Biotechnology: How Do We Use What We Know about Life?

Transcription

1 7 Biotechnology: How Do We Use What We Know about Life? Overview Designer babies, gene therapy, genetically modified crops, DNA evidence in the courtroom these are the phrases that come to mind when most people think of biotechnology. We are witnessing a revolution in science that could include all of these things and more a revolution that may have implications for our society that are beyond anything previously imagined. The term biotechnology actually refers to any technique that uses living organisms or substances from those organisms in agriculture, We shall never cease from exploration; and the end of all our exploring will be to arrive where we started, and know the place for the first time. T. S. Eliot, 1942 Chapter opening photo Computerized images of molecules are used to design medicines. 174

2 7-1 What Tools Are in the Biotechnology Tool Box? 175 industry, or medicine. Farming, for example, or the domestication of animals is a form of biotechnology. In recent years, however, the term has been used nearly synonymously with DNA technology the tools and techniques for manipulating the genetic material in organisms, ranging from bacteria to humans. It is this part of biotechnology that will be the focus of our discussion. We begin by taking a look at the tools that are used for manipulating DNA. Then we will explore just a few of the different applications of DNA technology, as well as some of the difficult ethical issues that arise when we begin to tinker with our genes. 7-1 What Tools Are in the Biotechnology Tool Box? By the end of the 1960s, that new breed of scientists who called themselves molecular biologists had constructed an accurate picture of the flow of information in prokaryotic cells. DNA in particular the sequence of A, T, C, and G acts as a template for constructing the intermediary molecule RNA, which encodes the information for assembling amino acids into proteins (Figure 7-1). Gene expression, or the selective production of particular proteins, is controlled by operons and regulatory feedback loops. to cell division replication prior DNA genetic information transcription mrna message translation PROTEIN cellular function Figure 7-1 By the 1960s, the flow of information within a cell from DNA to mrna to protein was understood. This is the central dogma of biology.

3 176 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? The big questions yet to be answered were how genes functioned in larger and more complex cell types the eukaryotes. Surprisingly, the answers to these questions, too, would come from experiments on microbes, namely bacteria and viruses. The techniques this new breed of biologists developed to learn about genes in eukaryotic cells have become the most powerful tools in the biotechnology toolbox. Bacteria and Viruses Play a Central Role in Biotechnology Why use microbes to study and manipulate eukaryotic DNA? Several features of bacteria make them particularly good for doing experiments (Figure 7-2). First, bacteria are small, easy to house, cheap to feed, and quick to multiply in the laboratory. Under the right conditions, bacteria can double their numbers in just 20 minutes and double again 20 minutes later. In a few short hours, a culture of bacteria can be grown to contain an enormously large numbers of cells. Second, the bacterial chromosome is simple compared with eukaryotic chromosomes. Bacterial DNA occurs as a simple closed loop with no complicating proteins such as those found in eukaryotic chromosomes. Third, bacteria often carry small extra loops of DNA, called plasmids, that are replicated and passed from one generation to the next when the cells divide. Plasmids are sometimes naturally released into the surrounding medium by one bacterium and taken up by another in a process called transformation. (In Chapter 5, Section 5-3, we saw that transformation was one of the phenomena that helped identify DNA as the genetic material. Griffiths transforming principle that converted harmless R-strain bacteria to virulent S-strain cells in mice was a plasmid.) Researchers quickly 1 Rapid growth rate Number of bacterial cells Time (hours) Bacterial chromosome 2 Bacterial chromosome lacks proteins "naked" DNA Figure 7-2 Microbes such as bacteria are useful for studying the function of genes in higher organisms like humans. This is because microbes grow rapidly in culture; their DNA is naked, that is, it lacks accessory proteins found in eukaryotic DNA; and because bacteria can be made to carry self-replicating rings of DNA called plasmids. 3 Bacterial cells can carry self-replicating plasmids Plasmids Bacterial chromosomes

4 7-1 What Tools Are in the Biotechnology Tool Box? 177 learned that they could add short stretches of DNA containing perhaps a gene or two and some regulatory sequences to bacterial plasmids and that these genetically engineered plasmids were readily taken up by bacterial cells and faithfully replicated with each round of bacterial cell division. Starting with a small stretch of eukaryotic DNA, a molecular biologist can use bacteria to grow bulk quantities of the stuff, all of it identical to the starting DNA. This is called gene cloning using bacteria to make multiple identical copies of a single stretch of DNA. Gene cloning was an important step in learning about eukaryotic genes, and it is a tool still used extensively in modern DNA technology. A genetically engineered plasmid is just one example of a cloning vector. A cloning vector is any vehicle that inserts a fragment of foreign DNA into the genome of a host cell.a virus, for example, can act as a cloning vector. Recall from Chapter 4 (Section 4-2) that viruses are little more than molecules of DNA or RNA housed in a protective protein coat. When a virus lands on a suitable host, its nucleic acid is injected into the host cell. Once inside, viral DNA either commandeers the host cell s machinery to make copies of itself or its DNA inserts itself into the chromosomes of the host to be replicated when the cell divides. Either way, the viral DNA is a genetic hitchhiker in the host cell, much like a genetically engineered plasmid is in a bacterial cell. Molecular biologists have used viruses as cloning vectors to insert foreign genes into various host cells, including those of humans. Viral cloning vectors, for example, are often used in gene therapy. In clinical trials, people born with two damaged copies of a gene have been purposely infected with a weakened virus that has been engineered to carry a healthy copy, or allele, of the damaged gene. The hope is that, once inside the human cells, the healthy human allele introduced by the virus will behave like a normal gene. This approach has met with only limited success, but more on that later. For now we shall examine the manner in which plasmids and viruses are engineered to carry foreign genes. In other words, how is DNA cut, pasted, and visualized? Molecular Tools Are Used to Manipulate DNA Prior to the advent of DNA technology, human proteins, such as insulin used in the treatment of diabetes and growth hormones used in the treatment of growth abnormalities, were prohibitively expensive or not available at all. Sufferers of these maladies were treated with proteins isolated from cows or pigs brought to slaughter. However, the differences between bovine (cow) or porcine (pig) proteins and human proteins meant that these animal proteins were less efficient in humans. Today, cultures of bacteria engineered to carry the human genes for these proteins produce vast quantities of insulin, growth hormone, and many other human proteins, cheaply and accurately (Figure 7-3). Genetic engineering, or the ability to precisely manipulate DNA sequences from widely different organisms, has revolutionized the pharmaceutical industry. Figure 7-3 Genetically engineered bacteria containing human genes are grown in bulk. Pharmaceutical companies harvest the human proteins, such as insulin and growth hormone, synthesized by these living factories.

5 178 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? We have already seen how to get foreign genes into a bacterial cell by using a cloning vector such as a plasmid or virus, but how does one get a fragment of DNA into the cloning vector? The size of native, uncut DNA from most sources is much too large to be inserted into a cloning vector. The DNA first must be broken into small fragments, each one small enough to be inserted into a plasmid or other cloning vector. In the early days of molecular biology, native DNA was bombarded with high-frequency sound waves. This treatment smashed the DNA into bits, but the result was always a wide assortment of fragments of random sizes. This made it impossible to replicate an experiment from one day to the next; each time a new cell s DNA was shattered with sound waves, the resulting collection of DNA fragments was different from the time before. Several important developments in the early 1970s, however, made the task of creating cloning vectors much easier. These were (1) the ability to cut DNA at specific places, reliably and consistently every time, and (2) the ability to combine any two pieces of cut DNA, regardless of the source of either piece. In other words, scientists discovered tools that acted like molecular scissors and molecular paste. Exploration Bigger and Better Vectors Plasmids and viruses are excellent vectors for getting small bits of DNA on the order of about 5,000 to 10,000 base pairs into host cells, but there are times when it is desirable to introduce much larger fragments of DNA into a cell for cloning. In recent years, several large vectors have been created that can insert as many as 35,000 to 45,000 base pairs into a bacterial host. These artificial vectors are called cosmids. Even larger pieces of DNA, up to 200,000 base pairs, can be cloned into yeast cells by using yeast artificial chromosomes, or YACs.What components must be engineered into a cosmid or a YAC so that the foreign DNA is properly replicated in its host cell? What other cloning vectors have been developed for getting large fragments of DNA into bacteria and other hosts? How have these cloning vectors been used in biotechnology? Molecular Scissors and Molecular Paste Billions of years ago, when prokaryotic cells ruled the planet, bacteria were subject to invasion by stray bits of nucleic acid, usually in the form of viruses. Bacteria that could resist viral infection had an advantage over those that could not; they were more successful than nonresistant bacteria. One strategy for fighting off viruses is to degrade their DNA by chopping it into small pieces. Bacteria have evolved an arsenal of enzymes, called restriction enzymes or restriction endonucleases, that do just that. Restriction endonucleases cut DNA at specific places, resulting in fragments that can be easily removed by the bacterial cell. But restriction endonucleases owe their recent fame to the ways in which molecular biologists use them to manipulate DNA in the laboratory. These enzymes serve as molecular scissors one of the most powerful tools in DNA technology. Since their discovery in the early 1970s, over 800 different restriction endonucleases have been identified and isolated from various bacteria. Each one of them recognizes a specific sequence of nucleotides on the DNA to be cut, called its recognition site (Figure 7-4). For example, the restriction endonuclease called EcoR1 (so named because it was the first restriction enzyme found in the bacterium Escherichia coli) has a recognition site that looks like...gaattc cttaag.... Whenever EcoR1 encounters DNA containing this particular sequence, the enzyme will cut both strands of the DNA as follows:...g AATTC......CTTAA G....

