis rely on methods which are entirely random in the mutations produced. Cells may be exposed to UV radiation or mutagenic chemicals, and mutants with desired characteristic are then selected. For example, Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
may be exposed to UV radition, then plated onto agar medium. The colonies formed are then replica-plated
Replica plating
In molecular biology and microbiology, replica plating is a technique in which one or more secondary Petri plates containing different solid selective growth media are inoculated with the same colonies of microorganisms from a primary plate , reproducing the original spatial...
, one in rich medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
, another in minimal medium, and mutants that have specific nutritional requirement can then be identified by its inability to grow in minimal medium and isolated.
A number of methods for generating random mutation in specific protein were later developed to screen for mutants with interesting or improved properties. This may be done by using doped nucleotides in oligonucleotides synthesis, conducting a PCR reaction in conditions that enhance misincorporation of nucleotide thereby generating mutants.
Site-directed mutagenesis
It is desirable that specific changes can be introduced to the DNA. Analogs of nucleotides and other chemicals were first used to generate localized point mutations. Such chemicals may be aminopurine which induces AT to GC transitionTransition (genetics)
In genetics, a transition is a point mutation that changes a purine nucleotide to another purine or a pyrimidine nucleotide to another pyrimidine . Approximately two out of three single nucleotide polymorphisms are transitions....
, while nitrosoguanidine,, bisulfite,, and N4-hydroxycytidine may induce GC to AT transition. These technique allows specific mutations to be engineered into a protein, however, they are not flexible in the kinds of mutants generated.
Current techniques for site-specific mutation commonly involve using mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
, or deletion or insertion
Insertion (genetics)
In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping...
of small stretches of DNA to be introduced at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.
The site-directed approach may be done systematically in such technique as alanine scanning
Alanine scanning
In molecular biology, alanine scanning is a technique used to determine the contribution of a specific residue to the stability or function of given protein. Alanine is used because of its non-bulky, chemically inert, methyl functional group that nevertheless mimics the secondary structure...
mutagenesis whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein.
Combinatorial mutagenesis
Combinatorial mutagenesis is a technique whereby large number of mutants may be screened for a particular characteristic. In this technique, a few selected positions or a short stretch of DNA may be exhaustively modified to obtain a comprehensive library of mutant proteins. One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic propertyImmunogenicity
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human or animal.- Immunogenicity :The ability to induce humoral and/or cell-mediated immune responses....
. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.
More recently, techniques such as phage display
Phage display
Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...
has been used to discover short peptide sequences of up to 12 residues with binding affinity for other proteins.
Insertional mutagenesis
In cancer research engineered mutations also provide mechanistic insights into the development of the disease. Insertional mutagenesis using transposons, retrovirus such as mouse mammary tumor virusMouse mammary tumor virus
Mouse mammary tumor virus is a milk transmitted retrovirus like the HTL viruses, HI viruses and BLV. It belongs to the genus betaretroviruses. MMTV was formerly known as Bittner virus, and previously the 'milk factor' referring to the extra-chromosomal vertical transmission of murine breast cancer...
and murine leukemia virus
Murine leukemia virus
The murine leukemia viruses are retroviruses named for their ability to cause cancer in murine hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses...
may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. Various insertional mutagenesis techniques may also be used to study the function of particular gene.
See also
- MutagenMutagenIn genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are therefore also likely to be carcinogens...
- MutationMutationIn molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
- TransfectionTransfectionTransfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
- DNA repairDNA repairDNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
- Mutation breedingMutation breedingMutation breeding is the process of exposing seeds to chemicals or radiation in order to generate mutants with desirable traits to be bred with other cultivars. Plants created using mutagenesis are sometimes called mutagenic plants or mutagenic seeds...
- CarcinogenesisCarcinogenesisCarcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...