Download doc Review of Lecture 27

Basic Genetics III
Lecture 26: Mutation I – types & consequences

See lecture notes
Lecture 27: Mutation II – repair & consequences
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Convention & DNA/RNA sequences
o When discussing gene sequences in practice we refer to the non-template strand
since it approximates the RNA & allows prediction of protein sequences
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Spontaneous mutations
o Do they even occur?
o Spontaneous mutation vs. adaptation
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Are mutations caused only by environmental changes?
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Luria-Delbruck fluctuation test (1943)
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10 years before Watson & Crick
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If treatment leads to adaptation, then resistance not developed until
phages are added – should see similar levels in all samples.
o Low frequency adaptation as opposed to low frequency
spontaneous mutation
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If spontaneous mutations exist, then resistance can arise at any
point before/during/after addition of phage so we would expect to
see widely varying [fluctuating] resistance between multiple
samples.
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Results consistent with the idea that mutations arise spontaneously
and do not follow addition of the phage
o Highly fluctuating number of phage-resistant cells in each
culture
o How do they affect us?
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Pretty rare, but can result in disease
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See table.
o Types of spontaneous mutation
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Point mutations caused by the chemical nature of the bases”
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Replication errors
o Tautomeric shifts
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Adenine
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Single bond to NH2 is replaced by a double
bond to NH
o 1 out of a million
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Hydrogen bonding pairs it to
a normal cytosine
Similar base pairs between:
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G and T
o Bases usually in keto form
o Rare enol & imino forms can mispair

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Less energetically favourable
Depurination
o One cell loses about 10,000 purine residues per day
o If this happens during DNA replication, DNA polymerase
will not know what base to substitute in the non-template
strand at the apurinic site
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Deamination
o Can lead to G-C to A-T transitions

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Cytosine becomes uracil when it is deaminated
Mismatches at a replication fork
o G-T
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Wobble-pair
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In the first generation of progeny, one strand has
replicated correctly, one strand has a mismatch
(mutant).
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In the second generation, three strands have
replicated correctly, one strand has a mismatch
(mutant).
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Other
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Replication slippage (DNA polymerase)
o Usually associated with repeated DNA sequences
o Can lead to additions and deletions
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Extra base loops out on the synthesized strand and
is stabilized by repetitive sequences
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Addition
Extra base loops out on the template strand and is
stabilized by repetitive sequences
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Deletion
o Trinucleotide repeat diseases in humans
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Fragile X syndrome – FMR-1 gene
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Most common cause of mental retardation
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Upstream region from gene
o Enhancer

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If >200 copies of CGG gene transcription is
silenced
Huntington disease
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Repeat in coding region of CAG (glutamine)
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Normal = 19-21 copies of CAG repeat
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Affected = Average 46 copies of CAG
repeat
o Affects transcription of the gene and
stability of the protein

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Kennedy disease
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Number of copies reflects severity
Similar mechanism to Huntingdon diseases
in androgen receptor
All three have neural degeneration
o Induced mutations and mutagens
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In the lab or environment
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Chemicals that affect the structure of DNA in different ways:
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Base analogs
o Chemicals that resemble nucleotides
o Can be integrated into DNA in place of nucleotides
o Less stable forms than bases, shift base-pairing affinities &
lead to changes
o 5-bromouracil (5-BU) – causes A-T  G-C or G-C  A-T
transitions
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Thymine analog
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Switches tautomeric forms more than
adenine
o 2-aminopurine (2-AP) – also causes transitions
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Analog of adenine
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Switches tautomeric forms more than
adenine
Base alteration – alkylating agents
o React with nucleotide bases and change their base pairing
properties
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Ethylmethane sulfonate (EMS) – adds ethyl groups
to nucleotide bases which leads to mismatched base
pairing
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Nitroguanidine (NG) – adds methyl groups to keto
oxygen of nucleotide base which leads to
mismatched base pairing
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G-C  A-T transitions
G-C  A-T transitions
Base alteration – intercalating agents
o Mimic basepairs and slide into double helix
o Can cause single insertion/deletions
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Falls out during replication
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DNA polymerase deletes or inserts a
nucleotide  leads to frameshift mutations
o Includes DNA dye ethidium bromide
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Used for visualizing DNA
o Three aromatic rings connected together, similar to
nucleotide basepairs
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Base damage – photoproducts
o UV light can cause interactions between adjacent
pyrimidines
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Thymine residues crosslink creating a cyclobutyl
ring (dimer)
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Problems for replication
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Covalent bonds
Changes shape of DNA and causes problems
for DNA polymerase
Base damage – other mutagens
o Aflatoxin B1
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From infected peanuts
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Leads to apurinic site
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One strand remains normal, one strand has a
random nucleotide added
Problem in developing countries
Mutagens in our environment
o See table.
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Mutagens in our environment – radiation
o See table.
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Ames test – detection of mutagenic agents
o Developed in the 1970s
o Way to detect mutagenic substances developed by Bruce
Ames
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Bacterial strain containing mutation making it
deficient in production of histidine
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Auxotroph
o An organism, such as a strain of
bacteria, that has lost the ability to
synthesize certain substances
required for its growth and
metabolism as the result of
mutational changes.
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Test if a chemical can revert with second mutation
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Can happen spontaneously, but if a chemical
is mutagenic, the number of reversals will
increase in its presence
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Compare with spontaneous mutation rate
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Initially only done with a bacterial gene, however,
many chemicals in the environment are not
mutagens themselves but reactant with chemicals in
the body to become mutagenic
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Solution was to add rat liver enzymes to see if
chemicals were metabolically activated
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Using different His- strains can test for nucleotide
changes versus frameshift
Mutations happen
o At quite a high frequency!
o How do organisms deal with it?
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If they all accumulated, over time, would be lethal.
o Very efficient DNA repair systems.
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See rest of lecture 27 for repair mechanisms.
Lecture 28: Transposable Elements I – types & mechanisms
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Pieces of DNA with capacity to move in genome
o Also source of spontaneous mutation
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Unexpected observations made in different systems
o Patchy kernel derived from single cell
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Some are patchy, cannot be explained by Mendelian genetics
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Unstable alleles
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Genetics and the detection of the unexpected: Barbara McClintock and unstable alleles of
maize
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First case:
o Found chromosome breakage
o Ds mutation in presence of Ac there is chromosome breakage
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Cells loose downstream traits on chromosome with Ds
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These include:
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Pigmented
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Plump
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Shiny
Second case:
o C-Ds reverts to C in the presence of Ac
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Rare unstable alleles
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Affect only a single gene distal to usual breakage point
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Suggest that Ds is mobile, but only in the presence of Ac
Couldn’t map Ac so seemed to be mobile itself
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Mapped in different places in different strains of corn
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Both explained by mobile elements
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Genetics and the detection of the unexpected: Hybrid dysgenesis in Drosophila
o Crosses of wild isolate males (P) x lab strain females (M)
o F1 progeny sterile – chromosome breakage, unstable mutations in germ line
o Unstable mutations of white from dysgenic cross reminiscent of what seen
before…
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Genetics and the detection of the unexpected: “Enlarged” mutant alleles in E.coli
o Unstable lac- and gal- mutants of E. coli
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Found to contain extra DNA
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Inserted into the genes causing the mutations
All explained by transposable elements
Lecture 29 : Transposable Elements II – transposable elements & genomes

See lecture slides
Lecture 30 : Genetic Dissection I – reverse genetics & functional dissection
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See notes
Lecture 31 : Genetic Dissection II – forward genetic analysis
Lecture 32: Transcription attenuation, bacteriophage lambda, and sigma factors