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Plasmids 4 FIND CHAP TOC ����������������Table 4.1
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Plasmids4 FIND CHAP TOC

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Table 4.1

Plasmids4 FIND CHAP TOC

AA

T

TA

A

T A A T T T T T T A T T A G T A T

A

A

TT A T T A A A A A A T A A T C A T

AA

T

TB

A

T A A T T T T T T A T T A G T A

T A T T A A A A A A T A A T C A T

A T

TT

Borrelia sp.

Africanswinefevervirus

T

T A T A A T T T T A T A T A T A T

A T A T T A T A T G T A T

A A

TAVacciniavirus A

A T A A T T T T A T A T T A A

T A C T A A A T A A A T A T T A T

A T G A T T T A T T T A T A A T A

A A

A T

Box 4.2

Plasmids4 FIND CHAP TOC

Nick

Relaxed, no supercoiling

Supercoiled, covalentlyclosed circular DNA

Figure 4.1

Plasmids4 FIND CHAP TOC

Covalentlyclosedcircular

Chemical structureof ethidium bromide

EtBrconcentration

Linear DNA

0 Low,~ 2 µg/ml

Higher Higher yet

Up to1 EtBr/2 bases

Negativelysupercoiled

Positivelysupercoiled

No morecan bindRelaxed

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr EtBr

EtBr

EtBrEtBr

EtBrEtBrEtBr

EtBr EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

EtBr

H2N NH2

N+Br–

C2H5

Figure 4.2

Plasmids4 FIND CHAP TOC

Protein and membrane

Linear and nickedcircular DNA

Covalently closedcircular DNA

RNABefore

centrifugationAfter

centrifugation

Figure 4.3

Plasmids4 FIND CHAP TOC

oriV oriV

oriV

oriV

oriV

oriV oriV oriVA

B

C

DSONick

3′ OH is primerfor Pol III

Covalent attachmentto 5′ phosphate

Pol III+ SSB

DNAligase

Nicking andrejoiningby Rep

TyrRep

Tyr

SSO

DSO

CCC DNASS DNA

+

SSO

SSO

RNA Pol

CCC DNAPol III

RNA primer

Figure 4.4

Plasmids4 FIND CHAP TOC

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Table 4.2

Plasmids4 FIND CHAP TOC

pRNAII

mob

bomoriV

cea

immkil

incnic

rop

Figure 4.5

Plasmids4 FIND CHAP TOC

5′

5′

3′3′

Rop dimer

5′

OriginpRNAI

pRNAII

RNA I

RNA II

RNA II

RNA I

RNA II

RNase H

“Kissing” complex

RNA-DNAhybrid allowsRNA IIprocessing

prop

5′

3′5′

5′

RNA II

Pol I

RNA I-RNA II duplex

No RNA II-DNA hybrid

No RNA II processing

No replicationReplication

3′ OH of RNA II primesDNA synthesis

5′

3′5′

3′

Figure 4.6

Plasmids4 FIND CHAP TOC

Noreplicationoccurs

CopB repressesprepA

A Plasmid genetic organization

B Replication occurs afterplasmid enters cells

CopA RNA

RNase III cleavage

CopA RNArepA mRNA

repA codonsTranslational couplingLeader peptide

p copB

RepAactivatesoriV

R1

Promoter

pcopB

prepA

pcopA

Gene products expressed

RepA and CopB

RepA

CopA antisense RNA

RepA

C

C′

Replication shutdown

oriV

repA

copB

oriV

repA

copB

p copB

p repA

p repA

pcopA

pcopAp cop

BprepA

CopB

oriV

repA

copB

+

Figure 4.7

