The Science

Mar 10, 2006
By BioPharm International Editors

At the heart of modern biotechnology is the ability to manipulate DNA, the astonishing molecule that contains the genetic code of all life on earth. But fermentation and cell culture are also the practical art of keeping cells alive and growing in an artificial environment. As a result, the science of cell culture also includes a healthy focus on the natural processes that keep cells alive.

Recombinant DNA

Though human beings have long understood that certain characteristics could be passed from parent to child, until a half century ago they could only guess at how that worked. But then in February 1953, two young Cambridge scientists named James Watson and Francis Crick came up with the key when they worked out the structure of the DNA molecule.

Scientists already knew that DNA was composed of four "base" molecules: adenine, thymine, guanine, and cytosine (A, T, G, and C). What Watson and Crick determined is that the DNA molecule consists of two long strands of bases twisted around each other like a corkscrew staircase. More important, if you looked at the bases as they linked up across the "rungs" of the ladder, A was always linked to T and G to C and vice versa. The two strands were complementary: Pull them apart, and each could recreate the original double strand by adding the missing bases.

Watson and Crick's model showed how DNA could transmit information: information that was written in the four-letter language of G, A, T, and C. A crucial discovery came later—roughly 10% of the DNA molecules were genes that contained specifications for the production of proteins. These proteins are written in three-base "codons," each of which stands for a particular amino acid.

Throughout the 1960s, researchers studied DNA partly by breaking it into manageable bits using restriction enzymes. These enzymes recognize and bind to a specific pattern of base pairs and break the DNA molecule precisely at that point. Sometimes these enzymes leave blunt ends (as EcoRV does) and sometimes "sticky" ends (as EcoRI and TaqI do). Other enzymes called ligases bind those frayed ends together.

In 1972, John Morrow, Herb Boyer, and Stanley Cohen used these techniques to conduct an ingenious experiment. Using restriction enzymes and ligase, they pasted a bit of DNA from an African frog into a plasmid (a ring-shaped DNA molecule found in bacteria). When the plasmid was taken up by an E. coli bacterium, it became part of the organism's DNA: It was passed on from generation to generation — and it continued to produce frog RNA.

Morrow, Boyer, and Cohen used the plasmid, in the language of biotechnology, as a cloning vector. The job of the cloning vector was to carry foreign DNA into the E. coli without disturbing the organism's ability to reproduce. Plasmids were the first successful cloning vectors, but there were limits to how much DNA could be pasted into plasmids. A plasmid can typically accommodate DNA that is about 10,000 base pairs long. A cosmid, which consists of DNA taken from a virus that naturally lives in bacteria, can accommodate about 45,000. New vectors have been developed that will hold much larger fragments of DNA: A yeast artificial chromosome (YAC) can hold 100,000 base pairs. Mammalian and other artificial (or synthetic) chromosomes have been developed for even larger genes.

The African frog experiment contained the roots of the biotechnology revolution: It showed that altered or recombinant DNA could be passed on by a host organism, and that genes from one species could continue to function even when spliced into the DNA of a totally different species. From there it wasn't too great a leap to the insight that fast-growing bacteria or yeast loaded with recombinant DNA could be used to economically produce proteins and other substances in large quantities.