Another process used by bacteria to obtain DNA fragments from other bacteria is conjugation (Figure 25.2). This genetic recombination process occurs between two living bacteria: a donor and a recipient. The donor and recipient form a cytoplasmic bridge by which single-stranded DNA of the donor crosses to the recipient. On rare occasions the DNA may be integrated into the recipient’s chromosome, but most often the DNA remains in the cytoplasm as a free-floating loop, called a plasmid (see Chapter 6, Bacteria and Archaea).

FIGURE 25.2 Conjugation in bacteria.
*(1)* A cytoplasmic bridge forms between the donor cell and the recipient; *(2)* the cells contact each other; *(3)* one strand of donor plasmid DA transfers to the recipient; and *(4)* a strand complementary to the plasmid is formed in both the recipient and donor cells.

A third form of recombination is transduction, which involves bacterial viruses called bacteriophages (see Chapter 7, Viruses). The virus enters a bacterial cell and integrates its DNA into the bacterial chromosome.

Viruses other than bacteriophages are also useful in genetic engineering experiments. Viruses (see Chapter 7) consist of nucleic acid fragments, either DNA or RNA, surrounded by a protein coat. They can replicate only in living cells, taking over the host cell machinery. Some viruses integrate their own DNA into the host cell’s DNA and become part of the cell’s genome. An example is herpesvirus, which integrates into a nerve cell genome and remains within that cell for years, causing recurrent herpes infections. DNA technologists realized that this was a way to bring genes into a cell of choice. The tools of genetic engineering include the following:

• Restrictions enzymes

• Gene libraries

• Plasmids

• Complementary DNA (cDNA)

• Polymerase chain reaction

Restriction Enzymes

In the 1950s Salvador Luria and colleagues discovered that E. coli could resist destruction by bacteriophages; the organism could “restrict” the replication of the virus. Later, in 1962 Werner Arber and his research group identified the mechanism by which E. coli could achieve this restriction. They showed that an enzyme system was able to cleave the phage DNA before it could get to the bacterial cytoplasm. Subsequently they isolated the DNA-cleaving enzyme, which they called endonuclease, now known as a restriction enzyme. The enzyme is capable of cutting viral DNA but does not cut the host DNA because the organism modifies the bacterial DNA by adding a methyl group to it. Since the work of these two research groups other restriction enzymes have been discovered. More than 800 restriction enzymes have been identified so far, recognizing more than 100 different nucleotide sequences. Restriction enzymes are named after the bacterium from which they have been isolated. The name includes three letters from the bacterium, one from the strain, and roman numerals indicating the order in which enzymes from the same type of bacterium were identified (Box 25.1).

A restriction enzyme scans a DNA molecule, recognizing and cutting DNA only at a particular sequence of nucleotides. Some enzymes cut the DNA straight across both of the strands of the double helix and therefore produce “blunt” ends (Table 25.1). On the other hand, many restriction enzymes cut in an offset fashion and the ends of the cut have an overhanging piece of single-stranded DNA. These ends are called “sticky” ends (Table 25.2) because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Every cleavage performed with the same enzyme will occur at the same restriction site, regardless of the source of the DNA. When such molecules from different DNA sources are mixed together they will join with each other by base pairing between their sticky ends (Figure 25.3, A). This combination can be made permanent by DNA ligase, an enzyme that forms covalent bonds along the backbone of each strand. The result of this reaction is a molecule of recombinant DNA (rDNA) (Figure 25.3, B). The ability to produce rDNA molecules has revolutionized the study of genetics and formed the foundation for the biotechnology industry.

TABLE 25.1

Examples of Restriction Enzymes That Produce “blunt” Ends

Enzyme	Source	Recognition Site
AluI	Arthrobacter luteus	5′-AG↓CT-3′ 3′-TC↑GA-5′

EcoRII Escherichia coli R245

5′-CC↓GG-3′

3′-GG↑CC-5′

HaeIII Haemophilus aegyptius

5′-GG↓CC-3′

3′-CC↑GG-5′

HpaI Haemophilus parainfluenzae

5′-GTT↓AAC-3′

3′-CAA↑TTG-5′

SmaI Serratia marcescens

5′-CCC↓GGG-3′

3′-GGG↑CCC-5′

TABLE 25.2

Examples of Restriction Enzymes That Produce “Sticky” Ends

Enzyme	Source	Recognition Site
BamHI	Bacillus amyloliquefaciens H	5′-G↓GATCC-3′ 3′-CCTAG↑G-5′
EcoRI	Escherichia coli RY13	5′-G↓AATTC-3′ 3′-CTTAA↑G-5′
HindIII	Haemophilus influenzae Rd	5′-A↓AGCTT-3′ 3′-TTCGA↑A-5′
PstI	Providencia stuartii	5′-CTGCA↓G-3′ 3′-G↑ACGTC-5′
SalI	Streptomyces albus	5′-G↓TCGAC-3′ 3′-CAGCT↑G-5′