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7
DNA Structure and Replication
DNA analysis identified the remains of the murdered members of the Romanov family.
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Learning OutLine
7.1 experiments identified the genetic Material A. Griffith Discovered that Bacteria Can Transfer Genetic Information B. Avery, MacLeod, and McCarty Showed that Genetic Information Is DNA C. Hershey and Chase Confirmed the Genetic Role of DNA 7.2 Dna is a Double Helix that encodes recipes for Proteins A. Biochemists and Physicists Discovered DNAs Structure B. DNA Contains the Information Needed for Lifes Functions 7.3 Dna replication Maintains genetic information A. Replication Requires Many Enzymes B. Mutations May Occur During Replication 7.4 PCr replicates Dna in a test tube 7.5 Dna Sequencing reveals the Order of Bases A. Sanger Used Fragments to Determine a DNA Sequence B. DNA Microarrays Speed Sequencing 7.6 Dna Profiling Has Many applications 7.7 investigating Life: genetic Messages from the Dead tell tales of ancient ecosystems
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7.1
The nucleic acid DNA (deoxyribonucleic acid) is one of the most familiar molecules, the subject matter of movies and headlines (figure 7.1). Fictional scientists reconstruct dinosaurs from DNA preserved in an ancient mosquitos gut. Criminal trials hinge on DNA evidence; the idea of cloning animals raises questions about the role of DNA in determining who we are; and DNA-based discoveries are yielding new diagnostic tests, treatments, and vaccines. More important than DNAs role in society today is its role in life itself. Of all the characteristics that distinguish the living from the nonliving, the one most important to the continuance of life is the self-replicating cell. At the molecular level, reproduction depends on DNA, a biochemical that has dual abilities. First, it directs the activities of the cell by controlling protein synthesis. Second, it manufactures an exact replica of itself, copying those instructions for the next generation of cells. The recognition of DNAs vital role in life was a long time in coming. By the early 1900s, researchers had recognized the connection between inheritance and protein. For example, the English physician, Archibald Garrod, noted that people with inherited inborn errors of metabolism lacked certain enzymes. Other researchers added supporting evidence: they linked abnormal or missing enzymes to unusual eye color in fruit flies and nutritional deficiencies in bread mold. But how were enzyme deficiencies and inheritance linked? Experiments in bacteria would answer the question.
Bacterium
DNA
a.
FIGURE 7.1 DNAThe Molecule in the Media. (A) DNA bursts forth from this treated bacterial cell, illustrating just how much DNA is tightly wound into a single cell. (B) Jurassic Park was a 1993 blockbuster movie in which fictional scientists recreated dinosaurs. The dinosaur DNA came from blood found in ancient mosquitoes entombed in amber.
b.
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Bacterial colonies
Injection
Results
Rough (strain R)
Mouse healthy
Smooth (strain S)
Mouse dies
S bacteria with a DNA-destroying enzyme (DNase) first, however, prevented the killing ability. Avery, MacLeod, and McCarty confirmed that DNA transformed the bacteria by isolating DNA from heat-killed type S bacteria and injecting it along with type R bacteria into mice. The mice died, and their bodies contained active type S bacteria. The conclusion: Type S DNA altered the type R bacteria, enabling them to manufacture the smooth coat necessary to cause infection. At first, biologists hesitated to accept DNA as the biochemical of heredity. They knew more about proteins than about nucleic acids. They also thought that protein, with its 20 building blocks, was able to encode many more traits than DNA, which includes just four types of building blocks. In 1950, however, U.S. microbiologists Alfred Hershey and Martha Chase conclusively showed that DNA not proteinis the genetic material.
Heat-killed strain S
Mouse healthy
Mouse dies
Heat-killed strain S
Hershey and Chase used a very simple system; they infected the bacterium Escherichia coli with a bacteriophage called T4. A bacteriophage is a virus that infects only bacteria; most consist of only a protein coat and a nucleic acid core (DNA in this case). We now know that when the virus infects the bacterial cell, it injects its DNA, and the protein coat remains loosely attached to the bacterium (figure 7.4). The
FIGURE 7.2 A Tale of Two Microbes. Griffiths experiments showed that a molecule in a lethal strain of bacteria (type S) could transform nonkilling bacteria (type R) into killers.