6 7-1 What Tools Are in the Biotechnology Tool Box? 179 Restriction enzyme EcoRI cuts here (before AATT) T G C A G A A T T C A T T C A C G T C T T A A G T A A G Pieces separate Each piece has a "sticky end" T A G C C G A T G C A A T T A A T T C G A T T T A A C G EcoRI cuts here (before AATT) Figure 7-4 Restriction enzymes, such as EcoR1 shown here, cut DNA at specific nucleotide sequences called restriction sites. A cut made with EcoR1 leaves sticky ends capable of forming hydrogen bonds with other DNA cut with the same enzyme. This is the basis for recombinant DNA technology. There are two important things to note about the EcoR1 restriction site: First, the nucleotide sequence of the recognition site is an inverse palindrome; in other words, the nucleotide sequence is the same when it is read forward as it is when it is read backward and upside down. This is typical of restriction enzyme recognition sites (Figure 7-5). Second, the enzyme cuts in a manner that leaves several unpaired nucleotide bases on both strands of the DNA. These unpaired bases are called sticky ends, a term that describes their tendency to find similar unpaired sequences and form hydrogen bonds with them. Imagine that two strands of DNA, one from a bacterium and one from a human, were both cut with EcoR1 in the same test tube (Figure 7-6). Some of the sticky ends of the bacterial DNA would find complementary sticky ends from other bacterial DNA molecules, but others would find partners from among the human DNA strands with Bacterial strain Enzyme name Recognition sequences and cleavage sites Bacillus amyloliquefaciens H Bam H1 G GATCC C C T A G G Escherichia coli Ry13 Eco R1 G A A T T C C T T A A G Providencia stuartii 164 Pst 1 C TGCAG GACGTC Serratia marcescens SB Sma H1 C C CGGG GGGCCC Rhodopseudomonas sphaeroides Rsa 1 GTAC CATG Figure 7-5 Five restriction enzymes and the DNA sequences of their restriction sites. This is a small sampling of the over 800 different restriction enzymes currently known.

7 180 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Bacterial chromosome Plasmid Plasmid DNA is isolated from bacterial cells. Plasmids are cut in precise spots using restriction enzymes. A recombinant DNA plasmid is made by grafting the gene of interest into the plasmid. Chromosomal DNA is isolated from a different organism. A gene of interest is removed from the DNA using the same restriction enzyme used to cut the plasmids. The plasmids are reintroduced into bacteria. Figure 7-6 Making a molecule of recombinant DNA. The bacteria are grown in culture, when they synthesize the desired substance. complementary sticky ends. Once such partnerships have formed by hydrogen bonding, the breaks within the sugar-phosphate backbones can be sealed by using another bacterial enzyme, DNA ligase. DNA ligase plays a role in normal DNA replication and heals naturally occurring breaks in DNA. But in the molecular biology laboratory, DNA ligase is used to join fragments of DNA from different sources that have been cut by using the same restriction endonuclease. In other words, DNA ligase acts as molecular paste. A DNA molecule formed from the DNAs of different organisms is called recombinant DNA. In this example, the recombinant DNA would be part bacterial and part human. Agarose Gel Electrophoresis: Visualizing Cut DNA Restriction endonucleases made it possible to cut DNA into fragments of consistent sizes, as long as the DNA is taken from the same source each time it is cut. But how do we know what sizes of fragments we have made in such an experiment? A simple technique, called agarose gel electrophoresis, serves the dual purpose of separating bits of DNA on the basis of their size and allowing us to visualize the different fragments and determine exactly how big each one is. Figure 7-7 shows a researcher examining an agarose gel. The gel itself is a jellylike slab made of a polysaccharide called agarose. Mixtures of DNA fragments are introduced at one end in depressions formed in the gel, called wells.the entire gel is placed in an electrical field with the anode (the positive electrode) at the end farthest from the wells.

8 7-1 What Tools Are in the Biotechnology Tool Box? 181 Figure 7-7 Fragments of DNA are separated by using agarose gel electrophoresis. A mixture of DNA fragments of different sizes has been separated on the basis of size. Each bright band in this photograph represents DNA fragments of a different size. DNA has a negative electrical charge, so the fragments are attracted to the anode. But the speed with which the fragments of DNA move through the gel depends on their size. Small fragments are hindered less by the agarose than large fragments are; hence, small fragments move faster and further in the gel. The researcher in Figure 7-7 is examining a series of bands on a gel, each one representing DNA fragments of a different size from those on every other band.the bands glow under ultraviolet light because the gel has been treated with a chemical that binds to DNA and fluoresces under a UV lamp. Imagine an experiment in which the DNA from a few of your cells, collected perhaps from rinsing your mouth out with salt water or scraping your cheek with a toothpick, is digested with a certain restriction endonuclease. The number of fragments that result will depend on the number of times the recognition site for that enzyme occurs just by chance in your DNA. The size of the different fragments that result will depend on how much DNA resides between each recognition site. When recognition sites are infrequent or far apart, the fragments will be large. If there are many sequences that match the recognition site or two or more recognition sites that are close together, the fragments will be small. The different sized fragments of DNA that result from this digestion give a characteristic pattern of bands when they are separated on an agarose gel (Figure 7-8). For most parts of your DNA, the pattern of bands that result from separating restriction fragments on an agarose gel will look exactly like the pattern of every other person. After all, DNA from every human being is about 99.9% identical to that from every other person. There are a few regions in the human genome, however, where the sequences of DNA are unique to each individual. When these regions are digested with certain restriction endonucleases and the fragments are separated on a gel, the pattern of bands is as unique to you as your fingerprints. This is the principle behind DNA fingerprinting, the powerful technique that has been used to identify individuals with astounding accuracy. We will have more to say about DNA fingerprinting in Section 7-3. Agarose gel electrophoresis allows us to visualize two things: First, the number of different DNA fragments in a mixture and second, the size, or number of DNA base pairs, of each fragment. Fragment size is estimated by comparing the position of different bands on the gel with the positions of standards DNA fragments whose size we knew before we started applied to a different well on the same gel. Recall that

9 182 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? - cathode long fragments Power Supply intermediate fragments short fragments + anode A mixture of DNA fragments is carefully added to the wells of an agarose gel. In this gel, three different mixtures are added to each of three different wells. The entire agarose gel is placed in an electrical field, with the anode farthest from the wells. The DNA migrates through the gel toward the anode. Smaller fragments migrate farthest. After the DNA fragments have been separated, the electrical field is removed. The number of bands indicates how many different-sized fragments of DNA were in the mixture. The position of the bands indicates how big each fragment is. Figure 7-8 Agarose gel electrophoresis separates fragments of DNA on the basis of their size or the number of nucleotides in the fragment. smaller fragments run farther down the gel toward the anode than larger fragments. In this way, a gel automatically sorts fragments of DNA by size. But one thing we could not know from examining a gel is which fragments contain genes or where any particular gene, say the gene for insulin, is found among all the fragments produced by the restriction endonuclease. To identify genes, we need to use some other techniques of molecular biology. One approach is to make a DNA library containing clones of all the different DNA fragments from a particular cell and then use a molecular probe to look for the particular book in our library, that is, the DNA fragment containing the gene of interest. DNA Libraries and Molecular Probes Suppose you wanted to engineer a bacterial cell to make large quantities of the human protein insulin. To begin, you might use a restriction endonuclease to cut all of the DNA from a human cell (or many identical human cells) into small fragments (Figure 7-9). One of these fragments contains the gene for insulin, but there is no way to determine by looking at them which one it is. In addition, although there are lots of DNA fragments, each one (including the one containing the insulin gene) is present in very low concentrations. To find the insulin gene, each fragment must first be cloned in a bacterial host to increase the number of copies. How is this done? Recall that by inserting DNA fragments into cloning vectors and allowing bacterial cells to take up the vectors, the bacteria will do the work of copying your DNA fragments each time they divide, a process called gene cloning. The entire collection of bacterial cells, which together contain all of the human DNA fragments, is called a DNA library. The term DNA library evokes an accurate analogy. Each fragment of DNA, human DNA in our example, is like a single volume in an enormous library containing about a million different books. The entire bacterial population containing all the different human DNA fragments is the library s book collection. Unfortunately, DNA fragments are not as carefully catalogued as they are in an actual library, so finding the volume of interest the human insulin gene will be a bit more difficult than searching a library catalog. For that we need to screen our DNA library.

10 7-1 What Tools Are in the Biotechnology Tool Box? 183 Bacterial chromosome Plasmid Purify human DNA containing insulin gene Purify plasmid DNA Treat both human DNA and plasmid DNA with the same restriction enzyme. human insulin gene The human DNA has many restriction sites. Hence, many different DNA fragments are formed. Only one contains the gene for insulin. The plasmid DNA has only one restriction site. This creates open circles of DNA with the same sticky ends as the human DNA fragments. The human fragments are joined with the plasmids to make recombinant DNA. Each plasmid has a different fragment of human DNA. Bacterial cells are transformed with the recombinant plasmids. One may contain the human insulin gene. Plasmid-free bacteria Figure 7-9 Making a DNA library from human DNA.

11 184 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Exploration Working Backward The DNA library just described is a genomic library, a set of cloned DNA fragments representing the entire genome of an organism (in our example, a human). Depending on the source of DNA, a genomic library may be small, or it may be enormous.a human genomic library, for example, requires about 1 million different bacterial clones, each containing a different plasmid. Such large libraries are often unwieldy and difficult to manipulate. It may be more feasible to make a library that includes only those genes that are expressed in a particular cell type or at a particular time. This would greatly reduce the number of fragments that are cloned because only a small portion of a cell s DNA is actually expressed. Such a library is called a cdna library, or a complementary DNA library. Using the World Wide Web, learn more about cdna libraries. How is a cdna library made? When would it be advantageous to use a cdna library to find a gene as opposed to a genomic DNA library? What elements are missing from a cdna library that are included in a genomic library? Screening a DNA Library Now that we have made a DNA library that includes fragments representing the entire human genome, we need to find the bacterial cell or cells that harbor the human DNA fragment containing the gene of interest. We must screen our library for the insulin gene. The basic steps involved in screening a DNA library are illustrated in Figure First the bacterial cells containing the plasmid are spread thinly onto a surface of rich, growth-supporting media, called agar, in a petri dish. Bacterial colony 1 Bacterial cells containing plasmids with fragments of human DNA are spread on a petri dish coated with growth-supporting agar. Each cell gives rise to an entire colony. 2 Representative cells are transferred to a thin membranous plastic. 4 The DNA adheres to the membranous plastic. 3 The cells are killed and the DNA is denatured (made single stranded), using a strongly basic solution. probe DNA Figure 7-10 Screening a DNA library. 5 The DNA is soaked in a solution containing a lot of excess probe DNA that is, DNA that is complementary to the target DNA. 6 When the excess probe is washed away, only the spot representing cells containing the target DNA is labeled with the probe. The original bacterial colony from which the labeled spot is derived is used to start a new culture. Each of the cells in the culture will contain a copy of the target DNA.

12 7-1 What Tools Are in the Biotechnology Tool Box? 185 (a) (b) (c) (d) Each cell will divide over and over, until it becomes a distinct colony of identical cells. If the cells are spread thinly enough, the surface of the agar becomes dotted with individual separate colonies, each derived from a single cell.within a colony, all the cells contain the same plasmid and hence the same fragment of human DNA. But different colonies are derived from different parent cells, so each colony represents a different fragment of human DNA. One (or perhaps a few) of the many bacterial colonies has DNA representing the target gene for human insulin.to find the target DNA, we must design a probe that will somehow point to the right colony.the probe that is used is another strand of DNA a synthetic piece which is complementary to the target DNA that we seek. For example, if the target DNA has the nucleotide sequence AGCCTAA...etc.,then the probe DNA is synthesized with the complementary sequence, TCGGATT...etc.,with the T s matched to A s and the G s matched to C s.this bit of probe DNA is also constructed with a built-in marker that can be detected by the experimenter. The probe DNA might be made with a radioactive isotope, for example, or with a fluorescent marker that will glow under certain kinds of light.when the cells in the bacterial colonies are broken and the DNA is denatured (double-stranded DNA is treated with a strong base to separate the strands), the probe will bind to the target DNA, forming a hybrid molecule half target DNA, half probe. Excess probe is washed away, and the places where it sticks represent colonies containing the target DNA (Figure 7-11). Because probes form hybrids with target DNA, this screening process is called nucleic acid hybridization.the original colonies that reacted with the probe can be used to start a new culture and to make the protein encoded in the cloned gene. Figure 7-11 Nucleic acid hybridization is used to find a target gene from a DNA library. (a) Special paper is pressed against the bacterial colonies in a petri dish. The cells on the paper are broken open and the DNA is denatured. The denatured DNA, shown here with a red backbone, sticks to the paper. (b) The paper is soaked in a solution containing probe DNA DNA whose nucleotide sequence is complementary to that of the target. Probe DNA, shown here with a green backbone, has a built-in marker. (c) The probe DNA only sticks to the target DNA; the excess is washed away. (d) If the marker is a radioactive isotope, scientists can find the target DNA by exposing the paper to photographic film. The dark spot corresponds to the target DNA bound to its radioactive probe.

13 186 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Murder Mystery: Solve the death of Isabel Dirula by analyzing hair follicles. Making Many Copies from Only a Few: The Polymerase Chain Reaction We have already seen how gene cloning can be used to amplify a stretch of DNA by using the replication machinery of a bacterial cell. Gene cloning and screening a DNA library requires many steps and several weeks to complete. For some applications, such as isolating a recombinant gene to make human insulin, it is necessary and desirable to go to such trouble. For other applications, however, a simpler and faster method of copying DNA works just as well. In 1983, a clever technique for making large quantities of DNA from a tiny sample was developed. It is called the polymerase chain reaction, or PCR.The use of PCR allows specific sequences of DNA to be targeted from an entire genome and amplified, or copied billions of times in a test tube, without first being cut with restriction enzymes or cloning. Figure 7-12 illustrates how PCR works. Before doing PCR, one needs to know what the target DNA is, in other words, which part of the total DNA one wishes to copy. In addition, the nucleotide sequences of about 20 bases on either side of the target DNA generally need to be known. These flanking sequences are used to make primers short DNA strands that are complementary to the flanking regions of the target DNA. 1 First, the starting DNA is melted so that the two strands of the double helix separate. This requires heating the DNA to about 90 C, close to the boiling point of water. After the DNA is melted, the temperature is lowered slightly, allowing the primers to find their complementary bases on the separated strands and thus form hybrids. Included in the PCR mixture are all of the four nucleotides as well as a heat-resistant DNA polymerase, an enzyme that uses the original target DNA as a blueprint to add nucleotides one at a time to each of the primers. (The DNA polymerase that is used in PCR comes from a heatloving bacterium found in thermal hot springs. This is necessary so that the high temperature required to melt the DNA does not denature and destroy the DNA polymerase.) The temperature is again lowered a bit more and the DNA polymerase commences by making new DNA that is based on the two templates that were created by melting the target DNA. Recall from Chapter 6 that DNA polymerase cannot initiate a new strand of DNA. It can only add nucleotides to a strand that is already started: Hence the need for DNA primers. The entire cycle takes a minute or so, and then it is repeated as many as 40 times. Each time the cycle is repeated, the number of copies of the target DNA that which lies between the two primers is doubled. In less than an hour, PCR can make billions of copies from only the tiniest amount of starting DNA. PCR has been an indispensable tool in DNA technology. For example, the DNA that is amplified in PCR is often inserted into a plasmid and cloned in a bacterial culture. This way, one only needs to keep the culture of bacteria alive and well in order to have a continuous supply of some specific stretch of DNA that can be used in further experiments. PCR is also useful in medical diagnostics. If, for example, it is known that persons with a certain DNA sequence at some specific genetic locus (some specific position in DNA) are prone to a disease, that genetic locus can be amplified by PCR and analyzed for the disease-causing sequence.treatment for the disease can be started early, improving the likelihood of a good outcome. PCR is also used to generate DNA fingerprints. DNA fingerprints identify individuals in forensic biology or establish genetic relationships between individuals. Because only a small amount of starting DNA is necessary, PCR is a powerful technique for analyzing crime scenes, where a criminal may inadvertently leave behind a hair strand, a tiny bit of blood, semen, saliva, or skin. PCR is also used when scientists want to determine the exact order of nucleotides in a particular stretch of DNA, a procedure known as DNA sequencing. 1 Primers are made by using a DNA synthesizer, a machine that can artificially produce short pieces of DNA with any desired sequence of nucleotides. Several commercial biotechnology companies make primers for about $7.00 per base.

14 7-1 What Tools Are in the Biotechnology Tool Box? The tiny bit of starting DNA is "melted" so that the two strands separate. 2 Primers are short DNA sequences that are complementary to the ends of the DNA. Primers are added to the mixture upon which new DNA strands can be made. The primers line up with their complementary bases on the separated strands. Primer Primer Nucleotides Heat-resistant DNA Polymerase 3 A heat-resistant DNA polymerase and nucleotides are part of the mixture. This enzyme adds nucleotides to the primers, using the original DNA as a template. This creates exact copies of the original DNA. 4 This process (steps 1 3) is repeated many times. Each cycle doubles the amount of DNA, until the original DNA has been amplified into many identical copies. Figure 7-12 The polymerase chain reaction makes billions of copies of small regions of DNA.

