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 workSchmeissner_JMB_1977

Horizontal red lines show where the mutations must lie—between the endpoints ofthe 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 asback-to-back dimer. This puts each binding domainin the right orientation withrespect to the inverted repeat(protein toe binds 5’ end, heelbinds 3’ end of the repeat)

Binding siteshave invertedrepeats

Inverted repeats areon the same sideof 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)xF’

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

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Correlation of physical and genetic maps

Answers “where are mutations located in a particular piece of genetic material”