Recombinant DNA Research

The research by Boyer and Cohen was made possible by discover­ies made two decades earlier by the American biologist James Watson (1928 - ) and the English chemist Francis Crick (1916-2004). In 1953, Watson and Crick announced that genetic information is stored in large, complex molecules known as deoxyribonucleic acid, or DNA. They showed how the characteristic arrangement of certain chemical groups, known as base pairs, might provide a mechanism by which genetic information is stored in DNA molecules. Later re­search showed that the unit of inheritance that had, for more than half a century, been called a gene, was actually nothing more or less than a particular sequence of base pairs in a DNA molecule. The research of Watson, Crick, and their successors has brought about a revolution in the biological sciences in which many of the pro­cesses that take place in living organisms are now explained and understood in terms of chemistry. Indeed, the now-familiar phrase new biology refers to the fact that much of the research in biology is actually chemical in nature.

The diagram on page 91 shows a segment of a DNA molecule. The molecule consists of two very long strands wrapped around each other in a configuration known as a double helix. The strands con­sist of alternating sugar and phosphate groups. The sugar present in DNA is deoxyribose. Attached to each phosphate group on each strand is one of four nitrogen bases. The bases are adenine (A), cyto-sine (C), guanine (G), and thymine (T).

The nitrogen bases are not arranged randomly on the two strands, but always occur in specific base pairs. An adenine always pairs with a thymine (A-T), and a cytosine always pairs with a guanine (C-

Sugar-phosphate backbone

Base

Hydrogen bonds

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Structure of a DNA molecule

-C-C-T-T- 1         1       1        1 A- G- i С 1 -T- i G- i G i -A- i A i -G i -T- 1 c­i c­i T- i A- i -G-G-A-A- ■T- С - G -A- C- С - T- T -C -A- G- G- A- ■T- © Infobase Publishing

Segment of a gene

G). The human genome is thought to contain about 30,000 to 40,000 genes. These genes range in size from a few hundred base pairs to more than 10,000 base pairs. A small segment of a gene might have a structure similar to the diagram above.

Over the last half-century, scientists have learned a great deal about the way in which DNA carries out a variety of essential func­tions in the cell, such as the production of proteins, and the way in which DNA molecules replicate themselves. They have discovered that organisms have evolved a variety of highly specialized chemical molecules (enzymes) that make possible these functions. One group of these molecules is known as restriction enzymes (REs) or restriction endonucleases. Restriction enzymes were discovered in the 1960s by the Swiss microbiologist Werner Arber (1929 - ). Arber found that bacteria had evolved a mechanism for protecting themselves from infections by bacteriophages, a type of virus that infects bacteria. He determined that bacteria contain enzymes that are able to recognize distinctive base pair patterns in the DNA of a bacteriophage. When these enzymes locate those base pairs in a strand of DNA, they cut the bonds that hold the base pairs together, essentially destroying the DNA and inactivating the bacteriophage.

Today, many hundreds of REs are known, each designed to scout out characteristic base pair patterns and cut those patterns in a spe­cific location. The chart on page 93 shows some examples of REs, the base pair patterns they recognize, and the point at which they make a cut in the base pair sequence. Notice that some REs cut the two DNA strands at points directly opposite each other forming two segments with blunt ends.

< SOME EXAMPLES OF RESTRICTION ENZYMES >
BASE PAIR
RESTRICTION ENZYME	BACTERIAL SOURCE	SEQUENCE RECOGNIZED AND POINT OF STAGGERED CUT*
EcoRI	Escherichia col	G|AATTC CTTAA|G
BamHI	Bacillus amylo-liquefaciens	G|GATCC CCTAG|G
Hind\\\	Haemophilus influenzae	A|AGCTT TTCGA|A
Sau3A1	Staphylo-co ccus aureus	N|GATC** NCTA|G
Taq \	Thermus	T|CGA
aquaticus	AGC|T
BLUNT CUT
Alu\	Arthrobacgter	AG|CT
luteus	TC|GA
Stu\	Streptomyces	AGG|CCT
tubercidicus	TCC|GGA
*Vertical line (|) represents point of cleavage.
** N represents any base.

The diagram below shows how the enzyme AluI, for example, would cut a DNA segment:

agg|cct agg cct tcc|gga tcc gga

Other REs, however, make staggered cuts, in which the portions cut on each strand are separated from each other by a small num­ber of base pairs. The diagram below shows how the enzyme EcoRI makes a staggered cut in a DNA segment:

g|aattc g aattc cttaa|g cttaa g

Plasmids are circular pieces of DNA that occur in bacteria and yeast. (N\H/Kakefuda/ Photo Researchers, Inc.)

