Biomedical Engineering Reference
In-Depth Information
same endonuclease or ones producing the same
ends. If the vector has more than one site for the
endonuclease, more than one fragment will be
produced. When the two samples of cleaved DNA
are subsequently mixed and ligated, the resulting
chimeras will, in all probability, lack one of the
vector fragments. It is advantageous if insertion of
foreign DNA at endonuclease-sensitive sites inactiv-
ates a gene whose phenotype is readily scorable,
for in this way it is possible to distinguish chimeras
from cleaved plasmid molecules which have self-
annealed. Of course, readily detectable insertional
inactivation is not essential if the vector and insert
are to be joined by the homopolymer tailing method
(see p. 40) or if the insert confers a new phenotype
on host cells.
pBR322, a purpose-built cloning vehicle
In early cloning experiments, the cloning vectors
used were natural plasmids, such as Col E1 and
pSC101. While these plasmids are small and have
single sites for the common restriction endonucle-
ases, they have limited genetic markers for selecting
transformants. For this reason, considerable effort
was expended on constructing,
in vitro
, superior
cloning vectors. The best, and most widely used of
these early purpose-built vectors is pBR322. Plasmid
pBR322 contains the
Ap
R
and
Tc
R
genes of RSF2124
and pSC101, respectively, combined with replication
elements of pMB1, a Col E1-like plasmid (Fig. 4.7a).
The origins of pBR322 and its progenitor, pBR313,
are shown in Fig. 4.7b, and details of its construction
can be found in the papers of Bolivar
et al.
(1977a,b).
(a)
(b)
R7268
Ap
R
1
R1
drd
19
Ap
R
Cm
R
Sm
R
Su
R
Km
R
4361/1
RSF 2124-derived
material
pSC101-derived
material
Eco
RI
pMB1
2
ColE1
5
pMB3
Ap
R
pSF2124
Ap
R
3
3145
pSC101
Tc
R
pMB8
6
pBR312
Ap
R
Tc
R
4
1763
6
pMB9
Tc
R
pMB1-derived material
7
pBR313
Ap
R
Tc
R
8
pBR322
Ap
R
Tc
R
Fig. 4.7
The origins of plasmid pBR322. (a) The boundaries between the pSC101, pMB1 and RSF2124-derived material. The
numbers indicate the positions of the junctions in base pairs from the unique
Eco
RI site. (b) The molecular origins of plasmid
pBR322. R7268 was isolated in London in 1963 and later renamed R1. 1, A variant, R1
drd
19, which was derepressed for mating
transfer, was isolated. 2, The
Ap
R
transposon, Tn
3
, from this plasmid was transposed on to pMB1 to form pMB3. 3, This plasmid
was reduced in size by
Eco
RI* rearrangement to form a tiny plasmid, pMB8, which carries only colicin immunity. 4,
Eco
RI*
fragments from pSC101 were combined with pMB8 opened at its unique
Eco
RI site and the resulting chimeric molecule
rearranged by
Eco
RI* activity to generate pMB9. 5, In a separate event, the Tn
3
of R1
drd
19 was transposed to Col E1 to form
pSF2124. 6, The Tn
3
element was then transposed to pMB9 to form pBR312. 7,
Eco
RI* rearrangement of pBR312 led to the
formation of pBR313, from which (8) two separate fragments were isolated and ligated together to form pBR322. During this
series of constructions, R1 and Col E1 served only as carries for Tn
3
. (Reproduced by courtesy of Dr G. Sutcliffe and Cold Spring
Harbor Laboratory.)
