7 Biotechnology - University of Phoenixmyresource.phoenix.edu/secure/resource/BIO100R3/BioInquiry Ch07.… · were treated with proteins isolated from cows or pigs brought to slaughter.However,the

174

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 beto arrive where we started, and knowthe place for the first time.

—T. S. Eliot, 1942

Chapter opening photo Computerizedimages of molecules are used to design medicines.

7

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 molecularbiologists had constructed an accurate picture of the flow of information in prokaryoticcells. DNA—in particular the sequence of A, T, C, and G—acts as a template for con-structing the intermediary molecule RNA, which encodes the information forassembling amino acids into proteins (Figure 7-1). Gene expression, or the selective pro-duction of particular proteins, is controlled by operons and regulatory feedback loops.

DNAgenetic information

mRNAmessage

PROTEINcellular function

transcription

translation

repl

icat

ion

prio

r to

cell division

Figure 7-1 ■ By the 1960s, the flow ofinformation within a cell from DNA tomRNA to protein was understood. This isthe “central dogma” of biology.

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 com-plex cell types—the eukaryotes. Surprisingly, the answers to these questions, too, wouldcome from experiments on microbes, namely bacteria and viruses. The techniques thisnew breed of biologists developed to learn about genes in eukaryotic cells have becomethe most powerful tools in the biotechnology toolbox.

Bacteria and Viruses Play a Central Role in BiotechnologyWhy use microbes to study and manipulate eukaryotic DNA? Several features of bacteriamake 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 con-ditions, bacteria can double their numbers in just 20 minutes and double again 20 minuteslater. In a few short hours,a culture of bacteria can be grown to contain an enormously largenumbers of cells. Second, the bacterial chromosome is simple compared with eukaryoticchromosomes. Bacterial DNA occurs as a simple closed loop with no complicating proteinssuch as those found in eukaryotic chromosomes. Third, bacteria often carry small extraloops of DNA, called plasmids, that are replicated and passed from one generation to thenext when the cells divide. Plasmids are sometimes naturally released into the surroundingmedium by one bacterium and taken up by another in a process called transformation. (InChapter 5, Section 5-3, we saw that transformation was one of the phenomena that helpedidentify DNA as the genetic material.Griffiths’ transforming principle that converted harm-less R-strain bacteria to virulent S-strain cells in mice was a plasmid.) Researchers quickly

0 1 2 3 4Time (hours)

Num

ber

of b

acte

rial c

ells

Bacterialchromosomes

Plasmids

Bacterial chromosome

"naked"DNA

Rapid growth rate

Bacterial chromosomelacks proteins

Bacterial cells cancarry self-replicatingplasmids

1

2

3

Figure 7-2 ■ Microbes such as bacteriaare useful for studying the function ofgenes in higher organisms like humans.This is because microbes grow rapidly inculture; their DNA is “naked,” that is, itlacks accessory proteins found ineukaryotic DNA; and because bacteria canbe made to carry self-replicating rings ofDNA called plasmids.


learned that they could add short stretches of DNA containing perhaps a gene or two andsome regulatory sequences to bacterial plasmids and that these genetically engineeredplasmids were readily taken up by bacterial cells and faithfully replicated with each roundof bacterial cell division. Starting with a small stretch of eukaryotic DNA, a molecularbiologist can use bacteria to grow bulk quantities of the stuff, all of it identical to the startingDNA. This is called gene cloning—using bacteria to make multiple identical copies of asingle stretch of DNA. Gene cloning was an important step in learning about eukaryoticgenes, 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 cloningvector is any vehicle that inserts a fragment of foreign DNA into the genome of a hostcell.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 pro-tein coat. When a virus lands on a suitable host, its nucleic acid is injected into the hostcell. Once inside, viral DNA either commandeers the host cell’s machinery to makecopies of itself or its DNA inserts itself into the chromosomes of the host to be replicatedwhen 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 genesinto various host cells, including those of humans.Viral cloning vectors, for example, areoften used in gene therapy. In clinical trials, people born with two damaged copies of agene have been purposely infected with a weakened virus that has been engineered tocarry a healthy copy, or allele, of the damaged gene. The hope is that, once inside thehuman cells, the healthy human allele introduced by the virus will behave like a normalgene. This approach has met with only limited success, but more on that later. For nowwe shall examine the manner in which plasmids and viruses are engineered to carryforeign genes. In other words, how is DNA cut, pasted, and visualized?

Molecular Tools Are Used to Manipulate DNAPrior to the advent of DNA technology, human proteins, such as insulin used in thetreatment of diabetes and growth hormones used in the treatment of growth abnor-malities, were prohibitively expensive or not available at all. Sufferers of these maladieswere treated with proteins isolated from cows or pigs brought to slaughter. However, thedifferences between bovine (cow) or porcine (pig) proteins and human proteins meantthat these animal proteins were less efficient in humans. Today, cultures of bacteriaengineered 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 widelydifferent organisms, has revolutionized the pharmaceutical industry.

Figure 7-3 ■ Genetically engineeredbacteria containing human genes aregrown in bulk. Pharmaceutical companiesharvest the human proteins, such as insulinand growth hormone, synthesized by theseliving factories.


We have already seen how to get foreign genes into a bacterial cell by using a cloningvector such as a plasmid or virus, but how does one get a fragment of DNA into thecloning vector? The size of native, uncut DNA from most sources is much too large tobe 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 earlydays of molecular biology, native DNA was bombarded with high-frequency soundwaves. This treatment smashed the DNA into bits, but the result was always a wideassortment of fragments of random sizes. This made it impossible to replicate anexperiment from one day to the next; each time a new cell’s DNA was shattered withsound waves, the resulting collection of DNA fragments was different from the timebefore. Several important developments in the early 1970s, however, made the task ofcreating cloning vectors much easier. These were (1) the ability to cut DNA at specificplaces, reliably and consistently every time, and (2) the ability to combine any two piecesof cut DNA, regardless of the source of either piece. In other words, scientists discoveredtools that acted like molecular “scissors” and molecular “paste.”

Bigger and Better Vectors Plasmids and viruses are excellent vectors for getting small bitsof DNA—on the order of about 5,000 to 10,000 base pairs—into host cells, but there are timeswhen it is desirable to introduce much larger fragments of DNA into a cell for cloning. In re-cent years, several large vectors have been created that can insert as many as 35,000 to 45,000base pairs into a bacterial host. These artificial vectors are called cosmids. Even larger piecesof DNA, up to 200,000 base pairs, can be cloned into yeast cells by using yeast artificial chro-mosomes, or YACs.What components must be engineered into a cosmid or a YAC so that theforeign DNA is properly replicated in its host cell? What other cloning vectors have been de-veloped for getting large fragments of DNA into bacteria and other hosts? How have thesecloning vectors been used in biotechnology?

Molecular Scissors and Molecular Paste Billions of years ago, when prokaryotic cellsruled the planet, bacteria were subject to invasion by stray bits of nucleic acid, usuallyin the form of viruses. Bacteria that could resist viral infection had an advantage overthose that could not; they were more successful than nonresistant bacteria. One strate-gy for fighting off viruses is to degrade their DNA by chopping it into small pieces. Bac-teria have evolved an arsenal of enzymes, called restriction enzymes or restrictionendonucleases, that do just that. Restriction endonucleases cut DNA at specific places,resulting in fragments that can be easily removed by the bacterial cell. But restrictionendonucleases owe their recent fame to the ways in which molecular biologists use themto manipulate DNA in the laboratory.These enzymes serve as “molecular scissors”—oneof the most powerful tools in DNA technology.

Since their discovery in the early 1970s, over 800 different restriction endonucleaseshave been identified and isolated from various bacteria. Each one of them recognizes aspecific sequence of nucleotides on the DNA to be cut, called its recognition site (Fig-ure 7-4). For example, the restriction endonuclease called EcoR1 (so named because itwas the first restriction enzyme found in the bacterium Escherichia coli) has a recognitionsite that looks like

. . . . .GAATTC. . . . .

. . . . .CTTAAG. . . . .

Whenever EcoR1 encounters DNA containing this particular sequence, the enzyme willcut both strands of the DNA as follows:

. . . . .G AATTC. . . . .

. . . . .CTTAA G. . . . .

