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
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
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
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