Thanksgiving Week … and beyond

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 MutationsMutation: an inheritable change in the DNA sequence of a chromosome. DNA replication in E. coli occurs with an error every ~ 109 bases. • - The E. coli genome is 4.6 x 106 bases. • an error occurs once per ~ 2000 replications. • - If a single colony has 107 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 wildtypeE. 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 37oC 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, • resistanceto RIF, in the majority of cases, has been associated with mutationswithin an 81-bp RIF resistance-determining region (RRDR) ofthe rpoB gene, which encodes the ß subunit of theRNA polymerase (1,342 bp). • RIF acts by binding to the ß subunitof the RNA polymerase, thus interfering with transcription andRNA elongation.

6. Part II: Selection for rifR mutants: • RifR 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 37oC 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 37oC 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). 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 LacZ: -galactosidase; Y: galactoside permease; A: transacetylase (function unknown), P: promoter; O: operator, LacI: repressor; PI and LacI are not part of the operon. IPTG: non-metabolizable artificial inducer (can’t be cleaved)

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 37oC 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 37oC overnight. Remove the next day and store at 4oC. • 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.