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Figure 5.1 Examples ofRestriction Enzyme Sites. The sites are shown whole on the left, and on the right they are shown after being cut by the enzyme. A. Restriction enzyme site for a restriction enzyme from Escherichia coli. B. Restriction enzyme site for a restriction enzyme from Haemophilus influenza.

very long stretches of DNA found in cells. Also, since a given restriction enzyme always cuts DNA at the same sequence, these DNA fragments are a collection of pieces that begin and end with the same base sequence. In addition, these short DNA fragments each contain a limited number of genes. Let us leave restriction enzymes for the time being and focus now on the second ingredient of cloning experiments: plas-mid vectors.

Back in the 1960s and 1970s, scientists discovered that bacterial cells often contained more than one piece of DNA. The majority of the DNA was associated with the bacterial chromosome, a single piece of DNA containing several million base pairs and carrying several thousand genes. However, many bacterial species also harbored a circular "minichromosome" that only contained a few thousand base pairs and carried just a few genes. These "minichromosomes" are called "plasmids." An important property of plasmids is that a bacterial cell can often carry multiple copies, sometimes hundreds, of these "minichromosomes." Another important feature is that, being small, plasmids often contain just a single cutting site for a number of restriction enzymes. Thus, cutting a circular plasmid with a restriction enzyme generates a linear molecule with complementary single-stranded ends (figure 5.2).

Figure 5.2 E. Coli Chromosomes. A diagram of an E. coli cell showing its main chromosome and a minichromosome or plasmid. Because plasmids are much smaller, a restriction enzyme often cuts it at only a single site.

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