Recombinant DNA Technology and Transgenic Animals

Figure 2

The first major step forward in the ability to chemically modify genes occurred when American biologist Martin Gellert and his colleagues from the National Institutes of Health purified and characterized an enzyme in Escherichia coli responsible for the actual joining, or recombining, of separate pieces of DNA (Zimmerman et al., 1967). They called their find "DNA-joining enzyme," and this enzyme is now known as DNA ligase. All living cells use some version of DNA ligase to "glue together" short strands of DNA during replication. Using E. coli extract, the researchers next showed that only in the presence of ligase was it possible to repair single-stranded breaks in λ phage DNA. (Discovered in 1950 by American microbiologist Esther Lederberg, λ phage is a virus particle that infects E. coli.) More specifically, they showed that the enzyme was able to form a 3'-5'-phosphodiester bond between the 5'-phosphate end of the last nucleotide on one DNA fragment and the 3'-OH end of the last nucleotide on an adjacent fragment. The identification of DNA ligase was the first of several key steps that would eventually empower scientists to attempt their own recombination experiments—experiments that involved not just recombining the DNA of a single individual, but recombining DNA from different individuals, including different species.

A second major step forward in gene modification was the discovery of restriction enzymes, which cleave DNA at specific sequences. These enzymes were discovered at approximately the same time as the first DNA ligases by Swiss biologist Werner Arber and his colleagues while they were investigating a phenomenon called host-controlled restriction of bacteriophages. Bacteriophages are viruses that invade and often destroy their bacterial host cells; host-controlled restriction refers to the defense mechanisms that bacterial cells have evolved to deal with these invading viruses. Arber's team discovered that one such mechanism is enzymatic activity provided by the host cell. The team named the responsible enzymes "restriction enzymes" because of the way they restrict the growth of bacteriophages. These scientists were also the first to demonstrate that restriction enzymes damage invading bacteriophages by cleaving the phage DNA at very specific nucleotide sequences (now known as restriction sites). The identification and characterization of restriction enzymes gave biologists the means to cut specific pieces of DNA required (or desired) for subsequent recombination.

Although Griffith and Avery had had demonstrated the ability to transfer foreign genetic material into cells decades earlier, this "transformation" was very inefficient, and it involved "natural" rather than manipulated DNA. Only in the 1970s did scientists begin to use vectors to efficiently transfer genes into bacterial cells. The first such vectors were plasmids, or small DNA molecules that live naturally inside bacterial cells and replicate separately from a bacterium's chromosomal DNA.

Plasmids' utility as a DNA shuttle, or vector, was discovered by Stanford University biochemist Stanley Cohen. Scientists had already established that some bacteria had what were known as antibiotic resistance factors, or R factor-plasmids that replicated independently inside the bacterial cell. But scientists knew little about how the different R factor genes functioned. Cohen thought that if there were an experimental system for transforming host bacterial cells with these R-factor DNA molecules, he and other researchers might be able to better understand R-factor biology and figure out exactly what it was about these plasmids that made bacteria antibiotic-resistant. He and his colleagues developed that system by demonstrating that calcium chloride-treated E. coli can be genetically transformed into antibiotic-resistant cells by the addition of purified plasmid DNA (in this case, purified R-factor DNA) to the bacteria during transformation (Cohen et al., 1972).

The following year, Stanley Cohen and his colleagues were also the first to construct a novel plasmid DNA from two separate plasmid species which, when introduced into E. coli, possessed all the nucleotide base sequences and functions of both parent plasmids. Cohen's team used restriction endonuclease enzymes to cleave the double-stranded DNA molecules of the two parent plasmids. The team next used DNA ligase to rejoin, or recombine, the DNA fragments from the two different plasmids (Figure 2). Finally, they introduced the newly recombined plasmid DNA into E. coli. The researchers were able to join two DNA fragments from completely different plasmids because, as they explained, "the nucleotide sequences cleaved are unique and self-complementary so that DNA fragments produced by one of these enzymes can associate by hydrogen-bonding with other fragments produced by the same enzyme" (Cohen et al., 1973).

Figure 3

Figure Detail

The same could be said of any DNA—not just plasmids—from two different species. This universality—the capacity to mix and match DNA from different species, because DNA has the same structure and function in all species and because restriction and ligase enzymes cut and paste the same ways in different genomes—makes recombinant DNA biology possible.

Today, the E. coli λ bacteriophage is one of the most widely used vectors used to carry recombinant DNA into bacterial cells. This virus makes an excellent vector because about one-third of its genome is considered nonessential, meaning that it can be removed and replaced by foreign DNA (i.e., the DNA being inserted). As illustrated in Figure 3, the nonessential genes are removed by restriction enzymes (the specific restriction enzyme EcoRI is shown in the figure), the foreign DNA inserted in their place, and then the final recombinant DNA molecule is packaged into the virus's protein coat and prepped for introduction into its host cell.

A fourth major step forward in the field of recombinant DNA technology was the discovery of a vector for efficiently introducing genes into mammalian cells. Specifically, researchers learned that recombinant DNA could be introduced into the SV40 virus, a pathogen that infects both monkeys and humans. Indeed, in 1972, Stanford University researcher Paul Berg and his colleagues integrated segments of λ phage DNA, as well as a segment of E. coli DNA containing the galactose operon, into the SV40 genome. (The E. coli galactose operon is a cluster of genes that plays a role in galactose sugar metabolism.) The significance of their achievement was its demonstration that recombinant DNA technologies could be applied to essentially any DNA sequences, no matter how distantly related their species of origin. In their words, these researchers "developed biochemical techniques that are generally applicable for joining covalently any two DNA molecules" (Jackson et al., 1972). While the scientists didn't actually introduce foreign DNA into a mammalian cell in this experiment, they provided (proved) the means to do so.