Mutations and Their Chemical Basis

Mutations and Their Chemical Basis

 Mutations and Mutagenesis

 Morphological mutations change the microorganism’s colonial or cellular morphology.
 Lethal mutations, when expressed, result in the death of the microorganism. Lethal mutations are recovered only if they are recessive in diploid organisms or conditional (see the following) in haploid organisms.


 Conditional mutations are those that are expressed only under certain environmental conditions. For example, a conditional lethal mutation in E. coli might not be expressed under permissive conditions such as low temperature but would be expressed under restrictive conditions such as high temperature.



 Biochemical mutations are those causing a change in the biochemistry of the cell. Since these mutations often inactivate a biosynthetic pathway, they frequently make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product.

 The mutant cannot grow on minimal medium and requires nutrient supplements. Such mutants are called auxotrophs, whereas microbial strains that can grow on minimal medium are prototrophs.



Mutations occur in one of two ways.

(1) Spontaneous mutations: arise occasionally in all cells and develop in the absence of any added agent.
(2) Induced mutations: on the other hand, are the result of exposure of the organism to some physical or chemical agent called a mutagen.






 Spontaneous Mutations

 Spontaneous mutations arise without exposure to external agents.
 This class of mutations may result from errors in DNA replication, or even from the action of transposons



 Generally replication errors occur when the base of a template nucleotide takes on a rare tautomeric form. Tautomerism is the relationship between two structural isomers that are in chemical equilibrium and readily change into one another.

 These tautomeric shifts change the hydrogen-bonding characteristics of the bases, allowing purine for purine or pyrimidine for pyrimidine substitutions that can eventually lead to a stable alteration of the nucleotide sequence. Such substitutions are known as transition mutations and are relatively common, although most of them are repaired by various proofreading functions



 In transversion mutations, a purine is substituted for a pyrimidine, or a pyrimidine for a purine. These mutations are rarer due to the steric problems of pairing purines with purines and pyrimidines with pyrimidines.



 Spontaneous mutations also arise from frameshifts, usually caused by the deletion of DNA segments resulting in an altered codon reading frame



 it is possible for purine nucleotides to be depurinated—that is, to lose their base. This results in the formation of an apurinic site, which will not base pair normally and may cause a transition type mutation after the next roundof replication.



 Cytosine can be deaminated to uracil, which is then removed to form an apyrimidinic site.

 Reactive forms of oxygen such as oxygen free radicals and peroxides are produced by aerobic metabolism. These may alter DNA bases and cause mutations.

o For example, guanine can be converted to 8-oxo-7,8-dihydrodeoxyguanine, which often pairs with adenine rather than cytosine during replication.


 Induced Mutations


 Mutagens can be conveniently classified according to their mechanism of action. Four common modes of mutagen action are incorporation of---
1. base analogs
2. specific mispairing,
3. intercalation bypass of replication

A. Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication. E.g-

 A widely used base analog is 5-bromouracil (5-BU), an analog of thymine. It undergoes a tautomeric shift from the normal keto form to an enol much more frequently than does a normal base.


B. Specific mispairing is caused when a mutagen changes a base’s structure and therefore alters its base pairing characteristics.e.g------
1.-methyl-nitrosoguanidine, an alkylating agent that adds methyl groups to guanine, causing it to mispair with thymine
2.-Hydroxylamine hydroxylates the C-4 nitrogen of cytosine, causing it to base pair like thymine.

C. Intercalating agents distort DNA to induce single nucleotide pair insertions and deletions
 Intercalating agents include acridines such as proflavin and acridine orange Other e.g-
UV radiation generates cyclobutane type dimers, usually thymine dimers, between adjacent pyrimidines ionizing radiation and carcinogens such as aflatoxin B1 and other benzo (a) pyrene derivatives

 The Expression of Mutations
 A mutation from the most prevalent gene form, the wild type, to a mutant form is called a forward mutation.
 Later, a second mutation may make the mutant appear to be a wild-type organism again. Such a mutation is called a reversion mutation because the organism seems to have reverted back to its original phenotype.
 True back mutation converts the mutant nucleotide sequence back to the wild-type sequence.
 The wild-type phenotype also can be regained by a second mutation in a different gene, a suppressor mutation, which overcomes the effect of the first mutation (table 11.2). If the second mutation is within the same gene, the change may be called a second site reversion or intragenic suppression.


 Point mutations
 Most mutations affect only one base pair in a given location and therefore are called point mutations.


Types of point mutations


 One kind of point mutation that could not be detected until the advent of nucleic acid sequencing techniques is the silent mutation.

 If a mutation is an alteration of the nucleotide sequence of DNA, mutations can occur and have no visible effect because of code degeneracy.

 A second type of point mutation is the missense mutation. This mutation involves a single base substitution in the DNA that changes a codon for one amino acid into a codon for another

 For example, the codon GAG, which specifies glutamic acid, could be changed to GUG, which codes for valine. The expression of missense mutations can vary. Certainly the mutation is expressed at the level of protein structure.


