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E.coli systems and recombination: Determinants of diversity: Overall aims ML. Nine/ten lectures with Key topics. Homologous recombination and DNA repair Role of methylation and repair. Role of Plasmids; control of replication, transfer and stability.
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Tn10 found on R100Cassette model for Plasmid evolution.
Many antibiotic resistance plasmids
such as R1, R6 and R100 are closely related to F- plasmids in the Enterobacteriaceae
e.g. F and R100 from Shigella flexneri
NO ENZYME EXPRESSIONDiscovery of Transposable DNA elements in bacteria
gal operon on defective lambda phage ; dgal
Melt an annealDiscovery of Transposable DNA elements in bacteria
Type Kbps Marker Inverted repeats Target dup’
Tn 1 5.0 ampr 38 5
Tn 3 5.0 ampr 38 5
5.0 NONE38 5
Tn 1721 5.0 tetr and INTEGRON system38 5
9bp duplication 9bp duplicationStructure of IS10 and composite Tn10 as an example
Active in transposition
Package into phage heads.
Infect recA, lac deletion, non-permissive
10% sectored and
90% blue or whiteDemonstration of IS10 conservative transposition
IS10 constructed into phage int-, replication deficient: needs permissive host
Plate on tet/Xgal plates for transposants
Therefore transposition must be conservative
IS10 transposase makes double stranded cuts
And can form circles via single stranded ligation
Only Mg+ needed in reaction
In vitro transposition shown using vectors
Rates of about 1 in 106 shown following
packaging and infection of host cells
Host factors such as;
Integration host factor(Ihf)
and supercoiled DNA needed
All transposons appear to be under negative regulation
This brings transposition recombinational frequencies
down to around 10-3 to 10-6
In E. coli the growth temperature greatly affects many
Higher frequencies at lower temperatures (below 37oC)
Especially IS1 and Tn3. Basis not known.
Negative control due to:
A. Repressor molecule Tn3 (earlier)
B. Antisense RNA (Tn10)
C. Methylation (Tn10 and many IS elements)
D. Transcriptional frameshift (IS1 specifically)
In Pin regionAntisense RNA and methylation: IS10R fromTn10
180 base overlap from Poutcauses
Transposition x10 higher in dam mutants
No expression when methylated
only after replication and hemimethylation
Combination leads to ONLY 0.25 molecules (1 per 4 cells)
of transposase (measured using cat gene fusions)
Many relatively NON specific in target preference
Often NO common features
Tn5 and IS1 prefer hot spot AT rich DNA
Tn7 has specific target
Tn10 shows some preference for a consensus
NGCTNAGCN but not clear cut.
“IMMUNITY” shown by Type II elements (Tn3)
Low probability of second transposition in a plasmid
E. coli chromosome shows strong “immunity”
Basis is not known
The relative role of transposons vs other recombinational
and mutational events.
A SPECTRUM of activities leads to variation
*10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8
* As frequency per cell per generation
The force that through the green fuse drives the flower
Drives my green age; that blasts the roots of trees
Is my destroyer.
And I am dumb to tell the crooked rose
My youth is bent by the same wintry fever
The force that drives the water through the rocks
Drives my red blood; that dries the mouthing streams
Turns mine to wax.
And I am dumb to mouth unto my veins
How at the mountain spring the same mouth sucks
Dylan Thomas 1914 - 1953