Virus (bacteriophage)
Protein coat
Cell wall Bacterial colonies Injection Results Cell membrane Viral DNA entering bacterial cell a. Virus
Mouse dies
+
DNase added
Heat-killed strain S
Mouse healthy
FIGURE 7.3 DNA Is the Transforming Principle. Avery, MacLeod, and McCarty identified Griffiths transforming principle as DNA. By adding enzymes that destroy either proteins (protease) or DNA (DNase) to the types of mixtures that Griffith used in his experiments, they demonstrated that DNA, and not protein, transforms bacteria.
A Virus (Bacteriophage) Infects a Bacterium. (A) A bacteriophage is a virus that infects only bacteria. It consists of a nucleic acid in a protein coat. The virus uses the protein coat to attach to and inject its DNA into a bacterial cell. (B) This photo shows several bacteriophages infecting a bacterium.
FIGURE 7.4
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viral DNA uses the bacterial cells energy and raw materials to manufacture more of itself. New virus particles then burst from the cell. Much of this information was not available in 1950. bacteriophages, p. 000; Focus on Model Organisms (E. coli), p. 000 Hershey and Chase wanted to know which part of the virus controls its replication: the DNA or the protein coat. The researchers labeled two batches of viruses, one with radioactive sulfur that marked protein, and the other with radioactive phosphorus that marked DNA. They used each type of labeled virus to infect a separate batch of bacteria and allowed several minutes for the virus particles to bind to the bacteria and inject their DNA into them (figure 7.5). Then they agitated each mixture in a blender, which removed the unattached viruses and empty protein coats from the surfaces of the bacteria. They poured the mixtures into test tubes and spun them at high speed. This settled the infected bacteria at the bottom of each test tube because they were heavier than the liberated viral protein coats. Hershey and Chase examined the contents of the bacteria that had settled to the bottom of each tube. In the
test tube containing sulfur-labeled virus, the virus-infected bacteria were not radioactive, but the fluid portion of the material in the tube was. In the other tube, where the virus contained radioactive phosphorus, the infected bacteria were radioactive, but the fluid was not. The blender experiments therefore showed that the part of the virus that could enter the bacteria and direct them to mass produce more viruses was the part with the phosphorus labelnamely, the DNA. The genetic material, therefore, was DNA and not protein. Summary A series of experiments revealed DNA, and not protein, to be the genetic material. Further experiments provided clues to its structure.
7.1
MASTERING CoNCEPTS
1. How did Griffiths research, coupled with the work of Avery and his colleagues, demonstrate that DNA, not protein, is the genetic material? 2. How did the Hershey-Chase blender experiments confirm Griffiths results?
Virus Blended and spun at high speeds to separate bacteria from viral protein coats
Virus Blended and spun at high speeds to separate bacteria from viral protein coats
Virus
FIGURE 7.5 DNAs Role Is Confirmed. Hershey and Chase used different radioactive isotopes to distinguish the viral protein coat from the DNA. Their experiments showed that the virus transfers DNA (not protein) to the bacterium, and that viral DNA alone could cause bacterial cells to produce viruses.
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7.2
The early twentieth century also saw corresponding advances in the study of the structure of DNA. By 1929, biochemists had discovered the distinction between ribonucleic acid (RNA) and DNA, the two types of nucleic acid. Later, they determined that nucleotides, the building blocks of nucleic acids, included sugars, nitrogen-containing groups, and phosphorus-containing components. Another important clue was the observation that DNA and RNA nucleotides always contain the same sugars and phosphates, but they may contain any one of four different nitrogen-containing bases.
a. Rosalind Franklin
b. Xray diffraction
FIGURE 7.6 Discovery of DNAs Structure. (A) Rosalind Franklin produced high-quality Xray images of DNA (B) that were crucial in the discovery of DNAs structure. (C) Maurice Wilkins, Francis Crick, and James Watson (first, third, and fifth from the left) shared the Nobel Prize in physiology or medicine for their now-famous discovery. Franklin had died in 1958, and by the rules of the award, she could not be included. FIGURE 7.7 Three Ways to Represent DNA. (A) This photo is a space-filling model of a DNA molecule that shows the threedimensional relationships of the component atoms. (B) This representation shows the helical structure of DNA. The sugar phosphate rails are identical in all DNA molecules and run in opposite directions. (C) The helix is unwound to show the base pairs and the sugarphosphate backbone in more detail.
