replication is either “relaxed” or “stringent,” a characteristic determined by the origin of
replication. Plasmids with stringently controlled replication have low copy number, repli-
cating alongside the host’s chromosome, once per cell cycle, while plasmids with
relaxed replication control have high copy number, replicating throughout the host’s cell
cycle, resulting in many hundreds of copies per cell. Whether replication is relaxed or strin-
gent, the rate of plasmid DNA synthesis is controlled to maintain harmony with the host’s
replication. In general, relaxed plasmid replication is controlled by the supply of an RNA
molecule, known asRNA II, which is required to prime (or start) DNA synthesis [for a
review, see Eguchi et al. (1991)]. The supply of RNA II is regulated by another RNA mol-
ecule, RNA I, which is complementary to the RNA II molecule. When these two molecules
hybridize, with the help of a protein known as theRop protein, the priming of DNA syn-
thesis is prevented. Therefore, plasmid replication is inhibited when RNA II is in short
supply. Stringently controlled plasmid replication uses a different mechanism. Here
plasmid copy number is regulated by the supply of the plasmid-encoded RepA protein, a
cis-acting protein, which negatively regulates its own transcription and positively regulates
the origin of replication [for a review, see Nordstrom (1990)]. Relaxed or high-copy-
number plasmids are used most often as vectors to produce large quantities of cloned,
recombinant DNA, while stringent or low-copy-number vectors are used to replicate
massive, unstable, foreign DNA fragments such as BACs, or genes that produce lethal
Figure 7.3.The restriction enzymeSacI recognizes a specific 6-nucleotide palindromic sequence
wherever it occurs in the DNA and cleaves the DNA asymmetrically at specific phosphodiester
bonds to produce 3^0 overhangs or “sticky ends.”
164 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION