Download High Frequency of Recombination (Hfr)

This Week
• Score Conjugation Plates,
• Start High Frequency of Recombination
(HFR) experiment,
• 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.
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
E. coli Map
• 0 minutes is at the
threonine,
• 100 minutes is
required to transfer
complete genome,
pp. 3 assignment
due 11/15/2005
car::tn10 (0.6)
tetR
car (0.6)
tetS
1st Selection: tetR, nalR
car::tn10 (0.6)
tetR
car (0.6)
tetS
1st Selection: tetR, nalR
Selection Scheme
Supplements
Marker Selected
met, cys
Plate A
proline, arginine, uracil
Plate B
proline, arginine, uracil,
methionine
cys
Plate C
proline, arginine, uracil,
cysteine
met
proline, arginine, uracil,
methionine, cysteine
no selection
Plate D
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 __