1. Isolation of Mutants; Selections, Screens and Enrichments
Carolyn Keeton
Turn In HW 1 in Front

2. Outline What causes mutations? Spontaneous Mutator Strains Mutagens
Considerations
Isolation of Mutants
Selections
Screens
Enrichments

3. Transitions vs. Transversions
Pu -> Pu
Py -> Py
G -> A
C -> T
Transversions
Pu -> Py
Py -> Pu
G -> T
C -> A
G -> C
C -> G
A, G Pu
T, C Py

4. What causes mutations?
Spontaneous- wide variety of mutations types substitutions, deletions, frameshifts, insertions
DNA replications errors- not repaired
Recombination –> rearrangements-> deletions and insertions (duplications)
DNA damage – radiation, metabolisms, free radicals
Transposable elements – insertions, usually rare, <106/gene/ generation

5. Types of Mutations
Missense
Insertion
Deletion
Frameshift

6. Mutator strains
Cells have mutation that affects DNA repair and metabolism (not WT)
Examples:
mutD = dnaQ= proofreading subunit of DNA Pol III
-Mutation rate increases 1000x
Substitutions, transitions, transversions, and frameshifts
mut L S H= mismatch repair
Mutation rate increases 100x
3. Several others – metabolism and repair
Repair
Mutation

7. Mutagens Chemical or physical agents Increase mutation frequency
How do they work?
1. Mispairing
2. Modify bases in DNA

8. Types of mutations produced
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
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)
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

9. 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)

10. 5BU

11. Hydroxylamine

12. 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

13. 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

14. 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

15. Intercalating Agents Insert between bases in DNA Causes frameshifts
Ex EtBR- carcinogen

16. 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

17. Types of mutations produced
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
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)
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

18. 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

19. 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

20. 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)

21. 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

22. 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 = 1/5000 probability of hit in a gene 5x 106 bp Assume 10% = phenotype (90% silent) x104 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) x =

23. *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 x x x x x x x x