Exploration


There are two important things to note about the EcoR1 restriction site: First, thenucleotide 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 back-ward 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 bothstrands of the DNA.These unpaired bases are called “sticky ends,” a term that describestheir 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 ofthe bacterial DNA would find complementary sticky ends from other bacterial DNAmolecules, but others would find partners from among the human DNA strands with

Restrictionenzyme EcoRIcuts here(before AATT)

EcoRIcuts here(before AATT)

C C CT T TAA AG G T TA

CC TT T AA A GG GT A A

Each piece hasa "sticky end" T T

A A

T T A A

CT AG G

CC TA G

C CT TA

G GT A A

Piecesseparate

Figure 7-4 ■ Restriction enzymes, such as EcoR1 shown here, cut DNA at specific nucleotidesequences called restriction sites. A cut made with EcoR1 leaves “sticky ends”capable offorming hydrogen bonds with other DNA cut with the same enzyme. This is the basis forrecombinant DNA technology.

Bacterial strain

Bacillus amyloliquefaciens H

Escherichia coli Ry13

Providencia stuartii 164

Serratia marcescens SB

Rhodopseudomonas sphaeroides

Enzyme name

Bam H1

Eco R1

Pst 1

Sma H1

Rsa 1

Recognition sequencesand cleavage sites

G A T C CGC C T A G G

A A T T CGC T T A A G

T G C A GCG A C G T C

C C G G GCG G G C C C

G T A CC A T G

Figure 7-5 ■ Five restriction enzymesand the DNA sequences of theirrestriction sites. This is a small sampling ofthe over 800 different restriction enzymescurrently known.

Bacterialchromosome

Plasmid

Plasmid DNAis isolated frombacterial cells.

Chromosomal DNAis isolated from adifferent organism.

A gene of interest is removedfrom the DNA using thesame restriction enzymeused to cut the plasmids.

The bacteria are grown in culture, when theysynthesize the desired substance.

The plasmidsare reintroducedinto bacteria.

A recombinant DNAplasmid is madeby grafting thegene of interestinto the plasmid.

Plasmids are cutin precise spotsusing restrictionenzymes.

Figure 7-6 ■ Making a molecule of recombinant DNA.


complementary sticky ends. Once such partnerships have formed by hydrogen bonding,the breaks within the sugar-phosphate backbones can be sealed by using another bacterialenzyme, DNA ligase. DNA ligase plays a role in normal DNA replication and healsnaturally occurring breaks in DNA. But in the molecular biology laboratory, DNA ligaseis used to join fragments of DNA from different sources that have been cut by using thesame restriction endonuclease. In other words, DNA ligase acts as “molecular paste.”A DNA molecule formed from the DNAs of different organisms is called recombinantDNA. In this example, the recombinant DNA would be part bacterial and part human.

Agarose Gel Electrophoresis: Visualizing Cut DNA Restriction endonucleases madeit possible to cut DNA into fragments of consistent sizes, as long as the DNA is taken fromthe same source each time it is cut. But how do we know what sizes of fragments we havemade 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 allow-ing 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 slabmade of a polysaccharide called agarose. Mixtures of DNA fragments are introduced atone end in depressions formed in the gel, called wells.The entire gel is placed in an elec-trical field with the anode (the positive electrode) at the end farthest from the wells.


Figure 7-7 ■ Fragments of DNA areseparated by using agarose gelelectrophoresis. A mixture of DNAfragments of different sizes has beenseparated on the basis of size. Eachbright band in this photographrepresents DNA fragments of adifferent size.

DNA has a negative electrical charge, so the fragments are attracted to the anode. Butthe 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, smallfragments move faster and further in the gel. The researcher in Figure 7-7 is examininga series of bands on a gel, each one representing DNA fragments of a different size fromthose on every other band.The bands glow under ultraviolet light because the gel has beentreated 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 perhapsfrom rinsing your mouth out with salt water or scraping your cheek with a toothpick, isdigested with a certain restriction endonuclease.The number of fragments that result willdepend on the number of times the recognition site for that enzyme occurs just by chancein your DNA. The size of the different fragments that result will depend on how muchDNA resides between each recognition site.When recognition sites are infrequent or farapart, the fragments will be large. If there are many sequences that match the recogni-tion site or two or more recognition sites that are close together, the fragments will besmall.The different sized fragments of DNA that result from this digestion give a char-acteristic 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 separatingrestriction fragments on an agarose gel will look exactly like the pattern of every otherperson. After all, DNA from every human being is about 99.9% identical to that fromevery other person. There are a few regions in the human genome, however, where thesequences of DNA are unique to each individual.When these regions are digested withcertain restriction endonucleases and the fragments are separated on a gel, the patternof bands is as unique to you as your fingerprints. This is the principle behind DNAfingerprinting, the powerful technique that has been used to identify individuals withastounding 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 ofdifferent DNA fragments in a mixture and second, the size, or number of DNA basepairs, of each fragment. Fragment size is estimated by comparing the position ofdifferent bands on the gel with the positions of standards—DNA fragments whose sizewe knew before we started—applied to a different well on the same gel. Recall that


A mixture of DNA fragments iscarefully 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. Smallerfragments migrate farthest.

After the DNA fragments havebeen separated, the electricalfield 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.

PowerSupply

cathode

anode

-

+

long fragments

intermediate fragments

short fragments

Figure 7-8 ■ Agarose gel electrophoresis separates fragments of DNA on the basis of theirsize or the number of nucleotides in the fragment.

smaller fragments run farther down the gel toward the anode than larger fragments. In thisway, a gel automatically sorts fragments of DNA by size. But one thing we could not knowfrom examining a gel is which fragments contain genes or where any particular gene, saythe gene for insulin, is found among all the fragments produced by the restrictionendonuclease. To identify genes, we need to use some other techniques of molecularbiology. One approach is to make a DNA library containing clones of all the differentDNA fragments from a particular cell and then use a molecular probe to look for the par-ticular “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 bacterialcell to make large quantities of the human protein insulin. To begin, you might use arestriction endonuclease to cut all of the DNA from a human cell (or many identicalhuman cells) into small fragments (Figure 7-9). One of these fragments contains thegene for insulin, but there is no way to determine by looking at them which one it is. Inaddition, although there are lots of DNA fragments, each one (including the onecontaining the insulin gene) is present in very low concentrations. To find the insulingene, each fragment must first be cloned in a bacterial host to increase the number ofcopies. How is this done? Recall that by inserting DNA fragments into cloning vectorsand allowing bacterial cells to take up the vectors, the bacteria will do the work ofcopying your DNA fragments each time they divide, a process called gene cloning. Theentire collection of bacterial cells, which together contain all of the human DNA frag-ments, is called a DNA library.

The term “DNA library” evokes an accurate analogy. Each fragment of DNA, humanDNA in our example, is like a single volume in an enormous library containing about amillion different books. The entire bacterial population containing all the differenthuman DNA fragments is the library’s book collection. Unfortunately, DNA fragmentsare not as carefully catalogued as they are in an actual library, so finding the volume ofinterest—the human insulin gene—will be a bit more difficult than searching a librarycatalog. For that we need to screen our DNA library.


Purify human DNAcontaining insulin gene

Purify plasmid DNA

Plasmid

Bacterial chromosome

The human fragments are joined with the plasmidsto make recombinant DNA. Each plasmid has adifferent fragment of human DNA.

Bacterial cells are transformed with the recombinant plasmids. One may contain the human insulin gene. Plasmid-free bacteria

Treat both human DNA and plasmid DNA with the same restriction enzyme.

The human DNA has many restriction sites. Hence, many different DNA fragments are formed. Only one containsthe gene for insulin.

The plasmid DNA has onlyone restriction site. Thiscreates open circles of DNAwith the same sticky endsas the human DNA fragments.

humaninsulingene

Figure 7-9 ■ Making a DNA library fromhuman DNA.


Representative cells are transferred to a thin membranous plastic.

2

The cells are killed and the DNA is denatured (made single stranded), using a strongly basic solution.

3

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.

1

The DNA adheres to the membranous plastic.

4

The DNA is soaked in a solution containing a lot of excess probe DNA—that is, DNA thatis complementary to the target DNA.

5 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 spotis derived is used to start a new culture. Each of the cells in the culture will contain a copy of the target DNA.

6

Bacterial colony

probe DNA

Figure 7-10 ■ Screening a DNA library.

Working Backward The DNA library just described is a genomic library, a set of clonedDNA 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.Ahuman genomic library, for example, requires about 1 million different bacterial clones, eachcontaining a different plasmid. Such large libraries are often unwieldy and difficult to manip-ulate. It may be more feasible to make a library that includes only those genes that areexpressed in a particular cell type or at a particular time.This would greatly reduce the numberof 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 librarymade? When would it be advantageous to use a cDNA library to find a gene as opposed to agenomic DNA library? What elements are missing from a cDNA library that are included ina genomic library?

Screening a DNA Library Now that we have made a DNA library that includesfragments representing the entire human genome, we need to find the bacterial cell orcells that harbor the human DNA fragment containing the gene of interest. We mustscreen our library for the insulin gene. The basic steps involved in screening a DNAlibrary are illustrated in Figure 7-10. First the bacterial cells containing the plasmid arespread thinly onto a surface of rich, growth-supporting media, called agar, in a petri dish.

