Isolation of Mutants; Selections,
Screens and Enrichments
Carolyn Keeton
Turn In HW 1 in Front
Outline
• What causes mutations?
– Spontaneous
– Mutator Strains
– Mutagens
– Considerations
• Isolation of Mutants
– Selections
– Screens
– Enrichments
Transitions vs. Transversions
Transition
Transversions
• Pu -> Pu
• Py -> Py
• Pu -> Py
• Py -> Pu
• G -> A
• C -> T
A, G Pu
T, C Py
•
•
•
•
G
C
G
C
-> T
-> A
-> C
-> G
What causes mutations?
1.
Spontaneous- wide variety of mutations types substitutions, deletions,
frameshifts, insertions
2.
DNA replications errors- not repaired
3.
Recombination –> rearrangements-> deletions and insertions
(duplications)
4.
DNA damage – radiation, metabolisms, free radicals
5.
Transposable elements – insertions, usually rare, <106/gene/ generation
Types of Mutations
1.
2.
3.
4.
Missense
Insertion
Deletion
Frameshift
Mutator strains
• Cells have mutation that affects DNA repair and metabolism (not WT)
Examples: 1. mutD = dnaQ= proofreading subunit of DNA Pol III
-Mutation rate increases 1000x
- Substitutions, transitions, transversions, and
frameshifts
Repair
Mutation
2. mut L S H= mismatch repair
- Mutation rate increases 100x
- Substitutions, transitions, transversions, and
frameshifts
3. Several others – metabolism and repair
Mutagens
• Chemical or physical agents
• Increase mutation frequency
• How do they work?
1. Mispairing
2. Modify bases in DNA
Types of Mutagens
Mutagen
Mechanism
Types of mutations produced
Spontaneous
DNA replication and repair errors,
spontaneous modification of nucleotides
All types of mutations produced
UV irradiation
Pyrimidine dimers induce error prone
repair (SOS)
Mainly G-C to A-T transitions, but all
other types of mutations including
deletions, frameshifts, and
rearrangements at somewhat lower
frequency
2-aminopurine (2AP)
Base analog
A-T to G-C and G-C to A-T transitions
Bromouracil
Base analog
G-C to A-T and A-T to G-C transitions
Hydroxylamine (NH2OH)
Alkylating agent, generates N4hudroxycytosine
G-C to A-T transitions when used in vitro
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG)
Alkylating agent, generates O6methylguanine
G-C to A-T transitions, multiple, closely
spaced mutations common
Ethylmethane sulfonate (EMS) (EMS)
Alkylating agent, generates O6methylguanine
G-C to A-T transitions
Ethylethane sulfonate (DES)
Alkylating agent, induces SOS response
G-C to T-A transversions, other base
substitution mutations
Nitrous acid
Oxidative deamination
G-C to A-T and A-T to G-C transitions,
deletions produced at a lower frequency
ICR-191
Intercalating agent, alkylacridine derivative
that stabilizes looped out bases by stacking
between them
Frameshifts, mainly additions or
deletions in runs of G or C
Mispairing
• Occurs during replication by incorporating the
wrong base
• Cells must be actively growing for these to
work
• Example 5BU (Thymine Analog), or 2AP (Adenine analog)
5BU
Hydroxylamine
Modify bases in DNA
• By mispairing
• Can use alkylating agents
• Ex. Diethylsulphate, methylates guanine to pair with thymine
during replication= results in GC to an AT bp
• Ex. Nitrosogunidine is very potent (can’t buy anymore)
– Thought to act at the replication fork
• Ex. Depurination induces SOS repair
HA
• Interacts directly with the DNA by modifying the base
• Modifies C to bp with an A instead of a G
• Specific for GC to AT
• Only works in vitro, no true revertants as unidirectional
• We have sequenced hundreds of mutants, only 1 was
not GC to AT
Altered nucleotide pools
• Alters concentration of nucleotides, increases
rate of misincorporation
• Exc BrUTP inhibits dCTP synthesis
– Increases T to G misincorporations
– Modifying the nucleotide pools = increases rate of
misincorporations
– Ex. PCR dNTP concentrations
Intercalating Agents
• Insert between bases in DNA
• Causes frameshifts
• Ex EtBR- carcinogen
Indirect Mutagenesis
• Occurs during repair of DNA damage
• Induce SOS pathway
•
•
•
•
Ex. Expose to UV light or MMS (alkylation)
Makes T-T dimers which block replication
Induces repair
High probability of a mutation during DNA synthesis past
lesion
• Results in substitutions, rearrangements. and frameshifts
Types of Mutagens
Mutagen
Mechanism
Types of mutations produced
Spontaneous
DNA replication and repair errors,
spontaneous modification of nucleotides
All types of mutations produced
UV irradiation
Pyrimidine dimers induce error prone
repair (SOS)
Mainly G-C to A-T transitions, but all
other types of mutations including
deletions, frameshifts, and
rearrangements at somewhat lower
frequency
2-aminopurine (2AP)
Base analog
A-T to G-C and G-C to A-T transitions
Bromouracil
Base analog
G-C to A-T and A-T to G-C transitions
Hydroxylamine (NH2OH)
Alkylating agent, generates N4hudroxycytosine
G-C to A-T transitions when used in vitro
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG)
Alkylating agent, generates O6methylguanine
G-C to A-T transitions, multiple, closely
spaced mutations common
Ethylmethane sulfonate (EMS) (EMS)
Alkylating agent, generates O6methylguanine
G-C to A-T transitions
Ethylethane sulfonate (DES)
Alkylating agent, induces SOS response
G-C to T-A transversions, other base
substitution mutations
Nitrous acid
Oxidative deamination
G-C to A-T and A-T to G-C transitions,
deletions produced at a lower frequency
ICR-191
Intercalating agent, alkylacridine derivative
that stabilizes looped out bases by stacking
between them
Frameshifts, mainly additions or
deletions in runs of G or C
How specific are mutagens?
• J. Miller had a collection of lacI amber
mutants- examine reversion to lacI+
•
•
•
•
EMS, NG, 2AP, UV = several sites in common
UV mostly changes at other sites
mutT different pattern: transversion
2AP had hot spots (regions prone to mutagenesis)
• Handout
Some Genes do not mutagenize well
• Ex rII gene of phage T4
• Benzer isolated 3000 spontanous mutants
• ½ were at 2 sites
• Need to do lots of work to isolate new
mutants
Choice of Mutagens
• NG: very powerful: initial test to see if you can
isolate mutants
• ICR: Only makes frameshifts
• HA: Only specific in vitro
• No true revertants (for HA) (Explain later)
Insertional Mutants
• Use a transposon with an antibiotic cassette
• Inserts into chromosomes
• Isolate mutant
• Can sequence the adjacent DNA to determine
what was disrupted
• Good for finding undiscovered targets
Zen of Mutagenesis
Should I isolate a spontaneous mutant or use a mutagen?
Assumptions (generous)
1 gene = 103 basepairs= 330 amino acids
Chromosome = 5 x 106 bp for E. coli
Assume 1. Equal random change to mutate any bp
2. Assume 1 hit/cell
103 bp
5x 106 bp
= 1/5000 probability of hit in a gene
Assume 10% = phenotype (90% silent)
1 x 1
=
5000
10
1
5x104
If screened 250 colonies a plate= 200 plates for 1 mutant
(Lots of work)
If use a mutagen that increases mutation rate 100x= 2 plates (but can get second site
mutants)
*Backcross*
• Very important when doing mutagenesis
• After isolating a mutant, a good geneticist would move it to a clean
background
• Verifies the mutation gives the correct phenotype
• Will explain the mechanism later
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