Plasmids4 FIND CHAP TOC

R1DnaA R2 R3 IR1 IR2 repA

repAincpar

mRNA–35 –10

ori

bp 200 400 600 800 1000 1200

Figure 4.8

Plasmids4 FIND CHAP TOC

High concentrationof plasmid;Plasmids coupled

Lowconcentrationof plasmid

No replicationRepli

catio

n

A

A

A A A

A

A AA A

Figure 4.9

Plasmids4 FIND CHAP TOC

R1

R3repA

A

A

R2

AR1

R3R1

R2R3

A

B

R2

Figure 4.10

Plasmids4 FIND CHAP TOC

DimerDimer

Monomer

Monomers

XerCD×

cer

cer

cer

cer

cer

cer cer

Figure 4.11

Plasmids4 FIND CHAP TOC

Figure 4.12

Plasmids4 FIND CHAP TOC

A

B

Figure 4.13

Plasmids4 FIND CHAP TOC

A

Unequaldistribution oftwo plasmids

Each plasmidtype regulatesits own copynumber

B

Unequal distributionof two plasmid types

A B

Plasmids regulate eachother’s replication

Unequaldistributionof A and B

Unequaldistributionof A and B

Cured oftype B

Cured oftype A

Figure 4.14

Plasmids4 FIND CHAP TOC

Medium withoutchloramphenicol

Spread ontoplate withoutchloramphenicol

Incubate

Replicate

andincubate

on plate withchloramphenicol

Incubate

Severalhundredcolonies

Fourcolonies

InoculateDilute

Figure 4.15

Plasmids4 FIND CHAP TOC

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Table 4.3

Plasmids4 FIND CHAP TOC

Medium with ampicillin

This Ampr colony contains the ori clone

Ligate

Ampr

AmprAmprAmpr

Am

pr

+

Transform and select Ampr colonies

ori

ori

Figure 4.16

Plasmids4 FIND CHAP TOC

PstIAmpr Tetr

BamHI

pBR3224.36 kb

ori

EcoRI

Figure 4.17

Plasmids4 FIND CHAP TOC

PstI

BamHI

BamHI

BamHI BamHIAmpr Tetr

Ampr

EcoRI

Digest plasmid and“foreign” DNA with BamHI

Mix and ligate

BamHI

BamHI

BamHI

BamHI

BamHI

BamHI

BamHI

BamHI

Tet r

Amp

r

Tet′

′Tetr

Am

pr

Figure 4.18

Plasmids4 FIND CHAP TOC

pUC18 multiple cloning site and primer binding regions: 364–480

M13/pUC forward sequencing primer T7/T3α sequencing primer450

AlwNI 1217

1000

AfiIII 806

EcoRI 450

HindIII 399

NarI 235NdeI 183

DrdI 91BglI 245

FspI 256PvuI 276PvuII 306

PvuII 628pUC18

2,686 bpTfiI 641

TfiI 781

DrdI 908

lacI'OPZ'

plac

Esp3I 51DraII 2674

AatII 2617BsmAI 2531

SspI 2501

XmnI 2294

BcgI 2215

ScaI 2177

PvuI 2066

FspI 1919

AvaII 2059

AvaII 1837BglI 1813

Cfr10I 1779

2000

Ampr

1

Multiplecloningsite

400

5′–GT AAAACGACGGAAAACGACGGAAAACG

ACGACGT TGTACGACGT TGT

CCAGT–3′Sse 8387 I

HindIII SphI PstI SalIAccIHincII

XbaI BamHI KpnI SstIBanII

EcoRI α-Peptide start–3′

–3′CCAGTGCCAA GCT TGCATGC ACTCTAGAGGCTGCAGGTCG ATCCCCGGGT ACCGAGCTCG AAT TCGTAAT CATGGTCATA GCTGT T TCCT–5′3′–GTACCAGTAT CGACAA

5′–CCCAGTC5′–CCCAGTCM13/pUC forward 23-base sequencing primer

BspMI

XmaISmaI

Figure 4.19

Plasmids4 FIND CHAP TOC

LEU2

Tetr

Ampr

2µm origin

YEp1310.7 kb

ori

Figure 4.20


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