15 188 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? DNA Sequencing In the next section, we will see some of the remarkable insights and powerful new approaches to medicine that have resulted from the Human Genome Project, an international effort to learn the base-by-base sequence of all 3 billion nucleotide bases that make up the human genome. While this massive research effort has dominated the science headlines, smaller scale DNA sequencing is done routinely in many laboratories. What kinds of information can be learned by sequencing DNA? There are many answers to this question. What follows are just a few. Knowing the base-by-base sequence of a stretch of DNA can teach us whether a fragment of DNA contains a gene, or whether it is noncoding DNA. Consider this: If you were to randomly create a sequence of the letters A,T, C, and G, then use what you know about RNA to transcribe that sequence into a corresponding RNA (pair every A with a U, every T with an A, every C with a G, and every G with a C), you could predict how your sequence might be translated by a ribosome in a cell. The genetic code, illustrated in Figure 6-12, enables us to predict how that random sequence would be read as codons groups of three ribonucleotides, each of which is translated into a single amino acid. A purely random sequence would result in a stop codon about every 20 codons. (Recall from Section 6-3 that the codons UAA, UAG, and UGA signal the ribosome to stop translation.) Stretches of DNA in which the code for a stop codon occurs every 20 codons are not likely candidates for genes. On the other hand, if you isolated an actual fragment of DNA, determined by sequencing that it stretched for about 400 bases or more and had only one stop codon at the end, you could be somewhat certain that you were looking at an actual gene. If the codes for a start signal (AUG) and a promoter sequence (Section 6-3) were also present, you could be nearly certain you had isolated a gene. 2 A DNA sequence containing the codes for a start signal, a sufficient length of amino acid-encoding triplets to form a protein, and a stop signal is called an open reading frame or an ORF (Figure 7-13). Computers designed to scan large amounts of DNA sequence easily identify ORFs and direct scientists to look more closely at stretches of DNA containing genes. Nucleotide sequences often give insights into what a gene does, that is, the cellular role of the protein encoded in that gene. This is possible because proteins with similar cellular Figure 7-13 The difference between an open reading frame and a random sequence of nucleotides shown as RNA. Notice that the open reading frame has an AUG start sequence, 107 codons that each translate into an amino acid, and a UGA stop signal at the end. A random sequence of nucleotides usually has a stop codon about every 20 codons. In this random sequence example, there are four stop codons and one AUG start codon embedded in the sequence. Computers can read long strings of sequences and pick out the open reading frames likely candidates for genes. Open Reading Frame AUG UUA CAG GUU CCA GCC GGA ACU CUA GAC ACU CUU AUA GAG CCC UCA ACC GGA GAA GCA UGG CUU CUA ACU CUC AGU UCG UUC UCG UCG GCG GAA CUG GCG ACG UCC CUG UCG CCC CAA GCA ACU CUA ACG GGG UCG CUG AAU UCA GCU CUA ACU CGC GUU AGG CUU ACA AAG ACC CCU GGC GUU CGU ACU ACC AUA AAC ACC CCA UUC CAA UUU CUA CGA AUU CCG ACU GCG UUA UUG UUA AGG CAA UGC GUC UCC ACC AAG GGC GAA CGA UUC CCU CAG CAA UCG CAA ACU CCG GCA UCU ACU AGA CGC CGG CCA UGA Random Sequence of RNA Nucleotides ACC CUG GGA UAA GUC GCU CUA GUC AUC GCU AUC GCC GGU CGA UGC AAU GCU UAC CUG GAU GUU AGU AAG AUG GUA AAU CCU GUA CGA CGA CAG UUG CGA UGA AAG CGA UCG ACG GCA AAG CCG UUA UUG CCG AUC CGC UAA CGA UCG AUC GCU CGA CGA AGU CAU CGA GUA CGC AUA CUA CGG AUC UAU CGA AGC CGC UAG CCG UUA GCA CCC GUA CCG AGU UCU GGU AUA CGC AAG AUC GCU AUC CGA AAG UGU CUA UAU CGC AUC GCU CGA AUC GUA UUC AGC AUC GCA UAG CCC GGA CCA UAU CCG AAG CGA UGC UAU CCC 2 Human DNA is complicated by the presence of regions of noncoding DNA interspersed with parts of genes. These problems have been largely overcome by a combination of good detective work and comparisons of human gene sequences with similar genes from organisms that do not have these intervening sequences.

16 7-1 What Tools Are in the Biotechnology Tool Box? 189 roles usually have similar DNA sequences.when the DNA sequence of one protein is compared with that of another and found to be similar, we say the two proteins have homology. (Homology also implies that the two genes are related in an evolutionary sense and that their similarity occurs because they are descended from a common ancestral gene.) One of the first things a scientist does after finding an ORF is to compare its nucleotide sequence with that of genes encoding proteins of known function. While a base-by-base comparison with published DNA sequences would be tedious if done by hand, fortunately this is not necessary. Several large databases containing all of the DNA sequences that have been published to date are available on the Internet.As you will see in the next Web exercise, the DNA sequences in these databases are freely available to anyone with access to the Internet. Mix and Match You and your colleagues have discovered a new organism on the bottom of the sea that looks like no other organism ever before reported. You are an excellent molecular biologist, however, and you decide to find out what kind of creature you have by sequencing some of its DNA and comparing the sequence you get with that of other organisms whose DNA has been sequenced. The more closely your sequence matches some known DNA, the more likely your organism is related to the source of the known DNA. How would you go about making the comparison with other organisms? Exploration DNA sequences from certain human genes can give insights into an individual s susceptibility to disease. For example, there are a few spots single bases in human DNA where the presence of one particular nucleotide has been correlated with a predisposition to heart disease. By sequencing these hot spots in a patient s DNA, doctors can determine those who are at risk for heart disease. These patients are advised to be extra careful in their diet and exercise habits. DNA sequences can teach us about how gene expression is regulated. All genes have regulatory sequences the promoter sequence, for example that influence how often a gene is transcribed and translated and how much protein is made from that gene. Sometimes the sequence of the regulatory regions near a gene gives insights into the level of transcription and translation. For example, there are some forms of the congenital blood disease called thalassemia that occur because the cells of afflicted individuals do not make enough hemoglobin. Thalassemia patients suffer from severe anemia, growth abnormalities, retardation, and, sometimes, early death.when the DNA encoding hemoglobin proteins in some thalassemia patients were sequenced, single-base mutations were found in the promoter region and other sequences regulating transcription of these genes. Identifying these single-base mutations has provided insight into how regulatory sequences modify gene expression. As we will see in the next section, large genome sequencing projects are teaching us how entire genomes have evolved, where genes are found within human DNA and that of other organisms, how those genes are regulated, and how we differ from each other and other species at the genetic level. Piecing It Together Biologists use molecular tools to manipulate the genetic material. Regardless of whether the questions involve prokaryotic or eukaryotic cells, the tools are nearly always derived from microbes. We know that 1. Restriction endonucleases enable biologists to cut DNA at specific sequences of nucleotides, called restriction sites. These enzymes act as molecular scissors, while another enzyme, DNA ligase, acts as molecular paste.

17 190 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? 2. Fragments of DNA are separated and visualized by using agarose gel electrophoresis. The relative positions of bands of DNA on an agarose gel indicate the sizes of the different fragments in a mixture. 3. DNA libraries are collections of fragments of DNA often from a single source, such as the entire genome of an organism.the fragments in a DNA library can be cloned by inserting them into bacteria. In this way, the fragments are copied each time the bacteria replicate their own DNA, and many copies of each DNA fragment are made. DNA libraries are screened for specific target genes by using probes. DNA probes are separate strands of DNA that are complementary to the target DNA and are labeled with a molecular marker. 4. The polymerase chain reaction (PCR) is a technique used to amplify short stretches of DNA. PCR is often used in identification, whereby individualistic regions of the human genome taken from two or more individuals are amplified and compared. 5. DNA sequencing, or determining the base-by-base order of nucleotides in a stretch of DNA, can help identify regions of DNA containing genes. In addition, comparisons of DNA sequences, either between individuals or between species, teaches us about our susceptibility to disease and how we evolved. 7-2 Why Sequence the Human Genome? If you were asked to make a list of the greatest scientific achievements of all time, what would your list look like? Would you include landing a man on the moon, perhaps? Or the discovery of penicillin? The invention of the wheel? Some present day scientists are hailing the sequencing of the human genome as worthy of just such a list. First conceived in 1985, the Human Genome Project (HGP) was begun in earnest in 1990 (Figure 7-14). By February of 2001, leaders of this massive international research effort presented the public with a near-finished draft of the nucleotide sequence of all 24 human chromosomes (22 autosomes and the X and Y sex chromosomes). While this accomplishment is at the heart of the HGP, it is only part of the story. The Human Genome Project Has Short-Term and Long-Term Goals The overall goal of the Human Genome Project (HGP) is to decipher the full set of genetic instructions in human DNA and to develop that set of instructions (as well as that from several other species) as a research tool for scientists. The project includes not only the base-by-base sequence, but also genetic maps of the 24 different human chromosomes. A genetic map is based on careful analysis of inheritance patterns of human traits.traits often disease traits, but any trait can be used are assigned to particular chromosomes and Figure 7-14 Much of the sequencing done for the Human Genome Project is automated. These machines run day and night and make DNA sequences available on the Internet as soon as they are determined.