Exploration


Each cell will divide over and over,until it becomes a distinct colony of identical cells. If thecells are spread thinly enough, the surface of the agar becomes dotted with individual sep-arate colonies,each derived from a single cell.Within a colony,all the cells contain the sameplasmid and hence the same fragment of human DNA. But different colonies are derivedfrom 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 tar-get gene for human insulin.To find the target DNA,we must design a probe that will some-how point to the right colony.The probe that is used is another strand of DNA—a syntheticpiece which is complementary to the target DNA that we seek. For example, if the targetDNA has the nucleotide sequence AGCCTAA . . . etc., then the probe DNA is synthe-sized with the complementary sequence, TCGGATT . . . etc., with the T’s matched to A’sand the G’s matched to C’s.This bit of probe DNA is also constructed with a built-in mark-er that can be detected by the experimenter. The probe DNA might be made with a ra-dioactive isotope, for example, or with a fluorescent marker that will glow under certainkinds 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 probewill 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 containingthe target DNA (Figure 7-11). Because probes form hybrids with target DNA,this screeningprocess is called nucleic acid hybridization.The original colonies that reacted with the probecan be used to start a new culture and to make the protein encoded in the cloned gene.

(a)

(c) (d)

(b)

Figure 7-11 ■ Nucleic acid hybridizationis used to find a target gene from a DNAlibrary. (a) Special paper is pressed againstthe bacterial colonies in a petri dish. Thecells on the paper are broken open and theDNA is denatured. The denatured DNA,shown here with a red backbone, sticks tothe paper. (b) The paper is soaked in asolution containing probe DNA—DNAwhose nucleotide sequence iscomplementary to that of the target. ProbeDNA, shown here with a green backbone,has a built-in marker. (c) The probe DNAonly sticks to the target DNA; the excess iswashed away. (d) If the marker is aradioactive isotope, scientists can find thetarget DNA by exposing the paper tophotographic film. The dark spotcorresponds to the target DNA bound toits radioactive probe.


Making Many Copies from Only a Few: The Polymerase Chain Reaction We havealready seen how gene cloning can be used to amplify a stretch of DNA by using thereplication machinery of a bacterial cell. Gene cloning and screening a DNA libraryrequires many steps and several weeks to complete. For some applications, such asisolating a recombinant gene to make human insulin, it is necessary and desirable togo to such trouble. For other applications, however, a simpler and faster method ofcopying DNA works just as well. In 1983, a clever technique for making large quanti-ties of DNA from a tiny sample was developed. It is called the polymerase chain re-action, or PCR.The use of PCR allows specific sequences of DNA to be targeted froman entire genome and amplified, or copied billions of times in a test tube, without firstbeing 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, whichpart of the total DNA one wishes to copy. In addition, the nucleotide sequences ofabout 20 bases on either side of the target DNA generally need to be known. Theseflanking sequences are used to make primers—short DNA strands that are comple-mentary to the flanking regions of the target DNA.1 First, the starting DNA is “melt-ed” so that the two strands of the double helix separate. This requires heating theDNA to about 90°C, close to the boiling point of water. After the DNA is melted, thetemperature is lowered slightly, allowing the primers to find their complementarybases on the separated strands and thus form hybrids. Included in the PCR mixture areall of the four nucleotides as well as a heat-resistant DNA polymerase, an enzymethat uses the original target DNA as a blueprint to add nucleotides one at a time toeach of the primers. (The DNA polymerase that is used in PCR comes from a heat-loving bacterium found in thermal hot springs. This is necessary so that the high tem-perature required to melt the DNA does not denature and destroy the DNApolymerase.) The temperature is again lowered a bit more and the DNA polymerasecommences by making new DNA that is based on the two templates that were creat-ed by melting the target DNA. Recall from Chapter 6 that DNA polymerase cannotinitiate a new strand of DNA. It can only add nucleotides to a strand that is alreadystarted: Hence the need for DNA primers. The entire cycle takes a minute or so, andthen it is repeated as many as 40 times. Each time the cycle is repeated, the numberof 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 ofstarting DNA.

PCR has been an indispensable tool in DNA technology. For example, the DNAthat is amplified in PCR is often inserted into a plasmid and cloned in a bacterialculture. This way, one only needs to keep the culture of bacteria alive and well inorder to have a continuous supply of some specific stretch of DNA that can be usedin further experiments. PCR is also useful in medical diagnostics. If, for example, it isknown 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 am-plified by PCR and analyzed for the disease-causing sequence. Treatment for the dis-ease can be started early, improving the likelihood of a good outcome. PCR is alsoused to generate DNA fingerprints. DNA fingerprints identify individuals in foren-sic biology or establish genetic relationships between individuals. Because only asmall amount of starting DNA is necessary, PCR is a powerful technique for analyz-ing crime scenes, where a criminal may inadvertently leave behind a hair strand, a tinybit of blood, semen, saliva, or skin. PCR is also used when scientists want to determinethe exact order of nucleotides in a particular stretch of DNA, a procedure known asDNA sequencing.

1 Primers are made by using a DNA synthesizer, a machine that can artificially produce short pieces of DNAwith any desired sequence of nucleotides. Several commercial biotechnology companies make primers forabout $7.00 per base.

Murder Mystery: Solve the death of Isabel Dirula

by analyzing hair follicles.


The tiny bit of starting DNAis "melted" so that the twostrands separate.

Primers are short DNA sequencesthat are complementary to the endsof the DNA. Primers are added to themixture upon which new DNA strandscan be made. The primers line up withtheir complementary bases on theseparated strands.

Pr imer

Pr imer

A heat-resistant DNA polymerase and nucleotidesare part of the mixture. This enzyme adds nucleotidesto the primers, using the original DNA as a template.This creates exact copies of the original DNA.

Heat-resistantDNA

Polymerase

Nucleotides

This process (steps 1–3) is repeated many times.Each cycle doubles the amount of DNA, untilthe original DNA has been amplified into manyidentical copies.

1

2

3

4

Figure 7-12 ■ The polymerase chain reaction makes billions of copies of small regions of DNA.


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 ofhuman gene sequences with similar genes from organisms that do not have these intervening sequences.

DNA Sequencing In the next section, we will see some of the remarkable insights andpowerful new approaches to medicine that have resulted from the Human GenomeProject, an international effort to learn the base-by-base sequence of all 3 billion nu-cleotide bases that make up the human genome. While this massive research effort hasdominated the science headlines, smaller scale DNA sequencing is done routinely inmany 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 frag-ment of DNA contains a gene, or whether it is noncoding DNA. Consider this: If you wereto randomly create a sequence of the letters A,T, C, and G, then use what you know aboutRNA 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 yoursequence might be translated by a ribosome in a cell.The genetic code, illustrated in Fig-ure 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. Apurely random sequence would result in a “stop” codon about every 20 codons. (Recallfrom Section 6-3 that the codons UAA, UAG, and UGA signal the ribosome to stop trans-lation.) Stretches of DNA in which the code for a stop codon occurs every 20 codons arenot likely candidates for genes. On the other hand, if you isolated an actual fragment ofDNA, determined by sequencing that it stretched for about 400 bases or more and had onlyone stop codon at the end, you could be somewhat certain that you were looking at an ac-tual 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 sequencecontaining the codes for a start signal, a sufficient length of amino acid-encoding tripletsto form a protein, and a stop signal is called an open reading frame or an ORF (Fig-ure 7-13). Computers designed to scan large amounts of DNA sequence easily identifyORFs 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 roleof the protein encoded in that gene. This is possible because proteins with similar cellular

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

Open Reading Frame

ACC CUG GGA UAA GUC GCUCUA GUC AUC GCU AUC GCCGGU CGA UGC AAU GCU UACCUG GAU GUU AGU AAG AUGGUA AAU CCU GUA CGA CGACAG 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 CUACGG AUC UAU CGA AGC CGC UAG CCG UUA GCA CCC GUACCG AGU UCU GGU AUA CGC AAG AUC GCU AUC CGA AAG UGU CUA UAU CGC AUC GCUCGA AUC GUA UUC AGC AUC GCA UAG CCC GGA CCA UAUCCG AAG CGA UGC UAU CCC

Random Sequence of RNA Nucleotides

Figure 7-13 ■ The difference between anopen reading frame and a randomsequence of nucleotides shown as RNA.Notice that the open reading frame has anAUG start sequence, 107 codons that eachtranslate into an amino acid, and a UGAstop signal at the end. A random sequenceof nucleotides usually has a stop codonabout every 20 codons. In this randomsequence example, there are four stopcodons and one AUG start codonembedded in the sequence. Computers canread long strings of sequences and pick outthe open reading frames—likelycandidates for genes.


roles usually have similar DNA sequences.When the DNA sequence of one protein is com-pared 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 thattheir similarity occurs because they are descended from a common ancestral gene.) One ofthe first things a scientist does after finding an ORF is to compare its nucleotide sequencewith that of genes encoding proteins of known function.While a base-by-base comparisonwith published DNA sequences would be tedious if done by hand, fortunately this is notnecessary. Several large databases containing all of the DNA sequences that have beenpublished to date are available on the Internet.As you will see in the next Web exercise, theDNA 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 bottomof the sea that looks like no other organism ever before reported. You are an excellent mol-ecular biologist, however, and you decide to find out what kind of creature you have by se-quencing some of its DNA and comparing the sequence you get with that of other organismswhose 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 goabout making the comparison with other organisms?