 Mutations also occur in the regulatory sequences responsible for the control of gene expression and in other noncoding portions of structural genes
 Constitutive lactose operon mutants in E. coli are excellent examples. These mutations map in the operator site and produce altered operator sequences that are not recognized by the repressor protein, and therefore the operon is continuously active in transcription. If a mutation renders the promoter sequence nonfunctional, the coding region of the structural gene will be completely normal, but a mutant phenotype will result due to the absence of a product


Type of Mutation Result and Example

Forward Mutations
Single Nucleotide-Pair (Base-Pair) Substitutions

At DNA Level




Transition Purine replaced by a different purine, or pyrimidine replaced by a different
pyrimidine (e.g., AT GC).



Transversion Purine replaced by a pyrimidine, or pyrimidine replaced by a purine


At Protein Level
Silent mutation Triplet codes for same amino acid:
AGG CGG
both code for Arg
Neutral mutation Triplet codes for different but functionally equivalent amino acid:
AAA (Lys) AGA (Arg)
Missense mutation Triplet codes for a different amino acid.
Nonsense mutation Triplet codes for chain termination:
CAG (Gln) UAG (stop)


Single Nucleotide-Pair Addition or Deletion: Frameshift Mutation -------Any addition or deletion of base pairs that is not a multiple of three results in a frameshift in reading the DNA segments that code for protein




Intragenic Addition or Deletion of Several to Many Nucleotide Pairs



Reverse Mutations

True Reversion AAA (Lys) -----forward-------GAA (Glu) ------reverse--- AAA (Lys)
wild type mutant wild type







Equivalent Reversion UCC (Ser) ------------forward UGC (Cys) ------------reverseAGC (Ser)
wild type mutant wild type





Suppressor Mutations


Intragenic Suppressor Mutations
Frame shift of opposite sign at site within gene. Addition of X
to the base sequence shifts the reading frame from the CAT
codon to XCA followed by TCA codons. The subsequent
deletion of a C base shifts the reading frame back to CAT.








Extragenic Suppressor Mutations
Nonsense suppressors Gene (e.g., for tyrosine tRNA) undergoes mutational event in its anticodon
region that enables it to recognize and align with a mutant nonsense codon
(e.g., UAG) to insert an amino acid (tyrosine) and permit completion of the
translation.







Physiological suppressors A defect in one chemical pathway is circumvented by another mutation—for
example, one that opens up another chemical pathway to the same product, or
one that permits more efficient uptake of a compound produced in small
quantities because of the original mutation










 Detection and Isolation of Mutants
 Suitable detection system for the mutant phenotype under study also is needed. Since mutations are generally
rare, about one per 107 to 1011 cells,

 Many proteins are still functional after the substitution of a single amino acid, but this depends on the type and location of the
amino acid.
For instance, replacement of a nonpolar amino acid in the protein’s interior with a polar amino acid probably will drastically alter the protein’s three-dimensional structure and
therefore its function.


 A third type of point mutation causes the early termination of translation and therefore results in a shortened polypeptide. Such mutations are called nonsense mutations because they involve the conversion of a sense codon to a nonsense or stop codon.





 Frame shift mutations arise from the insertion or deletion of one or two base pairs within the coding region of the gene. Since the code consists of a precise sequence of triplet codons, the addition or deletion of fewer than three base pairs will cause the reading frame to be shifted for all codons downstream



 The replica plating technique is used to detect auxotrophic mutants. It distinguishes between mutants and the wild-type strain based on their ability to grow in the absence of a particular biosynthetic end product

 A lysine auxotroph, for instance, will grow on lysine-supplemented media but not on a medium lacking an adequate supply of lysine because it cannot synthesize this amino acid.


 Carcinogenicity Testing

 The Ames test, developed by Bruce Ames in the 1970s, has been widely used to test for carcinogens. The Ames test is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the histidine biosynthesis operon.



 SUMMARY

• A mutation is a stable, heritable change in the nucleotide sequence of the genetic material, usually DNA.
• Mutations can be divided into many categories based on their effects on the phenotype, some major types are morphological, lethal, conditional, biochemical, and resistance mutations.
• Spontaneous mutations can arise from replication errors (transitions, transversions, and frameshifts), from DNA lesions (apurinic sites, apyrimidinic sites, oxidations), and from insertions.
• Induced mutations are caused by mutagens. Mutations may result from the incorporation of base analogs, specific mispairing due to alteration of a base, the presence of intercalating agents, and a bypass of replication because of severe damage. Starvation and environmental stresses may stimulate mutator genes and lead to hypermutation.
• The mutant phenotype can be restored to wild type by either a true reverse mutation or a suppressor mutation.
• There are four important types of point mutations: silent mutations, missense mutations, nonsense mutations, and frameshift mutations .
• It is essential to have a sensitive and specific detection technique to isolate mutants; an example is replica plating for the detection of auxotrophs (a direct detection system).
• One of the most effective isolation techniques is to adjust environmental conditions so that the mutant will grow while the wild-type organism does not.
• Because many carcinogens are also mutagenic, one can test for mutagenicity with the Ames test and use the results as an indirect indication of carcinogenicity.
• Mutations and DNA damage are repaired in several ways; for example: proofreading by replication enzymes, excision repair, removal of lesions (e.g., photoreactivation), postreplication repair (mismatch repair), and recombination repair.







JSR