18 7-2 Why Sequence the Human Genome? 191 to particular positions on chromosomes. Figure 5-5 is a genetic map in which the positions of many disease-causing genes have been pinpointed on human chromosome 11. Such maps will enable us to find new genes responsible for disease and may help to provide a strategy for prevention and treatment. Although it took more than a decade to accomplish, genetic maps of the human chromosomes and the base-by-base sequence of each are considered short-term goals of the project. The long-term goals understanding all of the genes, what they do, how they interact, and how they work together to make a human will undoubtedly occupy scientists throughout the next century. Some have estimated that this goal will take about a million person-years to accomplish. Even then, there will undoubtedly be many unanswered questions about human biology. In addition to the human genome, the genomes of several model organisms have been sequenced as part of the HGP.A model organism is a microbe, plant, or animal used to study some aspect of biology that is directly relevant to humans.we saw in Chapter 3 that fruit flies are often used to study basic genetic principles, and indeed the fruit fly is one of the species whose genome has been fully sequenced. Yeasts are even simpler eukaryotes that are often used to study how genes are regulated and expressed. The sequence of the yeast genome, completed in 1997, showed that at least half of the genes in yeast have counterparts in humans. Understanding the function of these yeast genes gives great insights into human biology at the molecular level, but without the problems associated with studying humans directly. Other model organisms (Figure 7-15) include the bacterium Esherichia coli, the mouse, the nematode worm Caenorhabditis elegans, and the mustard plant, Arabidopsis thaliana. (b) (a) (d) (c) Figure 7-15 Four of the model organisms widely used in laboratories to study basic biological mechanisms. (a) Drosophila, the fruit fly, (b) C. elegans, the nematode worm, (c) Arabidopsis, the mustard plant, and (d) yeast. The genomes of these organisms have been sequenced as part of the Human Genome Project.

19 192 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Exploration Supermodels The genomes of model organisms are proving to be almost as valuable to scientists as the human genome. Each of the model organisms whose genome has been sequenced is particularly useful for probing certain kinds of biological questions. For example, yeast are used to study gene expression transcription and translation and how it is regulated. What kinds of biological problems are answered by studies on the nematode, Caenorhabditis elegans? Why was the mustard plant, Arabidopsis thaliana, chosen as a good plant model? What other genomes are likely to be sequenced in the near future, and why? What We Have Learned from the Human Genome The human genome can be read as the story of the human species.while each one of us is much more than just the product of our genes, the genome helps to define us, collectively and individually, as members of the human family. Knowledge of the genome will touch all of us in real ways, and its benefits will impact nearly everyone in the world. So what does this widely hailed story have to tell us? The first lesson is about numbers.the number 24 is how many different kinds of chromosomes we have 22 autosomes and the X and Y sex chromosomes. Each of us actually has a total of 46; two copies of each of the autosomes and either one X and one Y if we are male or two X s if we are female.three billion nucleotide base pairs are found on those 24 chromosomes. These numbers were known before the HGP began. The HGP, however, taught us that within these 3 billion base pairs, there are somewhere between 20,000 and 25,000 genes. This is only about two or three times as many genes as the worm or the fruit fly have and is considerably fewer than early estimates of anywhere from 100,000 to 300,000 human genes. How do we account for the complexity of a human being with so few genes? The answer can be found in another number: It is estimated that about 50%, or half of human genes, actually encode more than one protein. By piecing together parts of genes in different combinations, the number of actual human proteins, and hence the complexity of a human, is much greater than that implied by a mere 20,000 to 25,000 genes. No one yet knows exactly how many different proteins it takes to make a human. We do know that only about 3% of the DNA in the human genome is actually coding DNA. The other 97% contains some regulatory sequences and a lot of DNA that has no known role. The second lesson is about the genes themselves. As of this writing, about 15,000 genes have been catalogued, and many more are being identified every day. Finding new genes will have important implications for understanding human biology and what can go wrong in disease states. Many disorders, for example, are characterized by abnormalities in the structure of individual chromosomes, seen by looking at stained chromosomes under a microscope. With the human genome sequence in hand, we can correlate those chromosomal abnormalities with the nucleotide sequences found at those damaged chromosomal positions. This helps us to define disease states, to predict candidates who are likely to suffer from disease on the basis of their nucleotide sequences, and to design treatment strategies for preventing or combating the disease. Pharmaceutical companies use nucleotide sequences to design therapeutic agents that can interact with disease-causing genes and ameliorate their effects. A third lesson we learn from the fully sequenced human genome is about the human family our diversity and evolution. The human genome points to a remarkable degree of similarity among individuals. If we compare the base-by-base sequence of DNA from any group of individuals, 99.9% of the DNA sequence is identical, regardless of the country of origin or ethnicity of the DNA donors. At the level of our DNA, there are more differences among individuals of any one ethnic group than there are between different groups. While we can attach ethnic labels to individuals Asian, African, European, or Native American, for example the Human Genome Project has taught us that race and ethnicity are mostly cultural concepts, not genetic ones.

20 7-2 Why Sequence the Human Genome? 193 Much can be learned, however, from the 0.1% of the genome wherein our DNA differs among individuals. We previously discussed that if you line up the DNA from two or more people and compare the nucleotides position by position, 99.9% of the nucleotides will be the same. The points at which they are not, where different nucleotides occupy the same position, are called single nucleotide polymorphisms, or SNPs (pronounced snips, Figure 7-16). SNPs account for most of the differences between individuals, and indeed, most of the genetic diversity of the human species. The human genome is estimated to contain one SNP for every 2,000 nucleotides, or a total of about 1.5 million SNPs. SNPs reflect past mutations that have been handed down through the generations. By tracing the lineage of different SNPs, researchers can learn a great deal about human origins, history, and evolution. Early results from the Human Genome Project indicate that humans originated in Africa and branched to other continents about 150,000 years ago. These genetic studies are providing important correlations with theories of human expansion put forth by anthropologists. The first draft of the human genome has already provided important insights, and a few surprises, about what it means to be human. As genome researchers continue refining the rough draft, others have already begun using the information to study individual genes. Each gene has its own story to tell, and in the future, genome research will unravel the plots of these stories, one by one.we hope to eventually learn the role of each gene and even the vast stretches of DNA that do not contain genes. Continued study of SNPs may uncover the genetic basis of our particular talents and susceptibilities and enable physicians to predict individual responses to medicines, environmental influences, and lifestyles. It is no wonder the completion of the HGP has been hailed as the beginning of a new era of human biology. The HGP Has Raised Ethical, Legal, and Social Issues Who owns genetic information? Should people be tested for genetic disorders if there is no possibility for treatment? Are we responsible for our behavior or can we attribute it to our genes? From the very beginning of the HGP in the 1980s, it was understood that the genome would raise serious questions far beyond the scientific scope of the project. For that reason, the two American agencies footing the lion s share of the bill for the HGP, the U.S. Department of Energy and the National Institutes of Health, set aside about 5% of the $3 billion genome budget to examine the ethical, legal, and social issues (abbreviated ELSI) raised by genome research. The ELSI branch of the HGP is the largest bioethics effort in the world. While the ethical concerns raised by the HGP are complex, they can be broken down into a few major categories. The first is the issue of privacy: Who should have access to an individual s genetic information and how should they be allowed to use it? Most agree that a person s genetic information is private. Employers, insurers, schools, and other agencies do not have a right to genetic information pertaining to employees, clients, or students.the potential for misuse, genetic discrimination, or stigmatization is too great. In February 2000, then President Clinton signed an executive order prohibiting any federal department or agency from using genetic information in hiring or promotion decisions. Several genetic nondiscrimination bills have also been introduced to the U. S. Congress. It is clear that the issue of genetic privacy will be decided by legislation (Figure 7-17). A second concern involves genetic testing. Genetic tests involve screening the nucleotide sequences of a small region of an individual s DNA to determine the presence of a specific sequence of nucleotides that signals a genetic disorder. For many disorders, genetic testing has dramatically improved lives. People with genes that predispose them to colon cancer or breast cancer, for example, can be watched closely by their doctors throughout their lives, greatly improving their chances of early diagnosis and effective

21 194 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? X Y 1 Karyotype showing all the human chromosomes in pairs. 2 Individual human chromosome shown duplicated just prior to cell division. 15 p Unduplicated chromosome number 11, showing the traits that have been mapped to one small region of this chromosome. q Amyloidosis Combined apoa-i/c-iii deficiency Hypertriglyceridemia (1 form) Hypoalphalipoproteinemia Macular dystrophy, vitelli form type Porphryia, acute intermittent Vitreoretinopathy, neovascular, inflammatory Vitreoretinopathy, exudative, familial Usher syndrome, type 1B Parathyroid adenomatosis 1 Centrocytic lymphoma 4 Fragment of DNA making up human chromosome number 11, sequenced into individual nucleotides....atatcggctagctagctagctattagcgatcggatcggatcgatctaggtcaccacattcggc......tatagccgatcgatcgatcgataatcgctagcctagcctagctagatccagtggtgtaagccg......c G A T C G G A T... Person #1...C T A T C G G A T... Person #2...C G A T C G G A T... Person #3...C G A T C G G G T... Person #4...C G A T C G G A T... Person #5 5 Region of DNA in which there are single nucleotide polymorphisms, or SNPs. Figure 7-16 The relationship among chromosomes, chromosome maps, DNA sequences, and single nucleotide polymorphisms.