DNA sequences from certain human genes can give insights into an individual’s sus-ceptibility to disease. For example, there are a few spots—single bases—in human DNAwhere the presence of one particular nucleotide has been correlated with a predispositionto heart disease. By sequencing these “hot spots” in a patient’s DNA, doctors can deter-mine those who are at risk for heart disease. These patients are advised to be extra care-ful in their diet and exercise habits.

DNA sequences can teach us about how gene expression is regulated. All genes haveregulatory sequences—the promoter sequence, for example—that influence how often agene is transcribed and translated and how much protein is made from that gene. Some-times the sequence of the regulatory regions near a gene gives insights into the level of tran-scription and translation.For example, there are some forms of the congenital blood diseasecalled thalassemia that occur because the cells of afflicted individuals do not make enoughhemoglobin. Thalassemia patients suffer from severe anemia, growth abnormalities, retar-dation,and,sometimes,early death.When the DNA encoding hemoglobin proteins in somethalassemia patients were sequenced,single-base mutations were found in the promoter re-gion and other sequences regulating transcription of these genes.Identifying these single-basemutations has provided insight into how regulatory sequences modify gene expression.

As we will see in the next section, large genome sequencing projects are teachingus how entire genomes have evolved, where genes are found within human DNA andthat of other organisms, how those genes are regulated, and how we differ from eachother and other species at the genetic level.

Piecing It Together

Biologists use molecular tools to manipulate the genetic material. Regardless of whetherthe questions involve prokaryotic or eukaryotic cells, the tools are nearly always de-rived from microbes. We know that

1. Restriction endonucleases enable biologists to cut DNA at specific sequences of nu-cleotides, called restriction sites. These enzymes act as molecular “scissors,” whileanother enzyme, DNA ligase, acts as molecular “paste.”

Exploration


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 thedifferent fragments in a mixture.

3. DNA libraries are collections of fragments of DNA often from a single source, suchas the entire genome of an organism.The fragments in a DNA library can be clonedby inserting them into bacteria. In this way, the fragments are copied each time thebacteria replicate their own DNA, and many copies of each DNA fragment aremade. DNA libraries are screened for specific target genes by using probes. DNAprobes are separate strands of DNA that are complementary to the target DNA andare labeled with a molecular marker.

4. The polymerase chain reaction (PCR) is a technique used to amplify short stretch-es of DNA. PCR is often used in identification, whereby individualistic regions of thehuman 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 stretchof DNA, can help identify regions of DNA containing genes. In addition, compar-isons of DNA sequences, either between individuals or between species, teaches usabout 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, whatwould your list look like? Would you include landing a man on the moon, perhaps? Orthe discovery of penicillin? The invention of the wheel? Some present day scientists arehailing the sequencing of the human genome as worthy of just such a list. First conceivedin 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 thepublic with a near-finished draft of the nucleotide sequence of all 24 human chromo-somes (22 autosomes and the X and Y sex chromosomes). While this accomplishmentis at the heart of the HGP, it is only part of the story.

The Human Genome Project Has Short-Term and Long-Term GoalsThe overall goal of the Human Genome Project (HGP) is to decipher the full set of ge-netic instructions in human DNA and to develop that set of instructions (as well as thatfrom several other species) as a research tool for scientists.The project includes not onlythe 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 thesequencing done for the HumanGenome Project is automated. Thesemachines run day and night and makeDNA sequences available on theInternet as soon as they aredetermined.

7-2 Why Sequence the Human Genome? 191

to particular positions on chromosomes. Figure 5-5 is a genetic map in which the positionsof many disease-causing genes have been pinpointed on human chromosome 11. Suchmaps will enable us to find new genes responsible for disease and may help to provide astrategy for prevention and treatment.

Although it took more than a decade to accomplish, genetic maps of the humanchromosomes and the base-by-base sequence of each are considered short-term goalsof the project. The long-term goals—understanding all of the genes, what they do, howthey interact, and how they work together to make a human—will undoubtedly occupyscientists throughout the next century. Some have estimated that this goal will take abouta million person-years to accomplish. Even then, there will undoubtedly be many unan-swered questions about human biology.

In addition to the human genome, the genomes of several model organisms havebeen sequenced as part of the HGP.A model organism is a microbe, plant, or animal usedto study some aspect of biology that is directly relevant to humans.We saw in Chapter 3that fruit flies are often used to study basic genetic principles, and indeed the fruit fly isone of the species whose genome has been fully sequenced.Yeasts are even simpler eu-karyotes that are often used to study how genes are regulated and expressed. The se-quence of the yeast genome, completed in 1997, showed that at least half of the genes inyeast have counterparts in humans. Understanding the function of these yeast genesgives great insights into human biology at the molecular level, but without the prob-lems associated with studying humans directly. Other model organisms (Figure 7-15)include the bacterium Esherichia coli, the mouse, the nematode worm Caenorhabditiselegans, and the mustard plant, Arabidopsis thaliana.

Figure 7-15 ■ Four of the model organisms widely usedin laboratories to study basic biological mechanisms.(a) Drosophila, the fruit fly, (b) C. elegans, the nematodeworm, (c) Arabidopsis, the mustard plant, and (d) yeast.The genomes of these organisms have been sequenced aspart of the Human Genome Project.

(a)

(b)

(c)

(d)


Supermodels The genomes of model organisms are proving to be almost as valuable to sci-entists as the human genome. Each of the model organisms whose genome has been sequencedis particularly useful for probing certain kinds of biological questions. For example, yeast areused to study gene expression—transcription and translation—and how it is regulated. Whatkinds 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 othergenomes are likely to be sequenced in the near future, and why?

What We Have Learned from the Human GenomeThe human genome can be read as the story of the human species.While each one of usis much more than just the product of our genes, the genome helps to define us, collec-tively and individually, as members of the human family. Knowledge of the genome willtouch all of us in real ways, and its benefits will impact nearly everyone in the world. Sowhat does this widely hailed story have to tell us?

The first lesson is about numbers.The number 24 is how many different kinds of chro-mosomes we have—22 autosomes and the X and Y sex chromosomes. Each of us actu-ally has a total of 46; two copies of each of the autosomes and either one X and one Yif we are male or two X’s if we are female.Three billion nucleotide base pairs are foundon those 24 chromosomes. These numbers were known before the HGP began. TheHGP, however, taught us that within these 3 billion base pairs, there are somewhere be-tween 20,000 and 25,000 genes. This is only about two or three times as many genes asthe worm or the fruit fly have and is considerably fewer than early estimates of any-where from 100,000 to 300,000 human genes. How do we account for the complexity ofa human being with so few genes? The answer can be found in another number: It is es-timated 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 humanproteins, and hence the complexity of a human, is much greater than that implied by amere 20,000 to 25,000 genes. No one yet knows exactly how many different proteins ittakes to make a human. We do know that only about 3% of the DNA in the humangenome is actually coding DNA. The other 97% contains some regulatory sequencesand a lot of DNA that has no known role.

The second lesson is about the genes themselves. As of this writing, about 15,000genes have been catalogued, and many more are being identified every day. Findingnew genes will have important implications for understanding human biology and whatcan go wrong in disease states. Many disorders, for example, are characterized by ab-normalities in the structure of individual chromosomes, seen by looking at stained chro-mosomes under a microscope. With the human genome sequence in hand, we cancorrelate those chromosomal abnormalities with the nucleotide sequences found atthose damaged chromosomal positions.This helps us to define disease states, to predictcandidates who are likely to suffer from disease on the basis of their nucleotide se-quences, and to design treatment strategies for preventing or combating the disease.Pharmaceutical companies use nucleotide sequences to design therapeutic agents thatcan interact with disease-causing genes and ameliorate their effects.

