Q: Why did each starting strain contain more than one mutation? Neither strain A nor strain B gave colonies on minimal agar. But after mixing, some (1 in 107) that were able to grow. These were recombinants that exchanged genetic material such that they became w.t. for all 5 loci: met, bio, thr, leu and thi. Q: Why did each starting strain contain more than one mutation? From:

Types of mutations that can give back a w.t. phenotype Reversion: A mutation that changes a mutation back to its normal, w.t. sequence. For example a codon can mutate to a stop codon and back to normal again: AGA  TGA  AGA Arg  stop  Arg Suppressor: A second mutation, somewhere else, that fixes the first mutation. For example, bacterial relA- mutants that can’t make ppGpp (an important signaling molecule) are very sick and often acquire a second mutation in rpoB (RNA polymerase subunit) that fixes mosts of the problems associated with the relA- mutation.

The strains were not merely cross-feeding each other. It was shown that physical contact was required for the production of recombinant strains. The strains were not merely cross-feeding each other. No recombinants when strains were put into a device like this. From:

William Hayes found that some E William Hayes found that some E. coli had transferable plasmids-he called these “F factors” F+ F- From:

Chromosomes can transfer when an F-plasmid integrates into the chromosome B In this picture the whole chromosome has been transferred. C Recombination between the incoming donor fragment and the recipient chromosome can occur. Some parts of the the linear piece will recombine, the rest will be degraded (draw on board) From:

Explain Hfr mapping

Transfer times can be used to map genes. Transfer starts at the integrated F element, and it is directional In the top picture, transfer has been going for 25 min. azi entered first and the gal gene has just entered the recipient strain. The bottom shows when recombinants show up. Note that strr/azi+ strains appear before strr/gal+ strains. Therefore azi is closer to the origin of transfer than is gal. From:

The order and direction of transfer depends on where the F element integrates, and which direction it is pointing Different sites and orientations lead to different transfer order From:

Sometimes the integrated F element comes out of the chromosome and brings flanking DNA with it. This can be to “complement” mutations by making diploids The excised F element (called an F’) carries the lac+ locus which can provide w.t. lac function when transferred into a lac- strain. The recipient of the F’ is diploid for the lac region (aka “merodiploid”) From:

How does this relate to Lederberg’s work on Lac?

Cis-trans test with mutations in two different complementation groups

Cis-trans test with mutations in the same complimentation group

inducers are often not broken down by the enzymes they induce! Inducers of the lac operon in E. coli J. Monod 1951 Note: Napthyl-galactoside is broken down by LacZ, but is not an inducer! Note: melibiose induced lacZ, but is not broken down by LacZ—strange! But, common- inducers are often not broken down by the enzymes they induce!

Example of lactose analogs

Example of Example of lactose analogs

Example of difference in induction by similar molecules The only difference between galactose and glucose is the position of the hydroxyl group on C4. This position is apparently very important for recognition by the a-gal induction system. The diagram shows induction of the a-galactosidase gene (AP units) when S. meliloti is exposed to galactose (top) and glucose bottom. Clearly galactose is a good inducer, but glucose is not. Why? The system that induces the a-galactosidase gene must require certain things in order to be effective. A close look at the two sugars shows what one of those things might be.

Lac is not induced in lacY mutants

Lactose doesn’t induce Lac in lacZ mutants

Formation of allolactose by LacZ

From Wilson_JMB_1964 Some 800 Lac negative mutants have been isolated in which the wild alleles appear, by standard complementation test, to be dominant over the mutated alleles. In two cases (Is18 and Is694) however, the mutated alleles turned out to be dominant over the wild allele.

The lac genes cannot be induced in a lacIs mutant Most lac- muations are recessive to w.t however some aren’t. Why is this not fully induced? lacIs maps to lacI From: Wilson_JMB_1964

More on lacIs This phenotype could be caused by a promoter mutant right? But the lacIs muation is trans-dominant, (ie it renders even w.t. cells Lac-) so it can’t be a promoter mutant. LacIs is a repressor that no longer recognizes inducer an is therefore always stuck to the operator, (unless the operator is mutant and doesn’t bind LacI—see last experiment in the table below). The F elements must carry the whole lac region, with the mutations noted.

Nonsense suppressor slide 1 Wild type cells

Nonsense suppressor slide II Mutant with nonsense mutation in gene x

Nonsense suppressor slide II Mutant with nonsense mutation in geneX and a nonsense suppressor mutation

Use of nonsense suppressors in phage genetics Domain 1

Map of pBluescript Domain 1

Genotype of E. coli strain XL1Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacIQ lacZM15 Tn10(tetr)]

E. coli LacZ tetramer Each monomer shown in a different color with one dimer reddish the other blue. Alpha fragments are green

alpha peptide at LacZ tetramer interface Domain 1

Example of The diagram

Example of The diagram