22 7-2 Why Sequence the Human Genome? 195 Figure 7-17 DNA technology and the Human Genome Project are expected to create a flood of new genetics-related legislation. This issue of the legal journal Judicature is devoted to issues relating to the HGP. Funding for this issue was provided by the ELSI (Ethical, Legal and Social Issues) program of the HGP. treatment. But what of those genetic disorders for which we have only limited treatment, such as Alzheimer s disease or Huntington s disease? Is it better or worse for healthy people to know that they are going to suffer an incurable disease in later life? And what if genetic tests for a devastating disease are not always accurate? Erroneous genetic information could ruin lives. There are no simple answers to these difficult questions. Of all these issues, however, none has been more controversial than the genetics of behavior. Since 1993, when National Cancer Institute scientist Dean Hamer announced that he had found a gay gene, interest in the genetics of complex human behaviors has soared. Could a certain sequence of nucleotides at a single position in human DNA make a person homosexual? What role does the environment play in complex human behaviors? Although the answers to these controversial questions are incomplete, most scientists agree that behaviors have both genetic and environmental components, although there is widespread disagreement on the relative contribution of each. Even when the genetic components are fully understood, most scientists believe that complex human behaviors will not be explained on the basis of a single gene, but will involve many different genes. One thing scientists do agree on: While each one of us is born with a certain genetic makeup, humans also have a remarkable degree of behavioral plasticity. We are capable of making rational choices; none of us is a slave to our genes. Owning Genes Who owns genes? Is it the first person to identify a stretch of DNA as containing a gene? Or the first to assign a role to that gene? Or are human genes part of the collective human heritage, owned equally by each of us and not subject to exclusive rights? The courts have ruled that DNA sequences can be patented, thus protecting the rights of those who invest large amounts of time and money identifying genes and their products. What is the general policy on patenting products of nature? What must a researcher accomplish to apply for a patent covering a gene or sequence of DNA? What are some of the arguments both for and against gene patenting? Exploration

23 196 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Piecing It Together With the completion of the Human Genome Project, researchers and clinicians are entering a new era of human biology. Here s what we know: 1. The Human Genome Project, or HGP, is a massive international effort to map, sequence, and understand all of the DNA found in a human being. The short-term goals, those of mapping the genes to specific chromosomal locations and sequencing the 3 billion base pairs of DNA, are near completion. The long-term goals, to understand what each region of human DNA does, will take many more years to accomplish. 2. The HGP has taught us that, at the genetic level, humans are far more similar to each other than we are different. Places where we tend to differ in single nucleotides are called SNPs, or single nucleotide polymorphisms. SNPs teach us about our individual susceptibilities, our genetic relatedness, and our evolutionary history. 3. The HGP has raised serious ethical, social, and legal questions concerning individual privacy and genetic ownership. Many of these issues will be addressed by legislation. 7-3 How Do We Use Biotechnology? Ashanthi DeSilva was born with a severely compromised immune system. The problem could be traced to a single gene, called ADA, which encodes an enzyme that is essential for the disease-fighting white blood cells. Both her maternally inherited allele and her paternally inherited allele for ADA were nonfunctional and hence Ashanthi was subject to repeated infections that other children s immune systems could easily conquer. Ashanthi s syndrome, called severe combined immunodeficiency disease, or SCID, occurs rarely, affecting only about 1 of every 150,000 children:ashanthi was one of the unlucky ones. Most babies born with SCID do not survive past childhood, succumbing to one or another ordinary childhood infection. Gene Therapy and Designer Drugs In 1990, a team of doctors from the National Institutes of Health collected some of fouryear-old Ashanthi s white blood cells. Using recombinant DNA techniques and a viral vector, they inserted a functioning copy of the ADA gene into the cultured cells. After allowing enough time for the gene to become established, the physicians injected the cells back into the little girl s bloodstream. Two years later, about 25% of Ashanthi s white blood cells were making the ADA enzyme. The first successful gene therapy had been accomplished. Not all gene therapy stories, however, have been as encouraging.attempts to cure cystic fibrosis, cancer, hemophilia, and other genetic disorders by using gene therapy have met with only limited success, and thus far the Food and Drug Administration (FDA), the U.S. agency responsible for approving new medicines for therapeutic use, has not approved gene therapy outside of experimental use. The idea of gene therapy is to treat diseases that result from a defective gene by inserting the correct form of the gene into a patient s cells, as shown in Figure When it works, gene therapy is a powerful remedy for many genetic disorders that are otherwise difficult to treat. The problem, however, has been getting the functioning gene into the cells that need it; in other words, finding safe and effective vectors.

24 7-3 How Do We Use Biotechnology? White blood cells are taken from the child suffering from SCID. White blood cells 2 A functional copy of the ADA gene is inserted. ADA gene 4 The cells are injected into the child, where they produce the enzyme needed to combat infection. 3 The recombinant cells containing the ADA gene are cultured until there are many such cells. Figure 7-18 Gene therapy for patients suffering from severe combined immune deficiency syndrome. Stem cells that give rise to white blood cells, the diseasefighting cells of the immune system, are harvested from the patient and genetically engineered to contain a functioning copy of the ADA gene. These cells are then cultured and returned to the patient. Most gene therapy protocols have relied on genetically engineered viruses to carry genes into the DNA of target cells. Viruses, however, present two problems. First, the human immune system has evolved over millions of years to combat them. For a virus to carry a good gene all the way to the nucleus of a target cell, it must first evade the immune system. Second, the virus itself must be genetically engineered so that it is not infective, yet it must still be capable of inserting its nucleic acid into the target cell. Researchers believe that engineering better viral vectors will overcome these problems and that gene therapy will be commonplace within a decade or two. Stem Cells Regardless of the nature of the vector, the best target cells for introducing new genes are stem cells. Stem cells differ from other kinds of body cells in two ways. First, stem cells are uncommitted. In other words, they are capable of dividing, and their progeny might give rise to any number of different kinds of cells. Second, stem cells can grow and divide in laboratory cultures indefinitely, unlike other kinds of human cells, which might divide a few times in culture, but inevitably cease dividing and die after a few generations. Human bone marrow contains stem cells that give rise to all of the different types of blood cells, including red blood cells and a variety of white infectionfighting blood cells. A very small percentage of circulating white blood cells are actually stem cells, as well. Both bone marrow and circulating stem cells have been targets for gene therapies directed against diseases of the blood and immune system. Bone marrow and circulating stem cells can divide to become any of the many different types of blood cells, but their fate is limited to only blood cells. A different kind of human stem cell, derived from embryos (Figure 7-19), has the capacity to give rise to any of the 210 different kinds of cells that make up a human. These cells are said to be totipotent having an unlimited capability to differentiate. Under the right conditions, cultures of embryonic stem cells might be coaxed into becoming complex human tissues, or even whole organs. Today, people suffering from diseases that destroy cells or tissues are dependent on organ donations. Unfortunately, there

25 198 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Nucleated human egg Transfer nucleus to enucleated human egg Allow egg to develop Embryonic stem (ES) cells Remove nonreproductive cells from adult Grow ES cells in culture Transplant differentiated cells back into the patient Muscle cells Induce ES cells to differentiate into different tissues Blood cells Liver cells Figure 7-19 A procedure used for growing fully differentiated tissue from embryonic stem cells. Nerve cells are many more people needing organs than there are organs available for transplantation. In the future, stem cell research may give rise to a new approach for treating these people. Embryonic stem cells may enable us to grow replacement cells, tissues, or possibly entire organs. Exploration The Stem Cell Debates Organ transplantation is only one of the many possible uses of stem cells in medicine. Cell therapy, or the introduction of healthy stem cells to replace damaged tissue, may also provide cures for diabetes, Parkinson s disease, Alzheimer s disease, and many other devastating disorders that result in tissue damage. But not everyone is in favor of using stem cells in biomedical research. Learn about the role of stem cell research in efforts of researchers to treat human disorders and the objections to these studies. What legislation has been passed regulating the use of stem cells in the laboratory? Designer Drugs At the molecular level, there is one basic model that describes how the body s biochemical system operates. One molecule aligns with a second molecule, fitting perfectly into a certain spot, and something happens as a result of the pairing a chemical reaction occurs, a nerve impulse is sent, an infection is initiated, or a cell dies. Regardless of the outcome, the basic principle involves two molecules joining together with an exact fit, as illustrated in Figure Pharmaceutical companies use this model to develop drugs specifically designed to interfere with molecular binding. In addition to the advances made in learning DNA sequences, modern biotechnology has enabled researchers to predict the precise shapes of molecules in cells. Three-dimensional images of proteins are displayed on computer

26 7-3 How Do We Use Biotechnology? 199 Figure 7-20 Computers are used to model the precise geometry of important biological molecules. These models can be used to design highly specific, potent medicines. screens, where they are rotated and studied from all angles. Careful studies of the binding sites on virtual (computerized) proteins have enabled chemical engineers to design virtual drugs that fit those sites and potentially interfere with binding. From these studies, it is a fairly routine matter to actually develop those drugs for therapeutic use. Antiviral drugs that have had some success in the treatment of AIDS, for example, work by blocking the binding sites of viral enzymes and preventing the virus from replicating. Drugs designed to interfere with the ability of viruses to bind to their target cells are being developed as cures for common ailments such as colds and flu. DNA Is Used in the Courtroom A small boy in India desperately wants to join his father in England. British immigration authorities, however, require that a prospective immigrant prove he is a blood relative of someone living in England before he is allowed to immigrate. Both father and son are anxious to be together, so they willingly contribute their DNA for testing a few drops of blood, some cells from the insides of their cheeks, or a few hairs to prove that they are, indeed, father and son. Figure 7-21 is a diagram of an agarose gel showing DNA from both father and son, as well as from the boy s mother. It is clear that the boy and the man are father and son, and they are happily reunited on British soil. (a) Mother Child Father (b) Mother Child Alleged Father Figure 7-21 Gel electrophoresis is used to determine genetic relatedness among individuals. In these agarose gels, five different regions of the DNA are used to determine the relatedness of a child and two parents. (a) For each allele found in the child, there is a corresponding allele in either the mother or the father. This child is the biological offspring of these two parents. (b) Half of the alleles of the child in this gel correspond to alleles found in the mother, indicating genetic relatedness, but the other alleles do not correspond to those from the alleged father. Hence, the child is not the biological offspring of this alleged father.