A third lesson we learn from the fully sequenced human genome is about the humanfamily—our diversity and evolution.The human genome points to a remarkable degreeof similarity among individuals. If we compare the base-by-base sequence of DNA fromany group of individuals, 99.9% of the DNA sequence is identical, regardless of thecountry of origin or ethnicity of the DNA donors. At the level of our DNA, there aremore differences among individuals of any one ethnic group than there are betweendifferent groups. While we can attach ethnic labels to individuals—Asian, African, Eu-ropean, or Native American, for example—the Human Genome Project has taught usthat race and ethnicity are mostly cultural concepts, not genetic ones.

Exploration


Much can be learned, however, from the 0.1% of the genome wherein our DNAdiffers among individuals. We previously discussed that if you line up the DNA fromtwo or more people and compare the nucleotides position by position, 99.9% of the nu-cleotides will be the same.The points at which they are not, where different nucleotidesoccupy the same position, are called single nucleotide polymorphisms, or SNPs (pro-nounced “snips,” Figure 7-16). SNPs account for most of the differences between indi-viduals, and indeed, most of the genetic diversity of the human species. The humangenome is estimated to contain one SNP for every 2,000 nucleotides, or a total of about1.5 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 humanorigins, history, and evolution. Early results from the Human Genome Project indicatethat humans originated in Africa and branched to other continents about 150,000 yearsago.These genetic studies are providing important correlations with theories of humanexpansion put forth by anthropologists.

The first draft of the human genome has already provided important insights, anda few surprises, about what it means to be human. As genome researchers continue re-fining the rough draft, others have already begun using the information to study indi-vidual genes. Each gene has its own story to tell, and in the future, genome research willunravel the plots of these stories, one by one.We hope to eventually learn the role of eachgene and even the vast stretches of DNA that do not contain genes. Continued study ofSNPs may uncover the genetic basis of our particular talents and susceptibilities andenable physicians to predict individual responses to medicines, environmental influ-ences, and lifestyles. It is no wonder the completion of the HGP has been hailed as thebeginning of a new era of human biology.

The HGP Has Raised Ethical, Legal, and Social IssuesWho owns genetic information? Should people be tested for genetic disorders if there isno possibility for treatment? Are we responsible for our behavior or can we attribute itto our genes? From the very beginning of the HGP in the 1980s, it was understood thatthe 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 theHGP, the U.S. Department of Energy and the National Institutes of Health, set asideabout 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 thelargest bioethics effort in the world.

While the ethical concerns raised by the HGP are complex, they can be brokendown into a few major categories. The first is the issue of privacy: Who should haveaccess 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 istoo great. In February 2000, then President Clinton signed an executive order pro-hibiting any federal department or agency from using genetic information in hiring orpromotion decisions. Several genetic nondiscrimination bills have also been introducedto the U. S. Congress. It is clear that the issue of genetic privacy will be decided by leg-islation (Figure 7-17).

A second concern involves genetic testing. Genetic tests involve screening the nu-cleotide sequences of a small region of an individual’s DNA to determine the presenceof a specific sequence of nucleotides that signals a genetic disorder. For many disorders,genetic testing has dramatically improved lives. People with genes that predispose themto colon cancer or breast cancer, for example, can be watched closely by their doctorsthroughout their lives, greatly improving their chances of early diagnosis and effective


1 Karyotype showing all the human chromosomes in pairs. 2 Individual human chromosomeshown duplicated just prior tocell division.

p 1

15

14

11

13

22

23

1

2

q

Parathyroid adenomatosis 1Centrocytic lymphoma

Macular dystrophy, vitelli form type

Porphryia, acute intermittent

Vitreoretinopathy, neovascular, inflammatory

Vitreoretinopathy, exudative, familial

Usher syndrome, type 1B

Amyloidosis

Combined apoA-I/C-III deficiency

Hypertriglyceridemia (1 form)

Hypoalphalipoproteinemia

5 Region of DNA in whichthere are single nucleotidepolymorphisms, or SNPs.

4 Fragment of DNA making up human chromosome number 11, sequenced into individual nucleotides.

...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 G T... Person #4


.....ATATCGGCTAGCTAGCTAGCTATTAGCGATCGGATCGGATCGATCTAGGTCACCACATTCGGC....

.....TATAGCCGATCGATCGATCGATAATCGCTAGCCTAGCCTAGCTAGATCCAGTGGTGTAAGCCG....

3 Unduplicated chromosome number 11,showing the traits that have been mappedto one small region of this chromosome.

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y

Figure 7-16 ■ The relationship among chromosomes, chromosome maps, DNA sequences, andsingle nucleotide polymorphisms.


Figure 7-17 ■ DNA technology andthe Human Genome Project areexpected to create a flood of newgenetics-related legislation. This issue ofthe legal journal Judicature is devotedto issues relating to the HGP. Fundingfor 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 healthypeople to know that they are going to suffer an incurable disease in later life? And whatif genetic tests for a devastating disease are not always accurate? Erroneous genetic infor-mation 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 ofbehavior. Since 1993, when National Cancer Institute scientist Dean Hamer announcedthat he had found a “gay” gene, interest in the genetics of complex human behaviors hassoared. Could a certain sequence of nucleotides at a single position in human DNAmake a person homosexual? What role does the environment play in complex humanbehaviors? Although the answers to these controversial questions are incomplete, mostscientists agree that behaviors have both genetic and environmental components, al-though there is widespread disagreement on the relative contribution of each. Evenwhen the genetic components are fully understood, most scientists believe that com-plex human behaviors will not be explained on the basis of a single gene, but will involvemany different genes. One thing scientists do agree on: While each one of us is bornwith a certain genetic makeup, humans also have a remarkable degree of behavioralplasticity. 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 con-taining a gene? Or the first to assign a role to that gene? Or are human genes part of the col-lective human heritage, owned equally by each of us and not subject to exclusive rights? Thecourts have ruled that DNA sequences can be patented, thus protecting the rights of those whoinvest large amounts of time and money identifying genes and their products. What is thegeneral policy on patenting products of nature? What must a researcher accomplish to applyfor a patent covering a gene or sequence of DNA? What are some of the arguments both forand against gene patenting?

Exploration


Piecing It Together

With the completion of the Human Genome Project, researchers and clinicians are en-tering 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-termgoals, those of mapping the genes to specific chromosomal locations and sequencingthe 3 billion base pairs of DNA, are near completion. The long-term goals, to under-stand 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 toeach other than we are different. Places where we tend to differ in single nucleotidesare called SNPs, or single nucleotide polymorphisms. SNPs teach us about our indi-vidual susceptibilities, our genetic relatedness, and our evolutionary history.

3. The HGP has raised serious ethical, social, and legal questions concerning individualprivacy 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 problemcould be traced to a single gene, called ADA, which encodes an enzyme that is essentialfor the disease-fighting white blood cells. Both her maternally inherited allele and herpaternally inherited allele for ADA were nonfunctional and hence Ashanthi was sub-ject to repeated infections that other children’s immune systems could easily conquer.Ashanthi’s syndrome, called severe combined immunodeficiency disease, or SCID, oc-curs rarely, affecting only about 1 of every 150,000 children:Ashanthi was one of the un-lucky ones. Most babies born with SCID do not survive past childhood, succumbing toone or another ordinary childhood infection.

Gene Therapy and Designer DrugsIn 1990, a team of doctors from the National Institutes of Health collected some of four-year-old Ashanthi’s white blood cells. Using recombinant DNA techniques and a viralvector, they inserted a functioning copy of the ADA gene into the cultured cells. Afterallowing enough time for the gene to become established, the physicians injected the cellsback into the little girl’s bloodstream. Two years later, about 25% of Ashanthi’s whiteblood cells were making the ADA enzyme. The first successful gene therapy had beenaccomplished.

Not all gene therapy stories, however, have been as encouraging.Attempts to curecystic fibrosis, cancer, hemophilia, and other genetic disorders by using gene therapyhave 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 thera-py is to treat diseases that result from a defective gene by inserting the correct formof the gene into a patient’s cells, as shown in Figure 7-18. When it works, gene therapyis 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 needit; in other words, finding safe and effective vectors.

7-3 How Do We Use Biotechnology? 197

White blood cells are taken from the child suffering from SCID.

1

A functional copy of the ADA gene is inserted.

2

The recombinant cells containing the ADA gene are cultured until there are many such cells.

3

The cells are injected into the child, where they produce the enzyme needed to combat infection.

4

White blood cells

ADA gene

Figure 7-18 ■ Gene therapy for patientssuffering from severe combined immunedeficiency syndrome. Stem cells that giverise to white blood cells, the disease-fighting cells of the immune system, areharvested from the patient and geneticallyengineered to contain a functioning copyof the ADA gene. These cells are thencultured and returned to the patient.

