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 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) Binding sites have inverted repeats 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 repressorCrp 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

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 glucose— Why is this?

cAMP alleviates diauxic growth

Adenyl cyclase activities of various E. coli strains

Effect of cAMP on cya mutants

Correlation of physical and genetic maps Answers “where are mutations located in a particular piece of genetic material”