Pseudoreversion: Intragenic suppressors

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Mutation Rates
A) Mutation rate is often different for different
phenotypes. B) Random nature of mutation
provides a way to calculate the mutation rate.
2
Poisson distribution
m, average number (or mean number) of mutations
per culture. When i0, pi e-m m -ln
(pi)
m -ln (no mutant events)/(total number of
trials)
3
Mutation Rate
Number of mutations per cell division.
m
a
Where,
N
m average number of hits per target
no-mutation tests
m -ln(
)
total tests

N, number of cells in each test

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Example of calculating mutation rate
To determine the frequency of ampicillin
resistant mutants, a fluctuation test was done
with 50 tubes each containing 108 cells. 42 of
the tubes contained no mutants. What is the
mutation rate to the ampicillin resistance in
this test? Answer
A Calculate the average number of hits per
tube m -ln(42/50) - ln (0.84) 0.17 B
Divide the average number of hits per tube by
the number of cells per tube am/N 0.17/108
1.7x10-9.
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Strategies for Finding Mutants

• Screen
• To determine the presence of a phenotype by
  testing for growth under different conditions.
• Usually was done by replica plating onto each
  type of plates.
• Selection
• Conditions where only mutant cells with a
  particular phenotype can grow.

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• Isolation of mutants in catabolic pathways
• Select for resistance to a toxic analog
• Select for mutants failure to grow on a
  particular carbon or nitrogen source
• Isolation of mutants in biosynthetic pathways
• Penicillin enrichment
• Screen for failure to grow without a particular
  nutritional supplement

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• Isolation of mutants in essential genes
• Screen for conditional lethal mutants
• Screen for mutants in diploids
• Isolation of mutants in defined loci
• Localized mutagenesis
• Allele replacement (e.g. null mutant or knock-out)

8
Epistasis and Crossfeeding Test
Epistasis A double mutant where one mutation
masks the phenotype of another mutation. A
mutation preventing an early step in a
biochemical pathway will be epistatic to a
mutation that prevents a step later in the same
pathway
A
C
B
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Crossfeeding
The order of a biosynthetic pathway with
diffusible intermediates can often be determined
by crossfeeding
trpE-
A
B
A
B
trpD-
A
B
trpB-
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Suppression of Mutation(Revertant)
True reversions Back to wild type Pseudo
reversions Intragenic suppressors (within
the same gene) Intergenic suppressors (differen
t genes)
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True Reversion
Base substitution mutations UGG -gt
UGA -gt UGG trp -gt stop -gt trp
Frameshift mutations UGG GGG
AAA ... -gt UGG GGG GAA A.. -gt UGG GGG AAA ...
trp gly lys ... trp
gly glu ... trp gly lys
... Insertion mutations precise deletion
("excision") of the added DNA sequence.
Deletion mutations cannot be restored.
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Pseudo-reversionIntragenic suppressors
Base substitution mutation suppression
1.Same codon / Same nucleotide UGG -gt
UGA -gt UGC trp stop cys
2. Same codon / Different nucleotide
UGG -gt UGA -gt CGA trp stop
arg
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Intragenic suppressors (continue)
3. Different codon in the same gene
CGA GAG CAU -gt CUA GAG CAU -gt CUA GAG CCU
arg glu his leu glu
his leu glu leu
(wild-type) (1 mutant)
(pseudo-revertant)
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3.a)Intragenic suppressors that affect protein
structure Example
mutation reversion
backcross Gly210Gly174--gt Glu210Gly174 --gt
Glu210 Cys174 --gt Gly210 Cys174
(active) (inactive) (active)
(inactive)
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3.b) Intragenic suppressors that affect protein
function Example mutation
reversion backcross
GAG ACG -------gt GAG ATG -------gt AAG
ATG -------gt AAG ACG Glu18 Thr44
Glu18 Met44 Lys18 Met44
Lys18 Thr44 (active)
(inactive) (active)
(active) Super-active mutant
(gained function)
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4) Intramolecular frameshift mutation
suppression 5' AUG UGG GGA CCC AAG GGU AGC
CCC ... 3' (wild-type) met trp
gly pro lys gly ser pro ...
5' AUG UGG GGG ACC CAA GGG UAG CCC C.. 3'
(mutant) met trp gly thr
gln gly stop 5' AUG UGG GGG ACC .AAG GGU
AGC CCC . 3' met trp gly thr
lys gly ser pro ...
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Pseudo-reversion-2Intergenic Suppressors

• Informational
• Nonsense suppressors
• Ribosomes mutations (rare)
• Interaction (compensate the change)
• Overproduction
• Functional suppression
• Bypass (activate new pathway).

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Nonsense Suppressors
Mutations in the DNA corresponding to the
anticodon of a tRNA that cause the anticodon to
pair with one of the stop (or "nonsense")
codons, UAG (Amber) UAA (Ochre) UGA
(Opal)
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Suppressor tRNA
Amber
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Amber suppressor
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Ochre Suppressor
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Opal suppressor
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Nonsense mutations and nonsense suppressors
Amber mutant in wild type strain
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Amber mutant in supD amber suppressor strain
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How does translation terminate in sup mutants?
A) Tandem stop codons at the end of genes. B)
Ochre codon (TAA) is the most common termination
signal.
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Some uses of nonsense suppressors

• Nonsense mutations are the examples of
  conditional mutations.
• Amino acid substitutions at a specific position
  in a protein.