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
Arg


TGA
stop


AGA
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 relAmutation.
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. coli had transferable plasmidshe called these “F factors”
F+
From:
F-
Chromosomes can transfer when an F-plasmid integrates into
the chromosome
A
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 of the lac operon in E. coli
J. Monod 1951
Note: melibiose induced lacZ, but is not broken down by LacZ—strange! But, commo
inducers are often not broken down by the enzymes they induce!
Note: Napthyl-galactoside is broken down by LacZ, but is not an inducer!
Example of lactose analogs
Example of Example of lactose analogs
Example of difference in induction by similar molecules
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 agalactosidase 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.
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.
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.
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).
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
alpha peptide at LacZ tetramer
interface
Domain 1
Example of
The diagram
Example of
The diagram