1. The ability to manipulate DNA has led to a new genetics. Restriction Endonucleases
In 1980, geneticists used the relatively new technique of gene splicing, which we will describe in this chapter, to introduce the human gene that encodes interferon into a bacterial cell’s genome. Interferon is a rare blood protein that increases human resistance to viral infection, and medical scientists have been interested in its possible usefulness in cancer therapy. This possibility was difficult to investigate before 1980, however, because purification of the large amounts of interferon required for clinical testing would have been prohibitively expensive, given interferon’s scarcity in the blood. An inexpensive way to produce interferon was needed, and introducing the gene responsible for its production into a bacterial cell made that possible. The cell that had acquired the human interferon gene proceeded to produce interferon at a rapid rate, and to grow and divide. Soon there were millions of interferon-producing bacteria in the culture, all of them descendants of the cell that had originally received the human interferon gene.
The Advent of Genetic Engineering
This procedure of producing a line of genetically identical cells from a single altered cell, called cloning, made every cell in the culture a miniature factory for producing interferon. The human insulin gene has also been cloned in bacteria, and now large amounts of insulin, a hormone essential for treating some forms of diabetes, can be manufactured at relatively little expense. Beyond these clinical applications, cloning and related molecular techniques are used to obtain basic information about how genes are put together and regulated. The interferon experiment and others like it marked the beginning of a new genetics, genetic engineering.
The essence of genetic engineering is the ability to cut DNA into recognizable pieces and rearrange those pieces in different ways. In the interferon experiment, a piece of DNA carrying the interferon gene was inserted into a plasmid, which then carried the gene into a bacterial cell. Most other genetic engineering approaches have used the same general strategy, bringing the gene of interest into the target cell by first incorporating it into a plasmid or an infective virus. To make these experiments work, one must be able to cut the source DNA (human DNA in the interferon experiment, for example) and the plasmid DNA in such a way that the desired fragment of source DNA can be spliced permanently into the plasmid. This cutting is performed by enzymes that recognize and cleave specific sequences of nucleotides in DNA. These enzymes are the basic tools of genetic engineering.
Discovery of Restriction Endonucleases
Scientific discoveries often have their origins in seemingly unimportant observations that receive little attention by researchers before their general significance is appreciated. In the case of genetic engineering, the original observation was that bacteria use enzymes to defend themselves against viruses.
Most organisms eventually evolve means of defending themselves from predators and parasites, and bacteria are no exception. Among the natural enemies of bacteria are bacteriophages, viruses that infect bacteria and multiply within them. At some point, they cause the bacterial cells to burst, releasing thousands more viruses. Through natural selection, some types of bacteria have acquired powerful weapons against these viruses: they contain enzymes called restriction endonucleases that fragment the viral DNA as soon as it enters the bacterial cell. Many restriction endonucleases recognize specific nucleotide sequences in a DNA strand, bind to the DNA at those sequences, and cleave the DNA at a particular place within the recognition sequence.
Why don’t restriction endonucleases cleave the bacterial cells’ own DNA as well as that of the viruses? The answer to this question is that bacteria modify their own DNA, using other enzymes known as methylases to add methyl (CH3) groups to some of the nucleotides in the bacterial DNA. When nucleotides within a restriction endonuclease’s recognition sequence have been methylated, the endonuclease cannot bind to that sequence. Consequently, the bacterial DNA is protected from being degraded at that site. Viral DNA, on the other hand, has not been methylated and therefore is not protected from enzymatic cleavage.
How Restriction Endonucleases Cut DNA
The sequences recognized by restriction endonucleases are typically four to six nucleotides long, and they are often palindromes. This means the nucleotides at one end of the recognition sequence are complementary to those at the other end, so that the two strands of the DNA duplex have the same nucleotide sequence running in opposite directions for the length of the recognition sequence. Two important consequences arise from this arrangement of nucleotides.