The induction of mutations is accomplished by treating cells with mutagens. The mutagens most commonly used are high-energy radiation such as X-rays, UV rays or specific chemicals; examples of these mutagens and their efficacy are :
The intercalating agents are another important class of DNA modifiers. This group of compounds includes proflavin, acridine orange , and a class of chemicals termed ICR compounds , base analoges such as 5-bromouracil, Amino purine, Ethyl methane sulfonate, Nitrous acid, Methyl methane sulfonate, Nitrosoguanidine, Acridine mustard.
Molecular Basis of Gene Mutation Mutations are changes in the DNA sequence of genes. Point mutations typically refer to alterations of single base pairs of DNA or of a small number of adjacent base pairs. Mutations in DNA caus e substitutions in protein
New mutations are categorized as induced or spontaneous
. Induced mutations are defined as those that arise after purposeful treatment with mutagens, environmental agents that are known to increase the rate of mutations
. Spontaneous mutations are those that arise in the absence of known mutagen treatment. They account for the "background rate" of mutation and are presumably the ultimate source of natural genetic variation that is seen in populations .
1- transition is the replacement of a base by the other base of the same chemical category (purine replaced by purine: either A to G or G to A; pyrimidine replaced by pyrimidine: either C to T or T to C).
2- transversion is the replacement of a base of one chemical category by a base of the other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replaced by pyrimidine: A to C, A to T, G to C, G to T).
Most mutations are transitions : interchanges of bases of same shape
Missense mutations in DNA cause replacement substitutions in protein Consequences depend on position of substitution in polypeptide
none : substitution not in active site or binding site
minor : substitution of same type (synonymous substitution) allozymes are proteins with minor changes in electric charge
major : substitution affects 20, 30, or 40 structure (nonsynonymous substitution) Ex.: Sickle-cell hemoglobin (HbS) glu - val in sixth residue of beta chain because GAG GUG in sixth codon of beta-chain mRNA
1- Silent substitutions : the mutation changes one codon for an amino acid into another codon for that same amino acid . 2- Missense mutations : the codon for one amino acid is replaced by a codon for another amino acid . 3- Nonsense mutations : the codon for one amino acid is replaced by a translation termination (stop) codon .
Insertion / Deletion ( indel ) mutations Gain or loss of one or more nucleotides produces: F rameshift mutations ( triplet reading frame offset ) 1- Single & double nucleotide indel => all downstream amino acids change (e.g.) nonsense mutation eventually (quickly) produced
2- Triplet indel - insertion / deletion of single amino acid produce milder consequences
3- Multiple triplets may produce major effects (e.g.) length mutations - larger indels (102~6 bps) may affect cytology of chromosomes (" Fragile X ", )
Variable numbers of trinucleotide repeats occur in human genetic disorders
e.g. Huntington's Disease Gene has (CAG) n repeat near 5' end # copies in affected individuals inversely correlated with age of onset predicted mRNA & huntingtin protein with (gln) n repeat have been isolated autosomal dominant inheritance: HD alleles ' dominate ' standard alleles phenotype is a consequence of presence of modified protein :
e.g. Fragile-X Syndrome [Martin-Bellyndrome] Most common form of inherited predisposition to mental retardation (CGG) n repeat occurs upstream from 5' end of FMR-1 gene
<> X-linked inheritance Asymptomatic, hemizygous fathers pass " fragile X " chromosome to daughters Heterozygous daughters transmit to 1/2 of sons, who show syndrome 1/2 of daughers are carriers Syndrome becomes more pronounced in successive generations expansioin of repeat occurs in female germline # CGG repeats Geno / Phenotype 6 ~ 54 standard ~ 55 ~ 200 carrier > 200 Fragile-X Syndrome
1- Regulatory and other noncoding sequences. Those parts of a gene that are not protein coding contain a variety of crucial functional sites. At the DNA level, there are sites to which specific transcription-regulating proteins must bind.
2- At the RNA level, there are also important functional sequences such as the self-ligating sites for intron excision in eukaryote mRNAs .
Mutagens act through at least three different mechanisms. They can replace a base in the DNA , alter a base so that it specifically mispairs with another base, or damage a base so that it can no longer pair with any base under normal conditions .
