Basic biology of plasmid and phage vectors - Principles of Gene Manipulation - page 51

The Pst I site in the Ap R gene is particularly useful,
because the 3

Plasmid pBR322 has been completely sequenced.
The original published sequence (Sutcliffe 1979) was
4362 bp long. Position O of the sequence was arbit-
rarily set between the A and T residues of the Eco RI
recognition sequence (GAATTC). The sequence was
revised by the inclusion of an additional CG base
pair at position 526, thus increasing the size of the
plasmid to 4363 bp (Backman & Boyer 1983, Peden
1983). More recently, Watson (1988) has revised the
size yet again, this time to 4361 bp, by eliminating
base pairs at coordinates 1893 and 1915. The most
useful aspect of the DNA sequence is that it totally
characterizes pBR322 in terms of its restriction sites,
such that the exact length of every fragment can be
calculated. These fragments can serve as DNA markers
for sizing any other DNA fragment in the range of
several base pairs up to the entire length of the plasmid.
There are over 40 enzymes with unique cleavage
sites on the pBR322 genome (Fig. 4.8). The target
sites of 11 of these enzymes lie within the Tc R gene,
and there are sites for a further two ( Cla I and Hin dIII)
within the promoter of that gene. There are unique
sites for six enzymes within the Ap R gene. Thus,
cloning in pBR322 with the aid of any one of those
19 enzymes will result in insertional inactivation of
either the Ap R or the Tc R markers. However, cloning
in the other unique sites does not permit the easy
selection of recombinants, because neither of the
antibiotic resistance determinants is inactivated.
Following manipulation in vitro, E. coli cells trans-
formed with plasmids with inserts in the Tc R gene
can be distinguished from those cells transformed
with recircularized vector. The former are Ap R and
Tc S , whereas the latter are both Ap R and Tc R . In
practice, transformants are selected on the basis
of their Ap resistance and then replica-plated on to
Tc-containing media to identify those that are Tc S .
Cells transformed with pBR322 derivatives carrying
inserts in the Ap R gene can be identified more readily
(Boyko & Ganschow 1982). Detection is based upon
the ability of the

tetranucleotide extensions formed on
digestion are ideal substrates for terminal transferase.
Thus this site is excellent for cloning by the homo-
polymer tailing method described in the previous
chapter (see p. 40). If oligo(dG.dC) tailing is used, the
Pst I site is regenerated (see Fig. 3.11) and the insert
may be cut out with that enzyme.
Plasmid pBR322 has been a widely used cloning
vehicle. In addition, it has been widely used as a
model system for the study of prokaryotic transcrip-
tion and translation, as well as investigation of the
effects of topological changes on DNA conformation.
The popularity of pBR322 is a direct result of the
availability of an extensive body of information on
its structure and function. This in turn is increased
with each new study. The reader wishing more
detail on the structural features, transcriptional sig-
nals, replication, amplification, stability and con-
jugal mobility of pBR322 should consult the review
of Balbás et al. (1986).

′

Example of the use of plasmid pBR322 as a vector:
isolation of DNA fragments which carry promoters

Cloning into the Hin dIII site of pBR322 generally
results in loss of tetracycline resistance. However,
in some recombinants, Tc R is retained or even
increased. This is because the Hin dIII site lies within
the promoter rather than the coding sequence.
Thus whether or not insertional inactivation oc-
curs depends on whether the cloned DNA carries a
promoter-like sequence able to initiate transcription
of the Tc R gene. Widera et al. (1978) have used
this technique to search for promoter-containing
fragments.
Four structural domains can be recognized within
E. coli promoters. These are:
• position 1, the purine initiation nucleotide from
which RNA synthesis begins;
• position -lactamase produced by Ap R cells

to convert penicillin to penicilloic acid, which in turn
binds iodine. Transformants are selected on rich
medium containing soluble starch and Tc. When
colonized plates are flooded with an indicator solu-
tion of iodine and penicillin,

β 12, the Pribnow box;

• the region around base pair

− 6 to −

35;
• the sequence between base pairs

−

35.
Although the Hin dIII site lies within the Pribnow
box (Rodriguez et al. 1979) the box is re-created on
insertion of a foreign DNA fragment. Thus when
insertional inactivation occurs it must be the region
from

− 12 and −

-lactamase-producing
( Ap R ) colonies clear the indicator solution whereas
Ap S colonies do not.

β

− 13 to − 40 which is modified.