Download High Frequency of Recombination (Hfr)

This Week
• Start Mt DNA experiment,
• Start High Frequency of Recombination
(HFR) experiment,
• Continue Arabidopsis Developmental
Screen.
Arabidopsis flower
• Wild-type,
– perianth,
• calyx (4 sepals),
• corolla (4 petals),
– androecium,
• 6 stamens,
– gynoecium,
• 2 carpels.
Model Flower System
Floral Transition
Mutants
Inhibitory Conditions
• Circadian clock mutants,
• Photoreceptor mutants,
• Hormone mutants,
• Homeotic mutants,
mutant
wild type
• Others.
Inducing Conditions
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
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
Inheritance
•
In mammals, 99.99% of mitochondrial DNA (mtDNA) is
inherited from the mother,
•
The sperm carries its mitochondria around a portion of its tail
and has only about 100 mitochondria compared to 100,000 in
the oocyte,
•
As the cells develop, more and more of the mtDNA from
males is diluted out.
–
less than one part in 104 or 0.01% of the mtDNA is paternal.
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 Mutation Rate
Electron leak from the ETC (1 3 %) result in the formation of superoxide.
Protection Pathway
Hydroxyl radical
production
Mitochondrial Control Region
• Hair follicle DNA extraction,
• PCR,
• Sequencing (at Cold Spring
Harbor),
• Sequence analysis here at
WWU.