Lecture 2 Mutations and Mutagenesis

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BTY 227 Molecular and Environmental
Biotechnology Lecture 4 MUTATIONS 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 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 Figure 1

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Nomenclature hisC gene of E.coli

• This gene codes for a protein called the His C
  protein enzyme involved in histidine
  biosynthesis
• Genotype of organism designated by 3 lowercase
  letters followed by a capital letter (italics)
  e.g. E. coli hisC
• 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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Effect of a Mutation on a Cell

• 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
• Figure 2

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Mutations may Result in Micro or Macrolesions

• May involve one (or a very few) base pairs
  POINT MUTATIONS or MICROLESIONS
• May involve many base pairs
• MACROLESIONS

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Point Mutations/ Microlesions

• Figure 3
• These involve one or two bases
• Result in
• Transitions (pu for pu or py for py)
• Transversions (pu for py or vice versa)
• Frameshift mutations one base inserted this
  changes the aa sequence of the protein

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Back Mutations (Reversions)

• Point mutations are reversible Figure 4
• REVERTANT is a strain in which the WT phenotype
  that was lost in the mutant is restored
• Two types of revertants
• Same site revertant (true revertant)
• Second site revertant- due to presence of a
  suppressor mutation (mutation that restores WT
  phenotype without altering the original mutation)
  (Pseudo revertant)

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Macrolesions

• Figure 5
• Include
• Deletions
• Duplications
• Insertions
• Inversions
• translocations

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Deletions

• 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

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Insertions

• Insertions can add many bases to a sequence
  Figure 5
• result from errors during genetic recombination
• Inactivate the gene
• May be due to insertion of DNA sequences (700
  1400 bp) called INSERTION SEQUENCES

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Translocations and Inversions

• Translocations large DNA segment moves to new
  location
• Inversions orientation of DNA segment reverses
• Figure 5

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Mutation Rates

• Spontaneous mutations occurs 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

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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

• 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

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5 Bromouracilan Example of a Base Analogue

• 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
• Figure 6

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Mutagens acting directly on DNA

• These mutagens change the structure of a base 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
• Figure 7

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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 radiation
  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
• Figure 8

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Detection of mutants

• Replica plating is one technique
• Here we demonstrate how to detect nutritional
  mutants organisms unable to synthesise leucine
• Plate a premutagenised culture on complete medium
  at concentration which will give rise to
  indiviual colonies
• Imprint these colonies on a replicating block
• Transfer colonies to fresh plates
• Complete medium
• Minimal medium containing all essential nutrients
  except leucine
• Colonies absent on minimal medium plate will be
  leu auxotrophs