transposition and site-specific recombination recipient ... · transposition and site-specific...
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Transposition and Site-Specific Recombination9 FIND CHAP TOC
Donor DNA
Target(recipient)DNA
DonorDNA
Transposon
Transposition
RecipientDNA
Transposon
Figure 9.1
Transposition and Site-Specific Recombination9 FIND CHAP TOC
TTargetDNA
IR
ORFAB
ATTTAA
40-bp inverted repeat
Transposase
3-bp target site
IS3
G A T G A C A T T A A C C T A T
ORFBORFAIRIR
A AA C T A C T G T A A T T
Transposition
G G A T A T T
A T TT A A
A T TT A A
Figure 9.2
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Tn5IS50 Kanr Bler Strr IS50
1,500 bp5,700 bp
Tn9IS1 Camr IS1
768 bp2,638 bp
Tn10IS10 Tetr IS10
1,400 bp9,300 bp
Figure 9.3
Transposition and Site-Specific Recombination9 FIND CHAP TOC
IRtnp
Transposase
A IS element
B Composite: 2 IS + gene A
Target DNA
IR
Transposase
A
Target
IR IRIR IR
A
Figure 9.4
Transposition and Site-Specific Recombination9 FIND CHAP TOC
IS10
IS10
ori
Tetr
Ampr
(a)
(a)
(b)
(b)
Figure 9.5
Transposition and Site-Specific Recombination9 FIND CHAP TOC
E
I
I
II
A
D
B
C
iii
Double-strand breaks at inside ends (i and ii) of transposon Tn10
Staggered break (I and II) in target DNA
E II
A
D
B
Cii
II D
B
Cii
i
IE
A
i
II
D
B
CiiI
i
Tetr gene is destroyed
OR
Ends I and i and ends II and ii are joined
Deletion
Inversion
Ends I and ii and ends II and i are joined
A
A
E
EC BD
Tetr
2
3
1
4
5
Figure 9.6
Transposition and Site-Specific Recombination9 FIND CHAP TOC
r determinan
t
RTFTetr
Camr Strr Sulr Ampr
Kan
r
IS3
IS3
IS2
IS1
IS1
Figure 9.7
Transposition and Site-Specific Recombination9 FIND CHAP TOC
IRTn3
IRA res R Ampr
RA
Terminal inverted repeats
Transposase
Resolvase and repressor of A and R transcription
Recombination site
Regulator of Merr and transposase
Mercury resistance
Ampicillin resistance
Inverted repeat
A
R
res
merR
Merr
Ampr
IR
γδres
Tn501A R res Merr merR
Tn21
In2
A R Sulr Strrres Merr merR
Figure 9.8
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Tn10 may hop onto F
Tn10
F
Tn10F
Tetr Strr
Strr×
Tn10F
Tetr Strr
Strr
Figure 9.9
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Amp
r*
Transp
osition No
transposition
Ampr Amps
Ampicillin-resistant colonies No ampicillin-resistant colonies
Plate on ampicillin
Counterselect donor
Figure 9.10
Transposition and Site-Specific Recombination9 FIND CHAP TOC
tpnA or IR
* tpnR or res
No transpositionin transconjugant
Donor
Transconjugant
Recipient
Donor Recipient
Cointegrate
Amp r
Tn
3
res
Amp r
Tn
3
Ampr Tn
3
Cointegrate
Amp r
Tn
3
A
mpr Tn3
Figure 9.11
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Amp
r*
Complemen
tation
No
complementation
Ampr Amps
Ampicillin-resistant colonies No ampicillin-resistant colonies
∆Amp
Figure 9.12
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Single-strand cuts at outside ends of Tn3
3′ ends prime replication through Tn3
Ligation of Tn3 ends to target ends
Staggered cuts 5 bp apart in target DNA
5-bp duplicated sequence flanking Tn3 insertion into target DNA
Cointegrate
3′
Detail of ends
res
Recombination at res ( )resolves cointegrate
3′ OH
3′ OH
3χ OH
5′ PO4
5′ PO45′ PO4
5′ PO4
3′ OH
1
1
2
3
2
5
4
3′
Figure 9.13
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Double-strand breaks at ends of transposon
Staggered break in target DNA
Ligation of transposon ends and target DNA
Repair replication of gaps in target DNA, generating duplication
Donor DNA ( ) destroyed
1
2
3
4
5
Figure 9.14
Transposition and Site-Specific Recombination9 FIND CHAP TOC
lacZ+
lacZ+
lacZ+
lacZ
lacZ
Denature
λ::Tn10 Nam Pam
A Prepare λ::Tn10
C Plate
B Infect at low MOIlacZ
lacZ+ lacZ +
lacZ
lacZ
lacZ
lacZ+
