Lecture 3 Mutations and Mutagenesis

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Lecture 3Mutations and Mutagenesis
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Wild types and mutants

• Wild type strain is a strain of an organism
  isolated from nature
• Is the usual (native) form of the organism
• Any organism may undergo a heritable change in
  the base sequence of its nucleic acid genome and
  become a MUTANT

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Some Definitions

• Mutant organism or strain whose genome carries
  a mutation
• Mutation an inheritable change in the base
  sequence of the genome of an organism
• Genotype the precise genetic makeup of an
  organism
• Phenotype the observable characteristics of an
  organism

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Nomenclature

• hisC gene of E.coli
• This gene codes for a protein called the His C
  protein enzyme in the biosynthetic pathway of
  histidine
• genotype of organism designated by 3 lowercase
  letters followed by a capital letter (italics)
• Mutations in hisC gene designated as hisC1, hisC2
• Phenotype of organism designated by capital
  letter followed by 2 lowercase letters followed
  by a or e.g. E.coli His
• His strain of E.coli is capable of making
  histidine, His- strain cant

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Types of Mutants
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Molecular Basis of Mutation

• Mutations can be spontaneous or induced
• Induced mutations result of exposure to physical
  or chemical agents called mutagens
• Spontaneous mutations occur as result of
• Errors in DNA replication
• DNA damage due to radiation/heat
• Action of transposons

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Mutations

• The phenotypic change accompanying a mutation
  depends on
• Where in the gene the mutation occurred
• What the nucleotide change was
• What product the gene normally encodes
• May involve one (or a very few) base pairs
  POINT MUTATIONS
• These result in base pair substitutions in the
  DNA or in the insertion/deletion of a base pair
• May involve many base pairs deletions,
  insertions, translocations and inversions

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Base-Pair Substitutions

• Possible effects in a gene encoding a protein

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Frameshift Mutation

• Shifts in reading frame caused by insertion and
  deletion of a base pair

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Back Mutations (Reversions)

• Point mutations are reversible
• REVERTANT is a strain in which the WT phenotype
  that was lost in the mutant is restored
• 2 types of revertants
• Same site revertant
• Second site revertant- due to presence of a
  suppressor mutation (mutation that restores WT
  phenotype without altering the original mutation)

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Mutations involving many Base Pairs

• Deletions can remove large regions of DNA
• Result in complete loss of function of gene
• Can span more than one gene
• These are NOT reversible by further mutations
• Insertions can add many bases to a sequence
• result from errors during genetic recombination
• Inactivate the gene
• May be due to insertion of DNA sequences (700
  1400 bp) called INSERTION SEQUENCES
• Translocations large DNA segment moves to new
  location
• Inversions orientation of DNA segment reverses

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Mutation Rates

• On average, spontaneous mutations occur at a rate
  of 10-6 per generation
• In a normal growing culture (108 cells per ml)
  there are probably a number of different mutants
  per ml of culture
• Rate of transposition is higher 10-4
• Nonsense mutations occur less frequently only a
  few codons can mutate to nonsense codons 10-8

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Mutagens

• Mutagens are chemical, physical or biological
  agents that increase the mutation rate i.e.
  induce mutations
• Can classify mutagens according to mode of
  action
• Incorporation of base analogues
• Direct reaction with DNA
• intercalation

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Base analogues

• Are similar to normal bases and can be
  incorporated into polypeptide chain during
  replication
• They have different base pairing properties and
  in subsequent replication events may form a
  stable mutation
• e.g. 5-bromouracil
• Exists in keto state but often tautomerises to
  its enol state
• Keto state bonds with adenine
• Enol state pairs with guanine
• End up with AT to GC transition mutant

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Mutagens acting directly on DNA

• These mutagens change a bases structure and
  alter the base pairing characteristics
• E.g. is methyl nitrosoguanidine (NTG), an
  alkylating agent which adds methyl groups to
  guanine, causing it to base pair wiyh thymine
• Induce mutations at higher frequency than base
  analogues because they are active even in
  nonreplicating DNA

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Intercalating agents

• These mutagens insert themselves between adjacent
  base pairs and push them apart
• During subsequent replication this abnormal
  structure leads to microinsertions/deletions and
  frameshifts
• E.g.s acridine orange, ethidium bromide

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Nonionizing Radiation

• Purine and pyrimidine bases absorb UV radiaton
  strongly (abs max is 260nm)
• Major effect of this radiation is the formation
  of dimers between adjacent pyrimidines
• During subsequent rounds of replication, DNA
  polymerase hesitates at the dimers and can insert
  incorrect nucleotides

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