Transcript High Frequency of Recombination (Hfr)

This Week
• Start High Frequency of Recombination
(HFR) experiment (Weds.),
– work to do Thursday, Friday and next week,
• Start Bacteria Mutagenesis experiment (Fri.)
• Continue Nasonia experiment.
High Frequency of Recombination
(Hfr)
...bacteria exhibiting a high frequency of
recombination,
– an alteration DNA sequence such that the genotype of
subsequent individuals differs from the parent,
…specifically, strains with a chromosome
integrated F factor that is able to mobilize and
transfer part of the chromosome to the F- cell.
Hfr Cells
F factor
...F factor
integration site,
...host (bacteria
chromosome)
integration site.
Bacterial Chromosome
Inserted F plasmid
-
F Pilus Attaches to F Cell
Hfr DNA is Cut
F factor and Chromosomal DNA
are Transferred
Recombination Requires
Crossing over
Double Crossover
DNA not Incorporated into
Chromosome are Digested
F factor inserts in different regions of the bacterial
chromosome,
Also inserts in different orientations.
Origin of Replication
Hfr
strain
H
1
2
3
Order of transfer
thr azi ton lac pur gal his gly thi
thr thi gly his gal pur lac ton azi
lac pur gal his gly thi thr azi ton
gal pur lac ton azi thr thi gly his
Indicates direction of transfer.
F factor
Aa
A
aA
Hfr
F-
Hfr DNA that is not incorporated in
the F- strand, and DNA that has
crossed out of the F- strand is
digested.
F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
A
Hfr
A
F-
Leading Gene: the first gene transferred is determined empirically.
E. coli Map
• 0 minutes is at the
threonine,
• 100 minutes is
required to transfer
complete genome,
car::tn10 (0.6)
tetR
car (0.6)
tetS
Selection (all): tetR, nalR
car::tn10 (0.6)
tetR
car (0.6)
tetS
1st Selection: tetR, nalR
car::tn10 (0.6)
tetR
car (0.6)
tetS
Met +, Cys + Met +, Cys - Met -, Cys - Met -, Cys +
car::tn10 (0.6)
tetR
car (0.6)
tetS
Met+ Cys+
Met+ CysMet- Cys+
Met- Cys -
Plates: A __ B __ C __ D __
Plates: A __ B __ C __ D __
Plates: A __ B __ C __ D __
Plates: A __ B __ C __ D __
car::tn10 (0.6)
tetR
car (0.6)
tetS
Met+, Cys+
Plates: A __ B __ C __ D __
car::tn10 (0.6)
tetR
car (0.6)
tetS
Met_, Cys+
Plates: A __ B __ C __ D __
car::tn10 (0.6)
tetR
car (0.6)
tetS
_
Met , Cys-
Plates: A __ B __ C __ D __
car::tn10 (0.6)
tetR
car (0.6)
tetS
+
Met , Cys-
Plates: A __ B __ C __ D __
Assignments
Bacteria II Lab Report (last page ho),
with maps, is due 5/28/10,
pp. 3 assignment (Bacteria II)
due 5/21/10
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.
Rifamycin, Rifampin, 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
…very short course.
LacZ: -galactosidase; Y: galactoside permease;
A: transacetylase (not required for lactose catabolism),
P: promoter; O: operator,
LacI: repressor; PI and LacI are not part of the operon.
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.
•
•
•
•
•
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.
• PART IV: SELECTION FOR LAC-  LAC+ REVERTANTS
• Grow up an overnight culture of your lac- strain in minimal medium
plus glucose.
• Next day do a viable cell count on the culture as described previously
and set up an experiment to identify revertants
– how would you select for revertants?
– how would you screen for revertants?
• REPORT DUE: 6/2/10