27 200 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? (a) Figure 7-22 (a) Variable number tandem repeats, or VNTRs, occur at specific sites on several human chromosomes. These regions of DNA are removed by using restriction enzymes whose cleavage sites occur in the DNA, flanking the VNTR region. The fragment of DNA that is removed varies according to the number of tandem repeats found in that region. (b) VNTR regions from six different individuals were analyzed by using an agarose gel. Recall that each individual has two copies of each chromosome. Individuals 1, 2, and 3 have two different VNTR alleles, which appear as two separate bands on the gel. Individuals 4, 5, and 6 each have identical alleles at the VNTR region on their two chromosomes. Only one band appears on the agarose gel. (b) DNA identification methods have proven to be important tools for establishing relatedness, identifying individuals in criminal and civil proceedings, and searching for missing persons. The principle of DNA typing, or DNA fingerprinting, is based on polymorphisms, or differences, in the DNA of different people. Although the DNA of individuals is 99.9% identical, there are certain places within the other 0.1% of the human genome in which there is a great deal of individuality. We have already learned about single nucleotide polymorphisms in Section 7-2, which account for most of the genetic differences among individuals, but DNA fingerprinting often relies on a different kind of polymorphism. Parts of the human genome that are noncoding, that is, do not contain genes, are filled with short DNA sequences, called core sequences, repeated perhaps hundreds or thousands of times side by side (Figure 7-22). The actual number of times a core sequence is repeated varies from person to person. Regions of the genome containing these repeats are called VNTRs or variable number tandem repeats. Because they do not contain any genes, mutations that arise within a VNTR are not weeded out by natural selection. Researchers have identified restriction enzyme sites that occur on either side of many different VNTRs that is, in regions where DNA sequences are highly conserved. Recall that a restriction endonuclease acts like molecular scissors, cutting DNA at very specific places. In this case, restriction enzymes cut at positions flanking a VNTR, removing a stretch of DNA whose length will depend on how many times the core sequence is repeated. Often it is not possible to identify an individual with certainty by using just one VNTR. Assume a crime has been committed and several hairs found at the crime scene contain DNA that does not belong to the victim.the police have a suspect, and

28 7-3 How Do We Use Biotechnology? 201 they find that one VNTR region of the suspect s DNA matches that of the hairs. Does that mean that they have proven the suspect s guilt? Not necessarily. The suspect s defense attorneys determine that within the general population, 1 person in every 25 shares the same pattern at that particular VNTR.Arguing that odds of 1 in 25 are not enough to send a man to jail, the suspect goes free. But if the police compared two different VNTR sites and found a match between the suspect and the crime scene evidence at both regions, the evidence becomes more damning. Even if both patterns are found in 1 of every 25 people in the general population, the likelihood that two people share the same VNTR pattern at two sites is 1 or 1 25 * 1 25 * 1 25, 625. This is an example of the product rule of probability.the product rule states that the joint probability that two independent events will occur is the product of the individual probabilities of each.thus, the probability that any random person in a population will have the same pattern at the first VNTR as that of the DNA found at the crime scene is 1 in 25. Likewise the probability that any random person has the second VNTR pattern is also 1 in 25. The probability that a random individual will have both of the VNTR patterns found at the crime scene is 1 in 625.The more different VNTRs that are compared between suspect and crime scene DNA, the more likely it is that a match indicates guilt. Currently the FBI examines 13 different VNTR sequences in matching evidence to suspects. Biotechnology Is Used on the Farm Over 200 years ago, in 1798,Thomas Malthus wrote an essay that warned of impending famine, pestilence, and war. Human population, he wrote, grows exponentially, like compound interest in a bank account, but farm output rises at a slower, arithmetic rate; the result, human population will inevitably and repeatedly outstrip its food supply. This was the essay that influenced Darwin when he wrote The Origin of Species. (See Chapter 2.) Malthus predicted chaos and misery by the time the world population reached 3 billion. Now in the early 21st century, the world population has reached 6.5 billion over twice the level at which Malthus predicted widespread misery. True to his predictions, a few countries have experienced famine, pestilence, and war. But many others have not. What Malthus failed to predict was that human ingenuity would improve crop yields and farm productivity, keeping pace with an expanding population. In the last half of the 20th century, world grain output rose by over 200%, but world population during the same period doubled. In the early 1990s, however, the growth in world grain production showed signs of decline. Unpredictable weather patterns and the growing resistance of insects and weeds to insecticides and herbicides combined to decrease farm productivity. Yet the world population continues to grow exponentially. Many believe that our ability to feed ourselves will depend on creating higher yield, genetically engineered farm products. Agricultural biotechnology is big business, second only to medicine in terms of applications and innovations using DNA technology. To achieve the goal of increasing the world s food production while simultaneously decreasing both the costs and the environmental damage related to insecticide and herbicide use, scientists have focused their efforts in three areas: (1) developing crops capable of fending off insect pests without the use of insecticides; (2) engineering plants with a greater yield that can grow in a wider range of climatic conditions, especially hot, dry climates; and (3) making crops that are resistant to herbicides, so that fields can be treated for plant pests without damage to the crops. All these innovations have involved DNA engineering. The European corn borer, a moth species whose larvae attack not only corn, but also sorghum, cotton, and several vegetable plants, costs the United States about $1 billion each year in lost crop. For nearly 50 years, farmers have been spraying cornfields with

29 202 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Figure 7-23 Corn being sprayed with Bacillus thuringiensis (Bt) to combat the European corn borer. By genetically engineering the pesticide gene directly into corn plants, the amount of crop spraying for pesticides has been reduced. Plant Research: Genetically engineer plants for drought resistance. a mist containing the natural bacterium, Bacillus thuringiensis, also known as Bt (Figure 7-23). Bt produces an enzyme that is nontoxic to plants and humans, but becomes toxic in the alkaline digestive tract of the corn borer. (Humans and other animals have acidic digestive tracts, an environment in which the Bt enzyme is harmless.) The bacterium is a natural pesticide. Scientists from the Monsanto Company, a world leader in genetically modified food crops, identified the gene in Bt responsible for making the enzyme. Through the use of DNA technology, this pesticide gene was introduced directly into the corn genome, creating a strain of naturally resistant corn plants. Corn borer larvae are poisoned when they eat leaves of the genetically modified plant. Advances like these have sharply reduced the use of chemical pesticides. Currently scientists are exploring ways of introducing drought-resistant genes into sorghum, wheat, and other cereal plants.the hope is that the geographic range of these crops can be expanded to include dry areas. While these technological advances could increase food production, opponents of genetically modified food crops express concern that we are opening a Pandora s box by irreversibly tampering with our food supply.what are the risks to people with severe food allergies? How are genetically modified crops harming other species and disturbing the ecological balance of the environment? These are serious, but as yet unsolved questions whose answers must be weighed against the benefits of heartier, more pest-resistant crops. Exploration Poisonous Progress? In 1999, researchers at Cornell University reported that pollen from Bt corn was toxic to monarch butterflies. Although monarchs feed exclusively on milkweed plants, corn pollen is airborne and can be blown into milkweed patches where the monarchs feed (Figure 7-24). This observation ignited heated debate over the use of transgenic crops. Is Bt corn a major threat to butterfly populations? Using the World Wide Web, learn more about the monarch butterfly and Bt corn, and the debate over genetically modified (GM) food in general. What other human activities threaten the monarch butterfly? How serious a threat is Bt corn to these insects? What else are opponents saying about GM food? Can Biotechnology Save the Environment? On March 24, 1989, the Exxon Valdez oil tanker ran aground in the Prince William Sound off Alaska, spilling more than 10 million gallons of oil into the pristine arctic waters. The death toll was estimated at 250,000 seabirds, 2,800 sea otters, 300 harbor seals, 250 bald eagles, up to 22 killer whales, and billions of salmon and herring eggs. The Exxon company spent over $2 billion cleaning up the spill, and a further $1 billion to settle civil and criminal charges related to the spill.

