EMS-treated culture


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 ~ 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.

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?


• 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.

Rifampin, Rifamycin, Rifampicin,



• 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.

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.

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)

Negative regulation of the



~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


• Sequence analysis here at


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.