Most gene therapy protocols have relied on genetically engineered viruses to carrygenes into the DNA of target cells. Viruses, however, present two problems. First, thehuman immune system has evolved over millions of years to combat them. For a virusto carry a good gene all the way to the nucleus of a target cell, it must first evade the im-mune system. Second, the virus itself must be genetically engineered so that it is not in-fective, 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 problemsand 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 introducingnew 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 theirprogeny might give rise to any number of different kinds of cells. Second, stem cells cangrow 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 afew generations. Human bone marrow contains stem cells that give rise to all of the dif-ferent types of blood cells, including red blood cells and a variety of white infection-fighting blood cells.A very small percentage of circulating white blood cells are actuallystem cells, as well. Both bone marrow and circulating stem cells have been targets forgene therapies directed against diseases of the blood and immune system.

Bone marrow and circulating stem cells can divide to become any of the manydifferent types of blood cells, but their fate is limited to only blood cells. A differentkind of human stem cell, derived from embryos (Figure 7-19), has the capacity togive rise to any of the 210 different kinds of cells that make up a human. These cellsare said to be totipotent—having an unlimited capability to differentiate. Under theright conditions, cultures of embryonic stem cells might be coaxed into becomingcomplex human tissues, or even whole organs. Today, people suffering from diseasesthat destroy cells or tissues are dependent on organ donations. Unfortunately, there


Removenonreproductive cells from adult

Transfer nucleusto enucleatedhuman egg

Nucleatedhuman egg

Allow eggto develop

Grow ES cells in culture

Induce ES cellsto differentiate into different tissues

Transplantdifferentiated cells back into the patient

Embryonicstem (ES) cells

Liver cells

Muscle cells

Nerve cells

Blood cells

Figure 7-19 ■ A procedure used forgrowing fully differentiated tissue fromembryonic stem cells.

are many more people needing organs than there are organs available for trans-plantation. In the future, stem cell research may give rise to a new approach fortreating these people. Embryonic stem cells may enable us to grow replacement cells,tissues, or possibly entire organs.

The Stem Cell Debates Organ transplantation is only one of the many possible uses of stemcells in medicine. Cell therapy, or the introduction of healthy stem cells to replace damagedtissue, may also provide cures for diabetes, Parkinson’s disease,Alzheimer’s disease, and manyother devastating disorders that result in tissue damage. But not everyone is in favor of usingstem cells in biomedical research. Learn about the role of stem cell research in efforts of re-searchers to treat human disorders and the objections to these studies. What legislation hasbeen passed regulating the use of stem cells in the laboratory?

Designer Drugs At the molecular level, there is one basic model that describes howthe 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 togetherwith an exact fit, as illustrated in Figure 7-20.

Pharmaceutical companies use this model to develop drugs specifically designed tointerfere with molecular binding. In addition to the advances made in learning DNAsequences, modern biotechnology has enabled researchers to predict the precise shapesof molecules in cells. Three-dimensional images of proteins are displayed on computer

Exploration


Figure 7-20 ■ Computers are used to model the precise geometry of important biologicalmolecules. 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 bind-ing sites on virtual (computerized) proteins have enabled chemical engineers to designvirtual 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.Antiviraldrugs that have had some success in the treatment of AIDS, for example, work by block-ing the binding sites of viral enzymes and preventing the virus from replicating. Drugsdesigned to interfere with the ability of viruses to bind to their target cells are beingdeveloped as cures for common ailments such as colds and flu.

DNA Is Used in the CourtroomA small boy in India desperately wants to join his father in England. British immigra-tion authorities, however, require that a prospective immigrant prove he is a blood rel-ative of someone living in England before he is allowed to immigrate. Both father andson are anxious to be together, so they willingly contribute their DNA for testing—a fewdrops of blood, some cells from the insides of their cheeks, or a few hairs—to prove thatthey are, indeed, father and son. Figure 7-21 is a diagram of an agarose gel showing DNAfrom both father and son, as well as from the boy’s mother. It is clear that the boy andthe man are father and son, and they are happily reunited on British soil.

Mother Child Father(a) Mother ChildAllegedFather

(b)

Figure 7-21 ■ Gel electrophoresis is usedto determine genetic relatedness amongindividuals. In these agarose gels, fivedifferent regions of the DNA are used todetermine the relatedness of a child andtwo parents. (a) For each allele found inthe child, there is a corresponding allele ineither the mother or the father. This childis the biological offspring of these twoparents. (b) Half of the alleles of the childin this gel correspond to alleles found inthe mother, indicating genetic relatedness,but the other alleles do not correspond tothose from the alleged father. Hence, thechild is not the biological offspring of thisalleged father.


1 2 3 4 5 6

(a)

(b)

Figure 7-22 ■ (a) Variable numbertandem repeats, or VNTRs, occur atspecific sites on several humanchromosomes. These regions of DNA areremoved by using restriction enzymeswhose cleavage sites occur in the DNA,flanking the VNTR region. The fragmentof DNA that is removed varies accordingto the number of tandem repeats found inthat region.(b) VNTR regions from six differentindividuals were analyzed by using anagarose gel. Recall that each individual hastwo copies of each chromosome.Individuals 1, 2, and 3 have two differentVNTR alleles, which appear as twoseparate bands on the gel. Individuals 4, 5,and 6 each have identical alleles at theVNTR region on their two chromosomes.Only one band appears on the agarose gel.

DNA identification methods have proven to be important tools for establishingrelatedness, identifying individuals in criminal and civil proceedings, and searching formissing persons.The principle of DNA typing, or DNA fingerprinting, is based on poly-morphisms, or differences, in the DNA of different people. Although the DNA of indi-viduals is 99.9% identical, there are certain places within the other 0.1% of the humangenome in which there is a great deal of individuality. We have already learned aboutsingle nucleotide polymorphisms in Section 7-2, which account for most of the geneticdifferences among individuals, but DNA fingerprinting often relies on a different kindof polymorphism. Parts of the human genome that are noncoding, that is, do not containgenes, are filled with short DNA sequences, called core sequences, repeated perhapshundreds or thousands of times side by side (Figure 7-22). The actual number of timesa core sequence is repeated varies from person to person. Regions of the genome con-taining these repeats are called VNTRs or variable number tandem repeats. Because theydo not contain any genes, mutations that arise within a VNTR are not “weeded out” bynatural selection.

Researchers have identified restriction enzyme sites that occur on either side ofmany different VNTRs—that is, in regions where DNA sequences are highly conserved.Recall that a restriction endonuclease acts like molecular scissors, cutting DNA at veryspecific 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 coresequence is repeated.

Often it is not possible to identify an individual with certainty by using just oneVNTR. Assume a crime has been committed and several hairs found at the crimescene contain DNA that does not belong to the victim. The police have a suspect, and


they find that one VNTR region of the suspect’s DNA matches that of the hairs. Doesthat mean that they have proven the suspect’s guilt? Not necessarily. The suspect’sdefense attorneys determine that within the general population, 1 person in every 25shares the same pattern at that particular VNTR. Arguing that odds of 1 in 25 are notenough to send a man to jail, the suspect goes free. But if the police compared twodifferent VNTR sites and found a match between the suspect and the crime sceneevidence at both regions, the evidence becomes more damning. Even if both patternsare found in 1 of every 25 people in the general population, the likelihood that two peo-ple share the same VNTR pattern at two sites is or This is anexample of the product rule of probability.The product rule states that the joint prob-ability that two independent events will occur is the product of the individual proba-bilities of each.Thus, the probability that any random person in a population will havethe same pattern at the first VNTR as that of the DNA found at the crime scene is 1in 25. Likewise the probability that any random person has the second VNTR patternis also 1 in 25. The probability that a random individual will have both of the VNTRpatterns found at the crime scene is 1 in 625.The more different VNTRs that are com-pared between suspect and crime scene DNA, the more likely it is that a match indi-cates guilt. Currently the FBI examines 13 different VNTR sequences in matchingevidence to suspects.

Biotechnology Is Used on the FarmOver 200 years ago, in 1798,Thomas Malthus wrote an essay that warned of impendingfamine, pestilence, and war. “Human population,” he wrote, “grows exponentially, likecompound 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. (SeeChapter 2.) Malthus predicted chaos and misery by the time the world populationreached 3 billion.

Now in the early 21st century, the world population has reached 6.5 billion—overtwice the level at which Malthus predicted widespread misery.True to his predictions, afew countries have experienced famine, pestilence, and war. But many others have not.What Malthus failed to predict was that human ingenuity would improve crop yieldsand farm productivity, keeping pace with an expanding population. In the last half of the20th century, world grain output rose by over 200%, but world population during thesame period doubled.