The earliest experiments on recombinant DNA (rDNA) were made possible in the early 1970s when Cohen and Boyer discovered that the research they were doing independently had overlapping signifi­cance. Cohen, at Stanford University, was investigating the mecha­nism by which the bacterium E. coli could be made to incorporate into its cell a plasmid known as pSC101 that conferred resistance to the antibiotic tetracycline. A plasmid is a circular loop of DNA found in prokaryotic cells, such as bacteria. Boyer, at the University of California at San Francisco, was studying REs. In 1972, the two bio­chemists began working together to develop methods for inserting modified plasmids created with REs into a variety of organisms.

In their first experiments, Boyer and Cohen worked with the plas­mid pSC101 (whose name means that it is a plasmid [p] discovered by Stanley Cohen [SC] with a specific designation [101]. pSC101 is a very simple plasmid containing a gene for replication and a gene that confers resistance to tetracycline. When inserted into another cell, these two genes mean that the plasmid will be able to replicate and that its presence can be detected because the cell will not die when exposed to tetracycline.

In the first step of their initial experiment, as shown in the diagram on page 96, Boyer and Cohen cut the pSC101 plasmid with the restric­tion enzyme EcoRI. The enzyme makes a staggered cut in the plasmid, as shown in the table on page 93. The ends of the cut are said to be "sticky" because they are able to pair with base pairs from any strand of DNA with a complementary base pair pattern. A new gene that confers resistance to the antibiotic kanamycin is then mixed with the cleaved plasmid. The kanamycin gene also has been cut by EcoRI and has sticky ends that are complementary to those of the cleaved plasmid.

To the mixture of cleaved plasmid and kanamycin gene they added DNA ligase, an enzyme that catalyzes the formation of hydro­gen bonds between two DNA fragments. In other words, the ligase brought about the formation of a "hybrid," or recombinant, DNA mol­ecule that contained DNA from both the original pSC101 plasmid and the kanamycin gene.

In the next step in the experiment, Boyer and Cohen mixed the altered plasmid with a colony of E. coli bacteria. In the Boyer-Cohen experiment (and others of its kind), the step was accomplished sim­ply by making a physical mixture of the altered plasmid and the bacteria on a petri dish. Two antibiotics, tetracyclin and kanamycin,

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Gene insertion procedure

were also added to the petri dish. Over time, some of the bacteria on the petri dish absorbed altered plasmids into their cell structures. These bacteria were transformed because they contained not only their own genes but also genes from the plasmid that they had ab­sorbed. Such organisms are sometimes called chimeras, after a crea­ture from Greek mythology with the head of a lion, the body of a goat, and the tail of a serpent. They are also called transgenic organisms because they contain genes from some other foreign organism.

This sheep-goat chimera was created by combining DNA from each species in an egg that was later implanted into a surrogate mother. (Geoff Tompkinson/Photo Researchers, \nc.)

As the altered bacteria reproduced, later generations carried with them the altered plasmids that provided them with immunity to the two antibiotics on the petri dish. Bacteria that had not taken up the altered plasmids had immunity to tetracycline, but not to kanamycin, so they were killed off. Bacteria that had taken up the altered plasmids had immunity to both antibiotics and were able to survive and reproduce. When Boyer and Cohen examined the petri dishes containing bacteria and two antibiotics, they found that some colonies were able to survive and reproduce, proving that they had incorporated the altered plasmids into their cell bodies.

Having successfully transferred DNA from one unicellular organ­ism to another unicellular organism, Boyer and Cohen decided to show that their technique was applicable to more complex organ­isms. They repeated the experiment described above, but used this time a gene from the South African toad, Xenopus laevis. That is, the gene was removed from the DNA of X. laevis cells and then in­serted into E. coli cells. To determine whether the bacterial cells incorporated the X. laevis DNA, they immobilized frog RNA on a nitrocellulose membrane and then added cell extracts from E. coli to the membrane. When cells from altered E. coli were used (that is, cells that had incorporated the X. laevis gene), the RNA bonded with E. coli extracts, while in cells from native E. coli (that which had not been altered with an X. laevis gene), no bonding was observed.

The plasmids in this electron microscope photograph contain segments of DNA that have been inserted into their circular structure. (Dr. Gopal Murti/Photo Researchers, \nc.)

The transfer of a gene from one organism to another by the Boyer-Cohen technique described above was certainly an intellec­tual tour-de-force. Yet, the general principles involved were rela­tively simple and straightforward. Much of the credit due Boyer and Cohen arises from their ability to find ways of carrying out the two or three basic steps involved in producing a recombinant organ­ism: finding ways to cleave a DNA sequence in just the right place;

learning how to join the DNA from two organisms (such as a frog and a plasmid); figuring out how to insert the hybrid DNA into the host organism (E. coli cells in the above example); and proving that insertion of DNA from one organism into a second organism had, in fact, actually occurred.