CSE 331 Computer Organization and Design Fall 2006 Week 11&12 (Thanksgiving Week)
Thanksgiving Week … and beyond
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Transcript of Thanksgiving Week … and beyond
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.