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Transcript
Standard recombination between an F’ and the chromosome Recombination between an F’ and a strain with a deletion (this doesn’t give Lac+) Recombination between an F’ and a strain with a deletion (this does give Lac+) Jeffery Miller made a series of ordered deletions of the lacI gene Ach!! about 1 amino acid apart! He used these map mutations in lacI An example from Miller’s work Schmeissner_JMB_1977 Horizontal red lines show where the mutations must lie—between the endpoints of the deletions to their immediate left and right LacI monomer LacI dimer bound to lacO LacI dimer bound to DNA w/ HTH shown Close up of LacI HTH bound to DNA Binding of regulators to DNA is often at inverted repeats Binding sites have inverted repeats Regulator proteins bind as back-to-back dimer. This puts each binding domain in the right orientation with respect to the inverted repeat (protein toe binds 5’ end, heel binds 3’ end of the repeat) Inverted repeats are on the same side of the helix (spaced ~10 bp apart) LacI dimer forms back to back Other Helix-Turn-Helix (HTH) DNA-binding proteins TraR repressor Crp activator Close up of Crp HTH bound to lac DNA Mutations that give rise to stop codons Frequencies of lacI mutations generated by different mutagens Locations of nonsense mutations in LacI Locations of nonsense mutations in LacI Activity of suppressed nonsense mutations Recombination frequencies between closely linked nonsense mutations lacI(amber1) x F’ lacI(amber2) How frequent are LacI+ relative to distance separating the two mutations? Would expect that mutations that are close together would recombine less that ones that are far apart Location of mutations in lacI that give different lacI phenotypes cAMP levels during growth of E. coli Effect of cAMP on LacZ induction Note that cAMP has less effect on induction on poor carbon sources than on gluco Why is this? cAMP alleviates diauxic growth Adenyl cyclase activities of various E. coli strains Effect of cAMP on cya mutants 1100 1100 5336 5336 Correlation of physical and genetic maps Answers “where are mutations located in a particular piece of genetic material”