In the early 1990s, however, the growth in world grain production showed signs ofdecline. Unpredictable weather patterns and the growing resistance of insects and weedsto insecticides and herbicides combined to decrease farm productivity. Yet the worldpopulation continues to grow exponentially. Many believe that our ability to feed our-selves will depend on creating higher yield, genetically engineered farm products.

Agricultural biotechnology is big business, second only to medicine in terms ofapplications and innovations using DNA technology. To achieve the goal of increasingthe world’s food production while simultaneously decreasing both the costs and theenvironmental damage related to insecticide and herbicide use, scientists have focusedtheir efforts in three areas: (1) developing crops capable of fending off insect pests with-out the use of insecticides; (2) engineering plants with a greater yield that can grow ina wider range of climatic conditions, especially hot, dry climates; and (3) making cropsthat are resistant to herbicides, so that fields can be treated for plant pests without dam-age to the crops. All these innovations have involved DNA engineering.

The European corn borer, a moth species whose larvae attack not only corn, butalso sorghum, cotton, and several vegetable plants, costs the United States about $1 bil-lion each year in lost crop. For nearly 50 years, farmers have been spraying cornfields with

1�625.1�25 * 1�25 * 1�25,


Figure 7-23 ■ Corn being sprayed withBacillus thuringiensis (Bt) to combat theEuropean corn borer. By geneticallyengineering the pesticide gene directly intocorn plants, the amount of crop sprayingfor pesticides has been reduced.

Plant Research: Genetically engineer

plants for droughtresistance.

a mist containing the natural bacterium, Bacillus thuringiensis, also known as Bt (Fig-ure 7-23). Bt produces an enzyme that is nontoxic to plants and humans, but becomestoxic in the alkaline digestive tract of the corn borer. (Humans and other animals haveacidic digestive tracts, an environment in which the Bt enzyme is harmless.) The bac-terium is a natural pesticide. Scientists from the Monsanto Company, a world leader ingenetically modified food crops, identified the gene in Bt responsible for making theenzyme. Through the use of DNA technology, this pesticide gene was introduced di-rectly into the corn genome, creating a strain of naturally resistant corn plants. Cornborer larvae are poisoned when they eat leaves of the genetically modified plant.

Advances like these have sharply reduced the use of chemical pesticides. Currentlyscientists 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 ex-panded to include dry areas.

While these technological advances could increase food production, opponents ofgenetically modified food crops express concern that we are opening a Pandora’s box byirreversibly tampering with our food supply.What are the risks to people with severe foodallergies? How are genetically modified crops harming other species and disturbing theecological balance of the environment? These are serious, but as yet unsolved questionswhose answers must be weighed against the benefits of heartier, more pest-resistant crops.

Poisonous Progress? In 1999, researchers at Cornell University reported that pollen fromBt corn was toxic to monarch butterflies. Although monarchs feed exclusively on milkweedplants, corn pollen is airborne and can be blown into milkweed patches where the monarchsfeed (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, andthe debate over genetically modified (GM) food in general. What other human activitiesthreaten the monarch butterfly? How serious a threat is Bt corn to these insects? What elseare 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 Soundoff 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, 250bald eagles, up to 22 killer whales, and billions of salmon and herring eggs. The Exxoncompany spent over $2 billion cleaning up the spill, and a further $1 billion to settle civiland criminal charges related to the spill.

Exploration


Figure 7-24 ■ The monarchbutterfly, shown here on swampmilkweed, feeds on the pollen ofmilkweed plants. The butterflies mayingest corn pollen that has beenblown to milkweed patches.Researchers from Cornell Universityreported that pollen from Bt cornmay be toxic to the monarchbutterfly.

Figure 7-25 ■ Workers sprayedfertilizer on oil-contaminated beaches tostimulate the growth of oil-eatingbacteria. Beaches that were sprayed(shown here on the right) returned tonormal more quickly than those thatwere not (shown on the left).

As part of the cleanup effort, some of the oil-strewn beaches were fertilized withchemicals designed to enhance the growth of naturally occurring bacteria capable ofdigesting hydrocarbons. Figure 7-25 shows the difference between a beach that wasfertilized and one that was not.The sludgy oil disappeared faster from the treated beach-es than from the untreated beaches, indicating that natural bacteria may be one of thebest treatments for oil-damaged shorelines.This approach to environmental cleanup, inwhich microorganisms are used to decompose toxic pollutants into less harmful com-pounds, is called bioremediation. The success of the Exxon Valdez experiment has ledscientists of the Environmental Protection Agency (EPA) to declare that bioremedia-tion “has the potential of saving money, being ecologically sound, destroying contami-nates, and allowing for the treatment of waste on site.The application of bioremediationwill be an important aspect of waste management now and into the future.”

Indeed, by the early 1990s, microbes capable of digesting other pollutants were beingtested. Polyethylene, polypropylene, polystyrene, and polyvinyl chloride are just a fewof 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 seaseach year, creating a devastating environmental problem. Many believe our best hopefor combating plastic pollution is developing microorganisms that can digest plasticsand convert them to harmless by-products.


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. Genetherapy, or the insertion of a healthy gene into the cells of a person lacking a func-tional allele for that gene, has been only partially successful.As new and better vec-tors are developed, researchers are optimistic that many formerly untreatabledisorders will be cured.

2. DNA identification methods, whereby stretches of the human genome that typicallydiffer among individuals are compared, has been a powerful tool for identifyinggenetically related people and pinpointing criminals.

3. New, heartier crops that are pest resistant or pesticide resistant are being developedby using DNA technology.

4. The future of bioremediation—the use of living organisms to decompose toxicpollutants—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 Pseudomonassyringae. The bacterium produces a protein that causes water in its vicinity to freeze assoon as the temperature drops to 0°C. Although this is, indeed, the freezing point ofwater outside of cells, water inside of cells can usually remain liquid at temperatures aslow as in the absence of the protein. When ice forms inside cells, the result is cel-lular death.With the ice-forming bacterium on their surface, a single chilly night duringthe growing season can freeze the strawberries, damaging the entire crop. The naturalbacterium was costing strawberry growers millions of dollars each year in lost crop.

In 1984, researchers engineered a form of Pseudomonas syringae in which the genefor the ice-forming protein was removed. The idea was to spray strawberries with a mistof these so-called ice-minus bacteria. The engineered bacteria would outcompete thenonengineered forms and protect the plants from freezing. It was the first time anyone hadproposed introducing a living, genetically engineered organism into the environment.

The response was rapid and vociferous. Social activists filed a lawsuit, and a federaljudge issued an injunction putting a stop to the spraying before it had even begun.Scientistsspent 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 engineeredbacteria proved both safe and effective. Since that time, thousands of genetically engi-neered 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 BiotechnologyScientists and nonscientists alike have been aware of potential risks of new technologiesthat involve genetic engineering. These risks fall into two major categories: (1) risks tohuman health and (2) risks to the environment.

-5°C

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 ascientific one in the early 1970s, when one scientist proposed introducing genes from atumor-promoting virus into the common gut bacterium E. coli.The purpose of the workwas to test the virus as a possible vector for transferring DNA from one species to an-other, but the obvious concern was that the bacterium could somehow escape the lab-oratory and transmit the virus to humans. These experiments precipitated twoheadline-making conferences to discuss the biohazards of DNA technology: One in 1974and a second in 1975. As an outcome of these conferences, the National Institutes ofHealth established a set of guidelines for recombinant DNA research. Today, scientistsinvolved in recombinant DNA research follow a strict set of laboratory procedures thatprotect the researchers and prevent recombinant organisms from escaping the labora-tory unless they are thoroughly tested and found to be harmless.

Recombinant DNA, however, is still not risk free. In a tragic gene therapy trial con-ducted at the University of Pennsylvania in 1999, 18-year-old Jesse Gelsinger died justfour days after being treated with a gene therapy drug. His death was attributed to mas-sive organ failure, a consequence of a serious inflammatory response to the treatment.Nonetheless, gene therapy may be the only hope for desperately ill patients. Comparedwith certain death, the risks of gene therapy become acceptable to many. Fears about theunpredictable outcomes, however, are one reason for the slow progress in this other-wise promising field of medicine.

Risks to the Environment Most public concerns about the hazards of biotechnologyare focused on genetically modified organisms (GMOs) and their potential effects onthe food supply and the environment. Critics of GMOs fear that we might be causingharm to other, unmodified species (Figure 7-26). For example, a genetically modifiedbacterium might outcompete the natural, indigenous bacterium for resources andthereby contribute to its extinction. Or a genetically modified crop might kill insects thatfeed on that kind of plant or its pollen. Another fear concerns the known ability ofgenes 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 herbicide-resistant 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 foodhas raised concerns from citizen groups,especially in Europe.


evidence that genetically engineered pesticide-resistant crops present risks to humanhealth or the environment, but their report also included a call for tighter regulationsof GMOs. Under increasing pressure from consumers, governments throughout theworld are requiring exporters to label bulk food shipments containing genetically mod-ified strains. Importing nations can decide for themselves whether they want these food-stuffs to enter their borders and whether the need for imported food outweighs thepossible risks of growing and eating GMOs.

