1. Thanksgiving Week … and beyond Mutagenesis Lab, –spontaneous vs. induced mutations –gain of function, –loss of function, –revertants. mtDNA analysis, Wrapping things up.

2. Spontaneous Mutations Mutation: an inheritable change in the DNA sequence of a chromosome. DNA replication in E. coli occurs with an error every ~ 10 9 bases. - The E. coli genome is 4.6 x 10 6 bases. –an error occurs once per ~ 2000 replications. - If a single colony has 10 7 bacteria, 5,000 cells carry a mutation, –or, one mutation every ~ 1,000 bases (across a colony), –or, a mutation in about every gene.

3. Induced Mutations Ethylmethane sulfonate (EMS), –EMS adds an ethyl group to G and T residues, allowing the modified base to base-pair inappropriately. Question: how much higher is the rate of mutation after mutagenic treatment?

4. Mutagenesis Part I: Viable cell counts Untreated culture Do a serial dilution of the untreated wildtype E. coli culture: Fill 7 tubes with 4.5 ml of sterile saline. Transfer 0.5 ml of the undiluted culture to one of the tubes. This is a 10 -1 dilution. Next make serial dilutions of 10 -2, 10 -3, 10 -4, 10 -5, 10 -6 and 10 -7. Always change pipets and mix well between dilutions. Plate 0.1 ml of the 10 -6 onto an L plate. Repeat for the 10 -7 dilution. Place the plates at 37 o C overnight. EMS-treated culture You will be given an EMS treated culture. Do a viable cell count on this culture using the same dilutions as described above.

5. Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal) Rifampin (RIF) is a first-line antituberculosis drug, –resistance to RIF, in the majority of cases, has been associated with mutations within an 81-bp RIF resistance-determining region (RRDR) of the rpoB gene, which encodes the ß subunit of the RNA polymerase (1,342 bp). –RIF acts by binding to the ß subunit of the RNA polymerase, thus interfering with transcription and RNA elongation.

6. Part II: Selection for rif R mutants: Rif R mutants: Rifampcin is a potent inhibitor of E. coli RNA polymerase. Mutants of E. coli that are resistant to this antibiotic have been isolated and shown to have an altered RNA polymerase. Untreated culture To select for spontaneous rifampicin- resistant mutations: Spread 0.2 ml of undiluted culture on an L plate that contains rifampicin (100  g/ml). Set up a total of 2 such plates. Place the plates at 37 o C overnight. EMS-treated culture To select for rifampicin-resistant cells: Spread 0.1 ml of each of the following dilutions on an L plate that contains rifampicin (100  g/ml): undiluted, 10 -1, 10 -2, 10 -3. Place the plates at 37 o C overnight.

7. Regulation of prokaryotic transcription 1.Single-celled organisms with short doubling times must respond extremely rapidly to their environment. 2.Half-life of most mRNAs is short (on the order of a few minutes). 3.Coupled transcription and translation occur in a single cellular compartment. Therefore, transcriptional initiation is usually the major control point. Most prokaryotic genes are regulated in units called operons (Jacob and Monod, 1960) Operon: a coordinated unit of gene expression consisting of one or more related genes and the operator and promoter sequences that regulate their transcription. The mRNAs thus produced are “polycistronic’—multiple genes on a single transcript.

8. The metabolism of lactose in E. coli & the lactose operon IPTG: non- metabolizable artificial inducer (can’t be cleaved) LacZ:  -galactosidase; Y: galactoside permease; A: transacetylase (function unknown), P: promoter; O: operator, LacI: repressor; P I and LacI are not part of the operon.

9. Negative regulation of the lac operon ~6,000 bp

10. Part III: Screen for lac - +  lac - mutants lac - mutants: Wild-type lac + colonies appear dark red on MacConkey indicator plates. Mutant colonies that are not capable of utilizing lactose as an energy source will appear as white colonies on MacConkey plates. Untreated culture Spread 0.1 ml of the 10 -5 dilution on a MacConkey plate. Also, spread 0.1 ml of the 10 -6 dilution on a MacConkey plate. Set up a total of 3 plates of each dilution. Place the plates at 37 o C overnight. Remove the plates from the incubator the next day. Score immediately for white colonies. Streak out each candidate lac - mutant on a MacConkey plate to confirm the lac - phenotype and to isolate single colonies. Place at 37 o C overnight. Remove the next day and store at 4 o C. EMS-treated culture Follow the instructions for the untreated culture. No Part IV

11. Mitochondrial DNA -16, 569 bp, -multiple copies per mt, -100 - 1000 mt per cell, -37 genes; -22 oxidative phosphorylation, -13 tRNA, -2 rRNA, -Mitochondrial Control Region.

12. Mitochondrial Control Region control region, –single promoter on each strand initiates transcription, –ori, D-loop, – replication loop topography, hypervariable region, –mutation rate 10x greater than genome.

13. Mitochondrial Control Region Hair follicle DNA extraction, PCR, Sequencing (at Cold Spring Harbor), Sequence analysis here at WWU. Link Out

14. Business Hfr report due Nov. 29, Mutagenesis “report” due in notebook Dec. 7th, Arabidopsis report due Dec. 7th, Take home final (Dec. 1), due Dec. 7th.