Lecture15S10 - BIS101/Engebrecht 5/3/2010 Lecture 15...

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BIS101/Engebrecht 5/3/2010 Lecture 15 Genetic Fine Structure (cont): I reviewed how Benzer performed complementation analyses in phage T4 and then talked about intragenic recombination. Complementation = the mixing of gene products in the same cell environment with out any change in the genomes. We have seen complementation with respect to the F1 progeny and also with respect to co-infection of mutant rII T4 in the nonpermissive host E. coli K. Recombination = pairing, exchange and the production of new genotypes. These originate by physical breakage and rejoining of chromosome segments. Intragenic recombination = recombination within a gene. Once Benzer knew that there were two different genes, he could ask about whether the different mutations isolated in one gene were the same or different. He did this by performing phage crosses (see Handout13 ). First he co-infected E. coli B with two mutant phages, he then collected the phage particles and used that to infect E. coli K. If the mutations were different but within the same gene, then at a certain small frequency, recombinant wild-type phage could be generated due to intragenic recombination (= recombination within a gene). You would be able to find these by plating on E. coli K. However, to perform all pairwise crosses would require setting up 9 million double infections (3000 x 3000) in E. coli B and then examining whether recombinant phage were produced by infecting E. coli K. He realized that this wouldn’t be very effective for mapping all of these mutations. Benzer did notice that some of his rII mutants were “revertible”, meaning that he could isolate wild-type phage from them (in molecular terms these were base substitution mutations; the spectrum of mutations is dependent on the mutagen used, at some frequency the base could be mutated back to the wild type sequence) but that some rII mutants were “nonrevertible”. He realized that the nonrevertible class represented deletions, meaning that information was lost and therefore you could not restore wild-type information by a reversion event. He then came up with a scheme to use the deletion mutations in mapping. First he mapped the deletions relative to each other. In this experiment, he co-infected two deletion mutants in rIIA in E. coli B, collected the phage and infected the phage in E. coli K. Only where there was a recombination event that resulted in a wild type phage would he see plaques. If so, this indicated that the deletions were non- overlapping. However, if the deletions were overlapping, no wild type recombinant phage would be formed. Doing pairwise crosses with the different deletions, Benzer could generate a map. To examine and map all of his rII mutants, Benzer used a series of different deletion mutations. Benzer set up crosses to map his different rII point mutations relative to the deletions (see Handout13 ). He performed the experiment by co-infecting the deletion phage with the different point mutations in the permissive host, E. coli B.
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This note was uploaded on 07/02/2010 for the course BIS 101 taught by Professor Simonchan during the Spring '08 term at UC Davis.

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Lecture15S10 - BIS101/Engebrecht 5/3/2010 Lecture 15...

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