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This Week

Explore MtDNA and HFR experiments in Arabidopsis flower models, focusing on recombination, mutations, and control regions. Learn about the role of F factor and chromosomal DNA transfer. Discover the intricate pathway of mitochondrial inheritance and mutation rates.

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This Week

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  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. mutant wild type 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. ...F factor integration site, ...host (bacteria chromosome) integration site. 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. Double Crossover 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 tonazi thr thi gly his

  13. Indicates direction of transfer. A a A 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. A A Hfr F- 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 tonazi 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 car::tn10 (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

  18. 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. Protection Pathway Hydroxyl radical production 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.

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