1- alkylation - addition of alkyl group (methyl CH3- or ethyl CH3-CH2-)) ethyl methane sulfonate (EMS) is a common laboratory mutagen
G - 6-ethyl G , pairs with T
2- deamination - conversion of amino into keto group
nitrous acid ( HNO2 ) is a common food additive / preservative
3- depurination - loss of purine ( A or G ) base in intact nucleotide produces an apurinic site
replacement of base may produce transversion [a common form of damage in 'ancient' DNA ] apyrimidinic sites are similar: loss of C or T
4- intercalation - base analogue wedged into DNA double helix [ acridine dyes resemble 3-ring base pair ] intercalated analogues read as 'extra' base: DNAPol "stutters" => frameshift mutations ( ethidium bromide is a fluorescent dye of DNA )
All of the bases in DNA can exist in one of several forms, called tautomers , which are isomers that differ in the positions of their atoms and in the bonds between the atoms.
The forms are in equilibrium.
The keto form is normally found in DNA
The enol forms of the bases are rare.
The complementary base pairing of the enols is different from that of the keto forms .
For example, assume that a guanine in DNA changes into its enol form at the moment at which it is copied in the course of replication, the enol form will bind to an incoming thymine . Hence we can represent the mutagenic process as follows, in which G* is the enol form of guanine :
Genetic diseases are highly polymorphic with respect to their mutational basis
PAH locus occurs near the end of short arm of Chromosome 12 ( 12p13.33 ) 14 exons produce a 2.4kb mRNA and a 452 amino acid polypeptide Of 66 alleles known to affect expression at the PAH locus
29% produce non-PKU hyperphenylalanemia 71% produce PKU : of these, 14% are splice-site mutations 22% are deletion mutations (single base whole exon) 18% are " non-sense " mutations 55% are miss-sense mutations ( 39% of total)
A number of these disorders are due to deletions or duplications of repeated sequences.
e.g. mitochondrial encephalomyopathies are a group of disorders affecting the central nervous system or the muscles. They are characterized by dysfunction of mitochondrial oxidative phosphorylation and by changes in mitochondrial DNA structure.
These disorders have been shown to result from deletions of DNA sequences that lie between repeated sequences.
e.g. Fabry disease . This inborn error of metabolism results from mutations in the X-linked gene encoding the enzyme a - galactosidase Many of these mutations are gene rearrangements, resulting from either deletions or duplications between short direct repeats. All these deletions occur either by a slipped mispairing mechanism.
A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat, as in the Fragile X syndrome.
For this reason, they are termed trinucleotide repeat diseases. Fragile X syndrome is the most common form of inherited mental retardation, occurring in close to 1 of 1500 males and 1 of 2500 females. It is manifested cytologically by a fragile site in the X chromosome that results in breaks in vitro. Fragile X syndrome results from changes in the number of a (CGG ) n repeat in the coding sequence of the FMR-1 gene .
The proposed mechanism for these repeats is a slipped mispairing during DNA synthesis.
However, the extraordinarily high frequency of mutation at the trinucleotide repeats in fragile X syndrome suggests that in human cells, after a threshold level of about 50 repeats, the replication machinery cannot faithfully replicate the correct sequence and large variations in repeat numbers result .
X-linked spinal and bulbar muscular atrophy (known as Kennedy disease ) results from the amplification of a three-base-pair repeat in this case, a repeat of CAG. Kennedy disease, which is characterized by progressive muscle weakness and atrophy, results from mutations in the gene that codes for the androgen receptor.
Normal persons have an average of 21 CAG repeats in this gene, whereas affected patients have repeats ranging from 40 to 52 .
Myotonic dystrophy , the most common form of adult muscular dystrophy, is yet another example of sequence expansion causing a human disease.
Susceptible families display an increase in severity of the disease in successive generations; this increased severity is caused by the progressive amplification of a CTG triplet at the 3 end of a transcript.
Living cells have evolved a series of enzymatic systems that repair DNA damage in a variety of ways.
The low spontaneous mutation rate is indicative of the efficiency of these repair systems.
Failure of these systems can lead to a higher mutation rate.
A number of human diseases can be attributed to defects in DNA repair, as we shall see later. Let's first examine some of the characterized repair pathways and then consider how the cell integrates these systems into an overall strategy for repair .
Several recessive human genetic diseases are known or suspected to be caused by defective genes in repair systems.
These defects often lead to an increased incidence of cancer because, as part of the general increased level of mutation, more mutations are produced in genes that can cause a cell to become cancerous.
One cancer-prone disease, xeroderma pigmentosum (XP), results from a defect in any one of eight genes involved in nucleotide excision repair. People suffering from this disorder are extremely prone to UV-induced skin cancer ) as a result of exposure to sunlight) and have frequent neurological abnormalities.