This Week

1
This Week

• Start Mt DNA experiment,
• Start High Frequency of Recombination (HFR)
  experiment,
• Continue Arabidopsis Developmental Screen.

2
Arabidopsis flower

• Wild-type,
• perianth,
• calyx (4 sepals),
• corolla (4 petals),
• androecium,
• 6 stamens,
• gynoecium,
• 2 carpels.

Model Flower System
3
Floral Transition Mutants
Inhibitory Conditions

• Circadian clock mutants,
• Photoreceptor mutants,
• Hormone mutants,
• Homeotic mutants,
• Others.

wild type
mutant
Inducing Conditions
4
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.

5
Hfr Cells
F factor
Bacterial Chromosome
Inserted F plasmid
6
F Pilus Attaches to F- Cell
7
Hfr DNA is Cut
8
F factor and Chromosomal DNA are Transferred
9
Recombination Requires Crossing over
10
DNA not Incorporated into Chromosome are Digested
11
F factor inserts in different regions of the
bacterial chromosome,
Also inserts in different orientations.
12
Origin of Replication
Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
13
F factor
A
a
Hfr
F-
Hfr DNA that is not incorporated in the F-
strand, and DNA that has crossed out of the F-
strand is digested.
14
F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
Leading Gene the first gene transferred is
determined empirically.
15
Hfr Order of transfer strain
H thr azi ton lac pur gal his gly thi 1
thr thi gly his gal pur lac ton azi
2 lac pur gal his gly thi thr azi ton
3 gal pur lac ton azi thr thi gly his
16
E. coli Map

• 0 minutes is at the threonine,
• 100 minutes is required to transfer complete
  genome,

17
pp. 3 assignment due 11/15/2005
cartn10 (0.6) tetR
rpoB (90) rifS
lac (7)
metB- (89)
car (0.6) tetS
rpoB- (90) rifR
metB (89)
cysG- (72)
cysG (72)
gyrA (48) nalR
gyrA- (48) nalS
1st Selection tetR, nalR
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• 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.

19
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.

20

• 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.

21
Mitochondrial Mutation Rate
Electron leak from the ETC (1 3 ) result in the
formation of superoxide.
22

• Mitochondrial Control Region
• Hair follicle DNA extraction,
• PCR,
• Sequencing (at Cold Spring Harbor),
• Sequence analysis here at WWU.