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

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Presentation on theme: "Isolation of Mutants; Selections, Screens and Enrichments Carolyn Keeton Turn In HW 1 in Front."— Presentation transcript:

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 Transition 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? 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, <10 6 /gene/ generation

5 Types of Mutations 1.Missense 2.Insertion 3.Deletion 4.Frameshift

6 Mutator strains Cells have mutation that affects DNA repair and metabolism (not WT) RepairMutation 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:

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

8 Types of Mutagens MutagenMechanismTypes 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 analogA-T to G-C and G-C to A-T transitions BromouracilBase analogG-C to A-T and A-T to G-C transitions Hydroxylamine (NH 2 OH) Alkylating agent, generates N 4 - hudroxycytosine G-C to A-T transitions when used in vitro N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) Alkylating agent, generates O 6 - methylguanine G-C to A-T transitions, multiple, closely spaced mutations common Ethylmethane sulfonate (EMS) (EMS) Alkylating agent, generates O 6 - 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 acidOxidative 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 Mutagens MutagenMechanismTypes 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 analogA-T to G-C and G-C to A-T transitions BromouracilBase analogG-C to A-T and A-T to G-C transitions Hydroxylamine (NH 2 OH) Alkylating agent, generates N 4 - hudroxycytosine G-C to A-T transitions when used in vitro N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) Alkylating agent, generates O 6 - methylguanine G-C to A-T transitions, multiple, closely spaced mutations common Ethylmethane sulfonate (EMS) (EMS) Alkylating agent, generates O 6 - 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 acidOxidative 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 Should I isolate a spontaneous mutant or use a mutagen? Assumptions (generous) 1 gene = 10 3 basepairs= 330 amino acids Chromosome = 5 x 10 6 bp for E. coli Assume 1. Equal random change to mutate any bp 2. Assume 1 hit/cell 10 3 bp= 1/5000 probability of hit in a gene 5x 10 6 bp Assume 10% = phenotype (90% silent) x10 4 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 =

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


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