Transcript EMS-treated culture

Thanksgiving Week
…and beyond
• Mutagenesis Lab,
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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 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 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 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; PI 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 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.
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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.