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

• Pu -> Pu• Py -> Py

• G -> A• C -> T

Transversions

• Pu -> Py• Py -> Pu

• G -> T• C -> A• G -> C• C -> G

A, G PuT, C Py

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. Missense2. Insertion3. Deletion 4. Frameshift

Mutator strains• Cells have mutation that affects DNA repair and metabolism (not WT)

Repair Mutation

1. mutD = dnaQ= proofreading subunit of DNA Pol III-Mutation rate increases 1000x- Substitutions, transitions, transversions, and

frameshifts

2. mut L S H= mismatch repair- Mutation rate increases 100x- Substitutions, transitions, transversions, and

frameshifts

3. Several others – metabolism and repair

Examples:

Mutagens

• Chemical or physical agents• Increase mutation frequency

• How do they work?1. Mispairing2. Modify bases in DNA

Types of MutagensMutagen 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 N4-hudroxycytosine G-C to A-T transitions when used in vitro

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Alkylating agent, generates O6-methylguanine

G-C to A-T transitions, multiple, closely spaced mutations common

Ethylmethane sulfonate (EMS) (EMS) Alkylating agent, generates O6-methylguanine 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-191Intercalating 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 MutagensMutagen 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 N4-hudroxycytosine G-C to A-T transitions when used in vitro

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Alkylating agent, generates O6-methylguanine

G-C to A-T transitions, multiple, closely spaced mutations common

Ethylmethane sulfonate (EMS) (EMS) Alkylating agent, generates O6-methylguanine 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-191Intercalating 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

Should I isolate a spontaneous mutant or use a mutagen?Assumptions (generous)

1 gene = 103 basepairs= 330 amino acidsChromosome = 5 x 106 bp for E. coli

Assume 1. Equal random change to mutate any bp2. Assume 1 hit/cell

103 bp = 1/5000 probability of hit in a gene5x 106 bp

Assume 10% = phenotype (90% silent) 1 1 15000 10 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)

Zen of Mutagenesis

x=

*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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