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Download High Frequency of Recombination (Hfr)
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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, Maps Lab Report (last page ho), with maps, is due 12/04/08, pp. 3 assignment due 11/25/2008 End of Lecture Help Section pp. 3 assignment due 11/25/2008 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 __