T P P P P P P P P C C A G
G
Nucleotide
a.
b.
G A
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G A C T
c.
T P C P G
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sugarphosphate backbones, are alternating units of deoxyribose and phosphate joined with covalent bonds. The ladders rungs are AT and GC base pairs joined by hydrogen bonds.
the carbon atoms in deoxyribose are numbered. Opposite ends of each strand are designated 3 prime (3) and 5 prime (5). At the same end of the double helix, one chain ends in the 3 carbon, while the opposite chain ends in the 5 carbon.
O: O: P O
Phosphate group a. Sugar (deoxyribose)
N O O N N
O NH NH2
Sugar
N N N NH2
Sugar
O N
H3C
O NH N O
NH2
Sugar
Base pair
N N P N
O NH NH2
NH2 N O P P
Thymine (T)
Adenine (A)
H3 C
O NH N O
NH2 N N
b.
FIGURE 7.8 DNA Base Pairing. (A) In a nucleotide, a nitrogenous base is joined to the sugar deoxyribose, which is also bonded to a phosphate. Guanine and adenine are purines, each composed of two organic rings. Cytosine and thymine are pyrimidines, each built of one six-membered ring. (B) In the DNA double helix, purines pair with pyrimidines. Specifically, adenine pairs with thymine through two hydrogen bonds, and cytosine pairs with guanine through three hydrogen bonds.
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5
P
DNA
P
5 CH2OH 4
OH H C H
P
C H
3
C OH
H
P
Deoxyribose
G A
C T
To illustrate DNAs function, suppose a cell in a female mammals breast is producing milk to feed an infant (see figure 3.15). One of the many proteins in milk is albumin. The steps below summarize the production of albumin, starting with its genetic recipe (figure 7.10): 1. Inside the nucleus, an enzyme first copies the albumin genes DNA sequence to a complementary sequence of RNA. 2. After some modification, the RNA emerges from the nucleus and binds to a ribosome in the cytoplasm. 3. At the ribosome, amino acids are assembled in a specific order to produce the albumin protein, dictated by the sequence of nucleotides in the RNA molecule.
7.3
Before a cell divides, its DNA must replicate so that each daughter cell receives the same set of genetic instructions. Clues to the self-replication mechanism came from Watson and Cricks report on DNAs chemical structure. The paper ends with the tantalizing statement, It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mech-
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FIGURE 7.9 Parallel, but opposite. The two strands of the DNA double helix are oriented in opposite directions. The 5 and 3 ends of each strand refer to the numbers that chemists assign to the carbon atoms in deoxyribose.
G A
C T
RNA DNA
Protein
FIGURE 7.10 Genes Code for Proteins. This simplified figure illustrates DNAs function. In the nucleus, enzymes copy a gene to a molecule of RNA. The RNA leaves the nucleus and binds to a ribosome, where the cell uses the sequence of nucleotides in RNA to create a particular sequence of amino acidsa protein.
In this case, the result is albumin, but the sequence of nucleotides in DNA specifies the sequence of amino acids in every other protein in the cell as well. The details of this process are the subject of chapter 12. Summary The DNA molecule is a double helix with sugar phosphate rails and pyrimidinepurine pairs as rungs. The two strands of a DNA molecule are oriented in opposite directions. Genes are portions of DNA that code for specific proteins or RNA molecules.
7.2
MASTERING CoNCEPTS
1. What are the components of DNA and its threedimensional structure? 2. What evidence enabled Watson and Crick to decipher the structure of DNA? 3. How does a gene encode a protein?
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1 Helicase separates strands. 3 5 2 Binding proteins prevent single strands from rejoining. 3 Primase makes a short stretch of RNA on the DNA template.
