• Mutagenesis Lab,
– spontaneous vs. induced mutations
– gain of function,
– loss of function,
– revertants.
• mtDNA analysis,
• Wrapping things up.

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.

– 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?

• 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.

(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 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 rifampicinresistant 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.

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.

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; P
I and LacI are not part of the operon .
QuickTime™ and a
GIF decompressor are needed to see this picture.
IPTG: nonmetabolizable artificial inducer (can’t be cleaved)

lac
~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 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

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

• 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.