lacZ+/lacZ+
λ cannotintegrate orreplicate
Tn10
Tn10
Transposition
Tetr colonies:16% sectoredcolonies
Medium withtetracyclineand X-Gal
∆ lacZ sup+
E. coli
Anneal
Packagein vitro
lacZ/lacZ lacZ/lacZ+
Figure 9.15
Transposition and Site-Specific Recombination9 FIND CHAP TOC
λ::Tn10lacZ+/lacZ
Targetchromosome
“Cut and paste”
Transposition
1
Colony
White/blue
Tetr lacZ+
lacZ
Tn10
λ
Tetr
Replicative(replication and
resolution)
OR
Lossof phage
lacZ +
lacZ
λ::Tn10
Tn10
Tetr
Tetr lacZ
lacZ+
White
Blue
Tetr
Lossof phage
λ::Tn10
Tetr lacZ+
lacZ
2
Figure 9.16
Transposition and Site-Specific Recombination9 FIND CHAP TOC
tra genes RP4
A
tra genes RP4
ColE1
Tn5
ColE1
Tn5
Tn5
oriT Tn51
Tn5
Tn5 hops intochromosome
Tn5 hops intoplasmid
+
Tn5
Tn5
Tn5
Tn5
Kanr transformant
2
3
4
B
Figure 9.17
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Tn5
PstI
PstI
PstI
PstI
Sal I
Hin
dIII
1.15 kb 1.15 kb3.3 kb
pAT1533.6 kb
PstI2.913 kb
0 HindIII 0.029 kb
Sal I 0.651 kb
Hin
dIII
0.6 kb 1.08 kb 0.92 kb 2.4 kb 0.6 kb
2.6 kb
Kanr
3 kb
HindIII fragments
PstI fragments
Sal I fragments
ori
Amp
r Tet r
Figure 9.18
Transposition and Site-Specific Recombination9 FIND CHAP TOC
c ner A
Immunityregulation
Host DNA in virion
Attachment sites
Invertible region (G segment)
Genes expressed in lysogen
Lysis Tail fiber genes
SU
G recombination
DNA modification
U′S′ attRattL
B lys gin momEarly genes Head genes Tail genes
Transposition
Box 9.1
Transposition and Site-Specific Recombination9 FIND CHAP TOC
BA
23,137
9,416
6,557
2,176
1,766
1,2301,033
653
4,361
2,3222,027
Unknow
n HindIII
Markers
Unknow
n PstI
Markers
2
1.4
19876
5
4
3
2
11 2 3 4
Distance moved (cm)
5 6 7
109876
54.6
43.45
3
Size
(kb
)
Figure 9.19
Transposition and Site-Specific Recombination9 FIND CHAP TOC
0.729
PstI
PstI
Hin
dIII
0.25
0.61.0
0.729
PstI
Hin
dIII
0.25
0.6
0.46
9
PstI
Figure 9.20
Transposition and Site-Specific Recombination9 FIND CHAP TOC
oriT
GCTGCTA C T T A C
oriT
T GAATCA C T T A C
Pint Ptet
int xis orf7 orf6 tetM orf13 orf14 orf15 orf16 orf17 orf19 orf20 orf21 orf23orf18
Pxis Porf9
orf9
Porf7
oriT
orf8
A Tn916
Tn916
B Donor cell
orf22
ACTTAC int xis tetM oriT
tetMxis
int
TGAATG
CGACGA
GCChromosomeChromosomeTGCT
1a
Excision
2 Transfer
Staggered cuts in chromosomal DNA
b
b′
Ligation
a Nick at oriT
c Replication in recipient
b Single-strand transfer to recipient
Porf7
Heteroduplexcouplingsequence
tetM
xis
int
CGACGA
Recipient cell
int xis tetM oriT
Tn916
Couplingsequence
Chromosome Chromosome
3a
IntegrationCuts at coupling sequence
b Crossover into chromosome
TGA
Resolvedleavingdeletion
Imprecise excisionfrom donor DNA
ATG
Box 9.2
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Transposon
Cut (EcoRl); ligate
EcoRl EcoRl
EcoRl
Ampr
Amp r
ori
ori
Bacterial DNA
Transposon
Figure 9.21
Transposition and Site-Specific Recombination9 FIND CHAP TOC
AmprlacZp
Figure 9.22
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Mudlac
Mudlac
lacp
Mu
Mudlac Mu
Mu
Mu
Select Ampr transductants
Ampr
Mudlac Mu
Figure 9.23
Transposition and Site-Specific Recombination9 FIND CHAP TOC
Ampr ori
Ampr ori
A
B
C
Mu
Figure 9.24
Transposition and Site-Specific Recombination9 FIND CHAP TOC
IRIn0
IRP(P2)
int sul1orf4
Phage-like integrase
Sulfonamide resistance
Trimethoprim resistance
Streptomycin and spectinomycin resistance
GTT
59-bp element
Strong promoter
orf5
IRIn3
IRP(P2)
int dhfr sul1orf4 orf5
In2inTn21
int aadA sul1orf4 orf5
IR IRP(P2)
int
sul1
dhfr
aadA
P(P2)
IS1326 IS1353
Figure 9.25
Transposition and Site-Specific Recombination9 FIND CHAP TOC
A
B
Salmonella Hin p H2 flagellin Repressorhin
H1 flagellin
H2 rh1
H1
H1 flagellinH1
Mu Sc Sv U U Sv
p
p
gin
P1 cin
E14
HinH2 rh1
pin
hin H2 rh1
p
p
p
Figure 9.26