3 5
3 Overall direction of replication 3 5 4 DNA polymerase adds DNA nucleotides to the RNA primer. Proofreading activity checks and replaces incorrect bases just added. 5 Leading (continuous) strand synthesis continues in a 5 to 3 direction. 3 5
C T A G
G A
3
C G G T A C A T
5
C
G G T A C A T
G A
FIGURE 7.11 DNA Replication Is Semiconservative. In this simplified view of DNA replication, DNA strands separate. New nucleotides form complementary base pairs with each exposed strand. DNA replication requires RNA primers and many enzymes, which are not shown here.
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G T
G A G C T
3 5
3 3 5
3 5
3 Each strand is a template that attracts and binds complementary nucleotides, A with T and G with C. 4 Each double-stranded DNA molecule consists of one parental and one daughter strand, as a result of semiconservative replication.
3 5
3 5
Okazaki fragment 3
3 5 3 5 3 5
3
5
FIGURE 7.12 DNA Replication Requires Many Enzymes. This process occurs simultaneously at many points along a cells DNA. Although these enzymes are depicted separately for clarity, in reality they form a single cluster.
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Enzymes copy DNA simultaneously at hundreds of points, called origins of replication, on a long DNA molecule. Replication proceeds in both directions at once from each origin of replication. DNA polymerase, however, can add new nucleotides only to the exposed 3 endnever the 5 endof a growing strand. DNA replication therefore proceeds continuously from each point of origin on one strand, but discontinuously (in short 5 to 3 pieces) on the other strand. The short pieces on the discontinuous strand are called Okazaki fragments. Of course, none of this occurs for free. DNA replication requires a great deal of energy because a large, organized molecule contains much more potential energy than do many individual nucleotides. Energy is required to synthesize nucleotides and to create the covalent bonds that join them together in the new strands of DNA. Many of the enzymes that participate in DNA replication, including helicase and ligase, also require energy in the form of ATP to catalyze their reactions. ATP, p. 85 DNA replication is incredibly accurate; after proofreading, DNA polymerase incorrectly incorporates only about 1 in a billion nucleotides. Other repair enzymes help ensure the accuracy of DNA replication by cutting out and replacing incorrect nucleotides. Nevertheless, mistakes occasionally remain. The result is a mutation, which is any change in a cells DNA sequence.
What causes skin cancer? Cancer is a family of disorders in which cells divide out of control (see chapter 8). Cancer has many forms, some inherited and others caused by exposure to radiation or harmful chemicals. Exposure to ultraviolet radiation from the sun or from tanning beds, for example, increases the risk of skin cancer. Several types of skin cancer exist; melanoma is the most dangerous because it can quickly spread to other parts of the body. Sometimes, cells fail to detect or repair the damaged DNA. The result is a genetic mutation (discussed further in chapter 12). If the mutation occurs in genes coding for proteins that control the pace of cell division, cells may begin dividing out of control, forming a tumor. Problems with the enzymes that repair DNA therefore predispose people to cancer. A rare disorder called xeroderma pigmentosum (XP) illustrates the vital role of these enzymes in protecting against skin cancer. For people with XP, exposure to sunlight is extremely dangerous. They lack the proteins that would otherwise repair damage caused by UV radiation in sunlight. People with XP therefore have a 2000-fold higher risk of developing skin cancer than the population at large. Have a Burning Question of your own? Submit it to marielle_hoefnagels@mcgraw-hill.com for possible inclusion in future editions of this book!
7.3 1. 2. 3. 4. 5.
MASTERING CoNCEPTS
Why does DNA replicate? What is semiconservative replication? What are the steps of DNA replication? What is a mutation, and why are mutations important? Why is DNA repair necessary?
