
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


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


IRtnp

Transposase

A IS element

B Composite: 2 IS + gene A

Target DNA

IR

Transposase

A

Target

IR IRIR IR

A

Figure 9.4


IS10

IS10

ori

Tetr

Ampr

(a)

(a)

(b)

(b)

Figure 9.5


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


r determinan

t

RTFTetr

Camr Strr Sulr Ampr

Kan

r

IS3

IS3

IS2

IS1

IS1

Figure 9.7


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


Tn10 may hop onto F

Tn10

F

Tn10F

Tetr Strr

Strr×

Tn10F

Tetr Strr

Strr

Figure 9.9


Amp

r*

Transp

osition No

transposition

Ampr Amps

Ampicillin-resistant colonies No ampicillin-resistant colonies

Plate on ampicillin

Counterselect donor

Figure 9.10


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


Amp

r*

Complemen

tation

No

complementation

Ampr Amps

Ampicillin-resistant colonies No ampicillin-resistant colonies

∆Amp

Figure 9.12


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


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


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


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


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


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


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


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


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


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


Transposon

Cut (EcoRl); ligate

EcoRl EcoRl

EcoRl

Ampr

Amp r

ori

ori

Bacterial DNA

Transposon

Figure 9.21


AmprlacZp

Figure 9.22


Mudlac

Mudlac

lacp

Mu

Mudlac Mu

Mu

Mu

Select Ampr transductants

Ampr

Mudlac Mu

Figure 9.23


Ampr ori

Ampr ori

A

B

C

Mu

Figure 9.24


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


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

Transposition and Site-Specific Recombination Recipient ...

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