Some Question the Ethics of BiotechnologyDevelopments in biotechnology will present us with some of the most important pub-lic policy challenges and ethical questions of the coming decades. In Section 7-2, we sawsome of the ethical issues raised by the Human Genome Project, including genetic pri-vacy, ownership of genes, and the role of genes in behavior. But these concerns are onlypart of the story. At the most basic level, critics of biotechnology question our right togenetically tinker with species that have evolved over billions of years.

Most opponents of DNA technology argue that altering genes is unnatural, that itbreaches fundamental boundaries between species. Supporters of biotechnology pointout that species boundaries are not all that clear. Genes have been moving from placeto place within genomes and even between species since the first life forms appearednearly 4 billion years ago. In addition, we have been creating new crops, livestock, anddomestic pets for thousands of years by selectively breeding only certain strains of wildspecies. DNA technology is the next logical step in selective breeding.

Another ethical argument against biotechnology states that we are interfering withthe 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 thecourse of evolution than biotechnology. Rabbits in Australia, starlings in North Ameri-ca, and smallpox, which arrived in the New World with the conquistadors in the 16thcentury, drove many indigenous populations to extinction.While interfering with naturalprocesses may be ethically debatable, it is certainly not new.

Concerns voiced at the beginning of the DNA revolution in the late 1960s and early1970s focused on the possibility that we would inadvertently make transgenic organ-isms that could become dangerous and uncontrollable. These fears have abated as thishas not come to pass and we have become more accustomed to hearing about DNAtechnologies and their great promise in medicine and agriculture.

Unlike the other discoveries described in this book that were motivated by scientificcuriosity, discoveries in the field of biotechnology have been primarily commercialundertakings. For a commercial enterprise to succeed, it must generate profit. Manypeople’s anxieties about the ethics of biotechnology reflect a distrust of big business. Someof these anxieties may be misplaced, but others undoubtedly have merit and require con-tinuing dialog among corporations, governments, and concerned citizens. Regardless ofhow one views the ethical imperatives of the new biology, it is incumbent upon all of usto 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 direc-tion 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:

Where Are We Now? 207

1. Some argue that tampering with the genetic program of humans may pose healthrisks to patients and researchers. In response to these arguments, the National In-stitutes of Health established guidelines for scientists working in the field of re-combinant DNA.

2. Biotechnology may involve risks to the environment. Some people are opposed totampering with the genomes of the plants and animals on which we rely for food.Such issues will require constant vigilance and continuing dialog among groups rep-resenting all views.

3. The power of new technologies has called into question the ethics of interfering withnatural processes. While opponents argue that we are overstepping the boundariesof what is right and natural, those who are in favor of biotechnology see it as the nextlogical step in improving human well-being.

WHERE ARE WE NOW?In 1997, Ian Wilmut, a soft-spoken biologist from the Roslin Institute near Edinburgh,Scotland, startled the world when he announced that he had done something no onebelieved possible: He had cloned a sheep named Dolly. Overnight, Dolly became themost famous animal in the world, and our view of what it is possible to accomplish in thelaboratory was forever changed.

Whole animal cloning is not to be confused with gene cloning, described earlier inSection 7-1. Gene cloning occurs when a snippet of DNA containing a gene is insertedinto a host cell, where it is copied each time the cell divides.Animal cloning is when twoor more genetically identical animals are produced from a single genome.

When a sperm and egg fuse at fertilization, the cell that results—the zygote—containsall of the genetic instructions for making an entire organism.There is a short period afterthe zygote begins to divide during which each of the embryonic cells is totipotent. Identicaltwins result when the first two cells of an embryo become separated: Each cell developsinto a separate, genetically identical embryo. Identical twins are natural clones. BeforeDolly, the prevailing view among scientists was that shortly after the first few cell divisions,embryonic cells lost their totipotency, becoming irreversibly committed to some specificcellular fate. But Ian Wilmut and Dolly proved that view wrong.

Wilmut began by injecting hormones into Scottish Blackface ewes to make them su-perovulate, that is, produce a large number of mature eggs ready to be fertilized. Heharvested an egg and, by using a fine needle, he destroyed its nucleus and with it, itsDNA. A different nucleus, taken from an adult cell of a Dorset ewe, was inserted intothe Scottish Blackface egg.The cytoplasm from the enucleated egg stimulated the Dorsetnucleus to regain its totipotency.The cell began to divide like a normal embryo.The em-bryo was implanted into the uterus of a Scottish Blackface ewe that had been preparedfor pregnancy, and after seven months of gestation, the Scottish Blackface gave birth toa bouncing baby Dorset lamb named Dolly (Figure 7-27).

Public reaction to Dolly’s birth was immediate and unprecedented.While no one wasparticularly concerned that Dolly the sheep was a clone, there was widespread fear thathuman cloning would soon follow. Political leaders from France, Germany, Switzerland,China, the United States, and the World Health Organization called for an immediateban on human cloning. Religious leaders and bioethicists joined the cry to ban experi-ments on humans. What is it about human cloning that causes us to question what itmeans to be human?

This issue in particular raises concerns about human individuality and the impor-tance of uniqueness to our self-conceptions and our dignity. However, we already knowfrom the case of identical twins, the only natural example of human clones, that a humanbeing is not simply the product of its genes. Experience, surroundings, interactions with


The embryo was transplanted tothe womb of a Scottish blackface ewe.

The two cells were fused with a mild electrical shock.

The fused celldivided like anormal embryo.

An egg was harvested from a Scottish blackface ewe. The DNA was removed with the use of a needle.

Adult cells from a Dorset ewe were taken from the sheep's udder.

Seven months later, Dolly,a Dorset lamb, was born.

Figure 7-27 ■ How Dolly was cloned.

Review Questions 209

REVIEW QUESTIONS

1. What is a cloning vector? How are cloning vectors used? Givethree examples of cloning vectors, indicating which ones are bet-ter for small fragments of DNA and which are better for largefragments.

2. Two methods for cutting DNA into small pieces are ultrasoundand restriction enzymes. Which method is better and why?

3. Assume you have taken the entire human genome and cut it intosmall pieces by using a restriction enzyme. Now you are inter-ested in finding the piece that contains the gene for hemoglobin.Describe how you might go about finding that gene.

4. What components are necessary for performing the polymerasechain reaction? When might you use this technique?

5. List three kinds of information that can be learned by sequenc-ing DNA. Also list three specific things that have been learnedfrom the sequence of the human genome.

6. Why are scientists interested in the DNA sequence of modelorganisms such as yeast, fruit flies, and plants?

7. Name one controversy that has resulted from the HumanGenome Project. Make an argument for one side of the contro-versy, then make an argument for the other side.

8. Would gene therapy be a good approach to curing the commoncold? Why or why not? What kinds of diseases are good targetsfor gene therapy? Why has gene therapy not lived up to its orig-inal promise?

9. How has biotechnology revolutionized the manner in whichpharmaceutical companies develop new drugs?

10. Draw a diagram of an agarose gel, showing the alleles from twoVNTR regions of a mother, a father, and their natural biologicalchild. How might the diagram differ if one of the parents were anadoptive parent instead of a biological parent?

11. Why does the FBI use 13 different VNTR regions instead of just1 VNTR region in identifying criminals with DNA evidence?

12. What are three goals of agricultural genetic engineering? Givean example of an engineered crop plant that achieves one ofthese goals.

13. Why are scientists hopeful about bioremediation? Begin youranswer by defining bioremediation and describing the circum-stances in which it might be used.

14. Make an argument against the use of genetically modifiedorganisms as crops. Also make a counterargument in favor ofusing GMOs.

others, and our own choices contribute to who we are. It is a fallacy to think that genet-ic likeness diminishes individuality or the value of the individual.The main worry aboutcloning is that the technology could create a society in which people become resourcesor biological commodities in ways that are morally objectionable. Even if human cloningwere undertaken with the best intentions—giving a child to an infertile couple, forexample—many believe that creating certain individuals by design is fundamentally atodds with regarding and valuing each human being as an end in itself.

Documents

7 Biotechnology - University of Phoenixmyresource.phoenix.edu/secure/resource/BIO100R3/BioInquiry Ch07.… · were treated with proteins isolated from cows or pigs brought to slaughter.However,the