Technology: The Inventions That Make Us Go Wow!

Discovering the Cut-and-Paste Enzymes  A diagram shows a circular grey plasmid in column 1 and a circular red plasmid in column 2. Both plasmids look like two concentric cir-cles; each circle represents a DNA strand. Different restriction en-zymes are used to cleave, or cut, plasmid 1 and plasmid 2. \"Sticky ends,\" or overhanging DNA ends without a complementary strand, are treated with an enzyme that digests single-stranded DNA. New complementary sticky ends are then added by terminal transferase. dATP is added to one plasmid, and dTTP is added to the other plas-mid to produce poly-A and poly-T sticky ends, respectively. After the addition of complementary sticky ends to plasmids 1 and 2, the two plasmids are mixed together, and the complementary sticky ends base pair. A recombined plasmid is shown in a single, center col-umn. The recombined plasmid is composed of two larger concentric circles; half of the circle is grey, and the other half is red. DNA poly-merase, shown as a blue enzyme encircling both DNA strands, is added to the new, recombined plasmid to insert missing nucleo-tides. DNA ligase, shown as a small yellow enzyme encircling one DNA strand, seals nicks in the sugar-phosphate groups to ensure the fragments from each plasmid are joined together.  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 col-leagues 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 bacteri-al cells have evolved to deal with these invading viruses. Arber's team discovered that one such mechanism is enzymatic activity pro-vided 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 cleav-ing the phage DNA at very specific nucleotide sequences (now known as restriction sites). The identification and characterization of

 

 

View Full-Size ImageFigure 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 re-combining, 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 Leder-berg, λ phage is a virus particle that infects E. coli.) More specifical-ly, they showed that the enzyme was able to form a 3'-5'-phosphodiester bond between the 5'-phosphate end of the last nu-cleotide on one DNA fragment and the 3'-OH end of the last nucleo-tide on an adjacent fragment. The identification of DNA ligase was the first of several key steps that would eventually empower scien-tists 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 spe-cies.