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

• Circadian clock mutants,

• Photoreceptor mutants,

• Hormone mutants,

• Homeotic mutants,

• Others.

Inhibitory Conditions

wild typemutant

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

Bacterial Chromosome

Inserted F plasmid

...F factor integration site,

...host (bacteria chromosome) integration site.

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 Order of transferstrain

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

F factor

Hfr F-

A

a

Indicates direction of transfer.

A

A

a

Hfr DNA that is not incorporated in the F- strand, and DNA that has crossed out of the F- strand is

digested.

F factor

Hfr F-

A

Leading Gene: the first gene transferred is determined empirically.

A

Hfr

A

F-

A

A transfers first.

A transfers last.

Hfr Order of transferstrain

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

E. coli Map

• 0 minutes is at the threonine,

• 100 minutes is required to transfer complete genome,

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

1st Selection: tetR, nalR

pp. 3 assignmentdue 11/15/2005

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.