30 7-3 How Do We Use Biotechnology? 203 Figure 7-24 The monarch butterfly, shown here on swamp milkweed, feeds on the pollen of milkweed plants. The butterflies may ingest corn pollen that has been blown to milkweed patches. Researchers from Cornell University reported that pollen from Bt corn may be toxic to the monarch butterfly. As part of the cleanup effort, some of the oil-strewn beaches were fertilized with chemicals designed to enhance the growth of naturally occurring bacteria capable of digesting hydrocarbons. Figure 7-25 shows the difference between a beach that was fertilized and one that was not. The sludgy oil disappeared faster from the treated beaches than from the untreated beaches, indicating that natural bacteria may be one of the best treatments for oil-damaged shorelines. This approach to environmental cleanup, in which microorganisms are used to decompose toxic pollutants into less harmful compounds, is called bioremediation. The success of the Exxon Valdez experiment has led scientists of the Environmental Protection Agency (EPA) to declare that bioremediation has the potential of saving money, being ecologically sound, destroying contaminates, and allowing for the treatment of waste on site. The application of bioremediation will be an important aspect of waste management now and into the future. Indeed, by the early 1990s, microbes capable of digesting other pollutants were being tested. Polyethylene, polypropylene, polystyrene, and polyvinyl chloride are just a few of the commonly used plastics that end up in landfills and on beaches or in the ocean. Several hundred thousand tons of nonbiodegradable plastics are dumped into the seas each year, creating a devastating environmental problem. Many believe our best hope for combating plastic pollution is developing microorganisms that can digest plastics and convert them to harmless by-products. Figure 7-25 Workers sprayed fertilizer on oil-contaminated beaches to stimulate the growth of oil-eating bacteria. Beaches that were sprayed (shown here on the right) returned to normal more quickly than those that were not (shown on the left).

31 204 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? Piecing It Together Depending on your perspective, biotechnology is either a boon or a bane to society. Here are some of the ways we are using biotechnology: 1. Biotechnology is being used to develop new drugs and therapies for disease. Gene therapy, or the insertion of a healthy gene into the cells of a person lacking a functional allele for that gene, has been only partially successful. As new and better vectors are developed, researchers are optimistic that many formerly untreatable disorders will be cured. 2. DNA identification methods, whereby stretches of the human genome that typically differ among individuals are compared, has been a powerful tool for identifying genetically related people and pinpointing criminals. 3. New, heartier crops that are pest resistant or pesticide resistant are being developed by using DNA technology. 4. The future of bioremediation the use of living organisms to decompose toxic pollutants may involve engineering microorganisms to digest toxins or pollutants. 7-4 What Are the Risks of Biotechnology and How Should We Address Them? Strawberries in the field are natural hosts to a surface bacterium called Pseudomonas syringae. The bacterium produces a protein that causes water in its vicinity to freeze as soon as the temperature drops to 0 C. Although this is, indeed, the freezing point of water outside of cells, water inside of cells can usually remain liquid at temperatures as low as -5 C in the absence of the protein. When ice forms inside cells, the result is cellular death. With the ice-forming bacterium on their surface, a single chilly night during the growing season can freeze the strawberries, damaging the entire crop. The natural bacterium was costing strawberry growers millions of dollars each year in lost crop. In 1984, researchers engineered a form of Pseudomonas syringae in which the gene for the ice-forming protein was removed. The idea was to spray strawberries with a mist of these so-called ice-minus bacteria. The engineered bacteria would outcompete the nonengineered forms and protect the plants from freezing. It was the first time anyone had proposed introducing a living, genetically engineered organism into the environment. The response was rapid and vociferous. Social activists filed a lawsuit, and a federal judge issued an injunction putting a stop to the spraying before it had even begun. Scientists spent three years testing the dangers of ice-minus bacteria on humans and the environment. Finally, in April of 1987, the first strawberry plants were sprayed.the genetically engineered bacteria proved both safe and effective. Since that time, thousands of genetically engineered organisms have been developed and used in agriculture, medicine, and research, but opponents argue that issues of safety have still not been adequately addressed. Some Question the Safety of Biotechnology Scientists and nonscientists alike have been aware of potential risks of new technologies that involve genetic engineering. These risks fall into two major categories: (1) risks to human health and (2) risks to the environment.

32 7-4 What Are the Risks of Biotechnology and How Should We Address Them? 205 Risks to Human Health Recombinant DNA first became a social issue as well as a scientific one in the early 1970s, when one scientist proposed introducing genes from a tumor-promoting virus into the common gut bacterium E. coli. The purpose of the work was to test the virus as a possible vector for transferring DNA from one species to another, but the obvious concern was that the bacterium could somehow escape the laboratory and transmit the virus to humans. These experiments precipitated two headline-making conferences to discuss the biohazards of DNA technology: One in 1974 and a second in As an outcome of these conferences, the National Institutes of Health established a set of guidelines for recombinant DNA research. Today, scientists involved in recombinant DNA research follow a strict set of laboratory procedures that protect the researchers and prevent recombinant organisms from escaping the laboratory unless they are thoroughly tested and found to be harmless. Recombinant DNA, however, is still not risk free. In a tragic gene therapy trial conducted at the University of Pennsylvania in 1999, 18-year-old Jesse Gelsinger died just four days after being treated with a gene therapy drug. His death was attributed to massive organ failure, a consequence of a serious inflammatory response to the treatment. Nonetheless, gene therapy may be the only hope for desperately ill patients. Compared with certain death, the risks of gene therapy become acceptable to many. Fears about the unpredictable outcomes, however, are one reason for the slow progress in this otherwise promising field of medicine. Risks to the Environment Most public concerns about the hazards of biotechnology are focused on genetically modified organisms (GMOs) and their potential effects on the food supply and the environment. Critics of GMOs fear that we might be causing harm to other, unmodified species (Figure 7-26). For example, a genetically modified bacterium might outcompete the natural, indigenous bacterium for resources and thereby contribute to its extinction. Or a genetically modified crop might kill insects that feed on that kind of plant or its pollen. Another fear concerns the known ability of genes to move from one species to another through cross breeding and hybridization. If we engineer a crop that is resistant to herbicides, for example, might the herbicideresistant genes find their way into closely related wild species, creating a new breed of superweeds? A 2000 report from the National Academy of Sciences found no Figure 7-26 Genetically modified food has raised concerns from citizen groups, especially in Europe.

33 206 CHAPTER 7 Biotechnology: How Do We Use What We Know about Life? evidence that genetically engineered pesticide-resistant crops present risks to human health or the environment, but their report also included a call for tighter regulations of GMOs. Under increasing pressure from consumers, governments throughout the world are requiring exporters to label bulk food shipments containing genetically modified strains. Importing nations can decide for themselves whether they want these foodstuffs to enter their borders and whether the need for imported food outweighs the possible risks of growing and eating GMOs. Some Question the Ethics of Biotechnology Developments in biotechnology will present us with some of the most important public policy challenges and ethical questions of the coming decades. In Section 7-2, we saw some of the ethical issues raised by the Human Genome Project, including genetic privacy, ownership of genes, and the role of genes in behavior. But these concerns are only part of the story. At the most basic level, critics of biotechnology question our right to genetically tinker with species that have evolved over billions of years. Most opponents of DNA technology argue that altering genes is unnatural, that it breaches fundamental boundaries between species. Supporters of biotechnology point out that species boundaries are not all that clear. Genes have been moving from place to place within genomes and even between species since the first life forms appeared nearly 4 billion years ago. In addition, we have been creating new crops, livestock, and domestic pets for thousands of years by selectively breeding only certain strains of wild species. DNA technology is the next logical step in selective breeding. Another ethical argument against biotechnology states that we are interfering with the order of life, altering the natural evolutionary process. In fact, transporting microbes, plants, and animals from one continent to another has had a much greater impact on the course of evolution than biotechnology. Rabbits in Australia, starlings in North America, and smallpox, which arrived in the New World with the conquistadors in the 16th century, drove many indigenous populations to extinction. While interfering with natural processes may be ethically debatable, it is certainly not new. Concerns voiced at the beginning of the DNA revolution in the late 1960s and early 1970s focused on the possibility that we would inadvertently make transgenic organisms that could become dangerous and uncontrollable. These fears have abated as this has not come to pass and we have become more accustomed to hearing about DNA technologies and their great promise in medicine and agriculture. Unlike the other discoveries described in this book that were motivated by scientific curiosity, discoveries in the field of biotechnology have been primarily commercial undertakings. For a commercial enterprise to succeed, it must generate profit. Many people s anxieties about the ethics of biotechnology reflect a distrust of big business. Some of these anxieties may be misplaced, but others undoubtedly have merit and require continuing dialog among corporations, governments, and concerned citizens. Regardless of how one views the ethical imperatives of the new biology, it is incumbent upon all of us to understand the power and the limitations of DNA technology. Without understanding, we cannot expect to have the kind of meaningful conversation that will inform the direction of new research and secure the best possible future for ourselves and our children. Piecing It Together Biotechnology may be the answer to feeding a hungry world and curing devastating disease, but opponents argue that the risks have not been fully addressed. Here s what we know: