Genetic Engineering: Cutting and Reassembling DNA

The very first step in a brave new world of genetic engineering was to learn how to cut and then reassemble DNA. It took the work of several geniuses, working across the world, to solve this problem.

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Werner Arber was born in Switzerland in 1929 and graduated from one of the world’s great universities, the Swiss Federal Institute of Technology, in Zurich. Arber studied bacterial viruses. Bacterial viruses are also called bacteriophages. They eat—“phage” comes from eat—bacteria. As a graduate student at the University of Geneva in the 1950s, he studied with a physics professor, and he watched this physics professor get converted from doing pure physics to doing biophysics, being interested in genetics. The DNA structure and the double helix had just been announced, and looking at genes in science was all the rage. So even physicists were catching the biology bug.

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Werner Arber

Arber’s Ph.D. thesis was on the phenomenon of bacteriophage restriction—a phenomenon in which a specific type of bacterial virus can only infect a specific genetic strain of host bacteria. The virus particle with its protein and DNA lands on the outside of the bacterial cell, its host. It injects its DNA into the cell, and this DNA of the bacterial virus then takes over the cell, and half an hour later, that cell, which was converted from a bacterial cell into a virus factory, is dead. The cell is dead, and hundreds of virus particles are released. Arber was specifically interested in the fact that certain viruses were restricted to certain host cells. Only certain host cells seemed to work for a particular virus. Other host cells didn’t.

In bacteriophage restriction, a specific type of bacterial virus infects a specific genetic strain of host bacteria. After the virus injects DNA into a bacterial cell, the DNA takes over and kills the cell, releasing hundreds of virus particles.
In bacteriophage restriction, a specific type of bacterial virus infects a specific genetic strain of host bacteria. After the virus injects DNA into a bacterial cell, the viral DNA takes over, turning the cell into a virus factory, which kills the cell and releases hundreds of new viruses.

Invasion of the Viral DNA

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Daisy Dussoix

Arber’s professors must have been really impressed with him because they hired him in 1960 as a junior professor at the university. In 1962, he and his graduate student, Daisy Dussoix, found that bacteria seemed to evade infection by viruses by chopping up the invading virus DNA into fragments. Arber proposed a hypothesis to explain this phenomenon, and he called this “virus restriction.”

First, host bacteria, Arber proposed, make an enzyme that recognizes a specific DNA sequence on viral DNA—catalyzing the chopping-up of the invading DNA.  Second, the bacteria have an enzyme that modifies their own DNA to make it resistant. Third, virus strains that are successful in infection must have mutations in DNA that make them resistant to the chopping enzyme. They fool the bacteria, and they take over. Arber’s hypothesis—all three aspects—was soon confirmed.

Hamilton Smith
Hamilton Smith

With the first aspect of this hypothesis—that there existed an enzyme that chopped up viruses—shortly after Arber published his hypothesis, Hamilton Smith and a team at Johns Hopkins University isolated and described the chopping enzyme from bacteria. Because it only cut DNA at certain sequences—namely, a sequence that was present in the bacteriophage—they called it a restriction endonuclease or a restriction enzyme; it cuts DNA where there is a certain sequence present.

Resisting Restriction Enzymes

The second aspect of Arber’s hypothesis was that the host cell modifies itself to make itself resistant. And, indeed, Arber, in his own laboratory in Switzerland, characterized this system that modifies its own DNA. There’s an enzyme. There’s a gene that codes for this enzyme in the bacterium that modifies its own DNA bases. It adds some chemical groups, and they’re no longer recognized by the restriction enzyme, so it doesn’t chop its own DNA.

The third aspect of his hypothesis was that successful virus strains must mutate so they’re no longer recognizable. And, indeed, these viruses had mutations in their DNA that altered the DNA base sequence so that it no longer had the site that the restriction enzyme recognized, and so it didn’t cut anymore. All three aspects were confirmed.

Enzymes in a Test Tube

This is all basic research. Scientists soon described other restriction enzymes that would cut DNA at other DNA sequence sites. Each of them was highly specific for a certain site that happened to be on a virus. Early in the 2oth century, it was recognized that a protein will fold in the same way it does inside the cell as if you put the protein in water. The cell is mostly water, so if you take a protein and you put it in water, it’ll fold the same way. It’s a spontaneous process. It’s a genetically determined sequence of amino acids that causes the protein to fold in its own specific way. You can study proteins outside of the cell; you can study enzymes in a test tube. In the same way, you can study restriction enzymes in a test tube. You can take them outside the bacteria, give them some DNA, and they chop it up if the DNA had that particular site.

This led to the first way of mapping DNA. If a restriction enzyme cut DNA wherever there was a sequence AATT, if you have a big piece of DNA, wherever there’s an AATT, it’ll cut. That was the first physical map of DNA in the 1970s. This was done at Johns Hopkins by a colleague of Hamilton Smith—who had done this restriction insight—named Daniel Nathans and his graduate student, Kathleen Danna.

Napkin Doodles Lead to Scientific Breakthrough!

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Stanley Cohen and Herbert Boyer

Meanwhile, on the West Coast, two scientists— Stanley Cohen at Stanford University and Herbert Boyer at the University of California at San Francisco— saw the publication of Nathans’s, Arber’s, and Smith’s works and wanted to follow it up. They thought—if we can take DNA and cut it, maybe we can put it back together again. Well, at Stanford University, another scientist had discovered that there is an enzyme that would catalyze just that. So then Cohen and Boyer apparently, by an anecdotal story, were sitting at a deli in Waikiki where they were at a conference. Ever the scientists, they weren’t out there on the beach surfing; they were at this deli doodling on a napkin, and they doodled two different DNAs, cut them with a restriction enzyme, and put them together in the test tube. And they said, gee, if we can do this with two different DNAs, we can do this with any chromosome, and we can swap chromosome pieces in the test tube.

The First Functional Recombinant DNA

They went back to the lab on the West Coast and tried the experiment using bacterial chromosomes from E. coli. They had two different strains of bacteria. One bacterium had resistance to antibiotic A. Another bacterial strain had resistance to antibiotic B. It had a gene that made it resistant to antibiotic B. They isolated chromosomes from both of these, put them in a test tube, and just as they had planned in the restaurant, they cut the chromosomes open with restriction enzymes and glued the two chromosomes together using this third enzyme.

They had to prove that these chromosomes had been glued together, and so they took some naive bacteria that didn’t have any bacterial resistance to antibiotics, and they put this new chromosome in with them. Lo and behold, these bacteria that never resisted anything now were resistant, in some cases, to both A and B. What had they done? It was 1973. These scientists had taken two chromosomes, cut them open, put them back together, and showed that they were functional in a cell. They had created genetically functional recombinant DNA, the recombination of the two different genomes. It was a revolutionary discovery. It meant that genes from any sources in nature could be taken out of a cell in a laboratory setting and swapped and spliced beside one another.

From the lecture series Understanding Genetics: DNA, Genes, and Their Real-World Applications
Taught by Professor David Sadava, The Claremont McKenna, Pitzer, and Scripps Colleges
Photo of Werner Arber courtesy of Jane Gitschier, University of California, San FranciscoGitschier J (2014) The Inventiveness of Nature: An Interview with Werner Arber. PLoS Genet 10(12): e1004879. doi:10.1371/journal.pgen.1004879
Photo of Hamilton Smith courtesy of Jane GitschierPLoS Genetics.
Photo of Herbert Boyer courtesy of Chemical Heritage Foundation.

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