Thanksgiving Week …and beyond

• Mutagenesis Lab,– spontaneous vs. induced mutations– gain of function,– loss of function,– revertants.

• mtDNA analysis,

• Wrapping things up.

Spontaneous Mutations Mutation: 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.

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?

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 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.

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.

• 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.

Regulation of prokaryotic transcription1. 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.

The metabolism of lactose in E. coli & the lactose operon

QuickTime™ and aGIF decompressor

are needed to see this picture.

IPTG: non-metabolizable artificial inducer (can’t be cleaved)

LacZ: -galactosidase; Y: galactoside permease;

A: transacetylase (function unknown),P: promoter; O: operator,

LacI: repressor; PI and LacI are not part of the operon.

Negative regulation of the lac operon

~6,000 bp

• 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

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.

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.

Mitochondrial Control Region

• Hair follicle DNA extraction,

• PCR,

• Sequencing (at Cold Spring Harbor),

• Sequence analysis here at WWU.

Link Out

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.