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7.4
An extremely powerful and useful tool, the polymerase chain reaction (PCR), taps into the cells DNA copying machinery to rapidly produce millions of copies of a DNA sequence of interest (figure 7.13). PCR is useful whenever a small amount of DNA would provide information if it was mass-produced. Thanks to this tool, a single hair or a few skin cells left at a crime scene can yield enough genetic material for DNA profiling. PCR rapidly replicates a selected sequence of DNA in a test tube. The requirements include the following: A target DNA sequence to be replicated. Taq polymerase, a heat-tolerant DNA polymerase produced by Thermus aquaticus, a bacterium that inhabits hot springs. (Other heat-tolerant polymerases can also be used.) Two types of short, laboratory-made primers that are complementary to opposite ends of the target sequence. The primers are necessary because DNA polymerase can only attach nucleotides to an existing strand. A supply of the four types of DNA nucleotides. PCR occurs in an automated device called a thermal cycler that controls key temperature changes. In the first step of PCR, heat separates the two strands of the target DNA. Next, the temperature is lowered, and the short primers attach to the separated target strands by complementary base pairing. Taq DNA polymerase adds nucleotides to the primers and builds sequences complementary to the target sequence. The newly synthesized strands then act as templates in the next round of replication, which is initiated immediately by raising the temperature to separate the strands once more. The number of pieces of DNA doubles with every round of PCR, so that after just 20 cycles, a million-fold increase occurs in the number of copies of the target sequence. PCR finds a wide variety of applications. Its greatest strength is that it works on crude samples, such as a bit of brain tissue on the bumper of a car, which in one criminal case led to identification of a missing person. In forensics, PCR is used routinely to establish genetic relationships, identify remains, convict criminals, and exonerate the falsely accused. When used to amplify the nucleic acids of microorganisms, viruses, and other parasites, PCR is important in agriculture, veterinary medicine, environmental science, and human health care. In genetics, PCR is both a crucial basic research tool and a way to identify disease-causing genes. PCRs greatest weakness, ironically, is its exquisite sensitivity. A blood sample contaminated by leftover DNA from a previous run or by a stray eyelash dropped from the person running the reaction can yield a false result. Summary Polymerase chain reaction (PCR) uses primers, nucleotides, and heat-tolerant DNA polymerase to generate copies of DNA.
Target sequence
3 Temperature is lowered, and primers from the solution attach to the target sequence.
FIGURE 7.13 PCR Amplifies a Specific DNA Sequence. In the polymerase chain reaction, primers bracket a DNA sequence of interest. A heat-stable Taq DNA polymerase uses these primers, and plenty of nucleotides, to build up millions of copies of the target sequence.
7.4
MASTERING CoNCEPTS
1. How do target DNA, primers, nucleotides, and Taq DNA polymerase interact in PCR? 2. Why is PCR useful?
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7.5
The uses of DNA sequences, from individual genes to entire genomes, seem endless. Researchers can apply sequence information to everything from identifying corpses, to predicting protein sequences, to determining evolutionary relationships. How do investigators get the DNA sequence information they need?
Primer
1 Four solutions contain unknown DNA sequence, primers, normal nucleotides (A,C,G, and T), labeled nucleotides, replication enzymes, and a small amount of terminator nucleotide. Terminator A added Terminator C added Terminator T added Terminator G added
A* ATGCGCA*
ATGC* ATGCGC*
AT* ATGCGCAT*
3 Samples are transferred to a gel between two glass plates. Electrodes are connected to both ends of the gel. 4 During electrophoresis, negatively charged phosphate groups are attracted to the positive electrode, causing the DNA fragments to move through the gel. The smaller the fragment, the faster it moves.
Power source
Power source
5 The fragments are read off by size, and the original sequence can be deduced.
G T A C G C G T A C A T G C G C A T
FIGURE 7.14 Determining the Sequence of DNA. In the Sanger method of DNA sequencing, complementary copies of an unknown DNA sequence are terminated early because of the addition of chemically modified terminator nucleotides. Placing the fragments in order by size reveals the sequence.
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DNA microarray of all possible 6-base combinations immobilized on surface Copies of fluorescently tagged DNA segment of unknown sequence
C C A T G T C C G A C C G T T A T T C T T T T C A A C A C G T C T G G G C C A T C G T A A C C A G C T T G A T T T
a.
C T C T C A T G A C
G G
A T C
T A G
b.
G G
A T C
T A G
C C A
C G T T C T T T T C A A C A C G T C T G G G C C A T C G T A A C C A G C T T G A T T T
C A T G A C T C
T G
T A T
fluorescent label are then also placed on the microchip. The copies stick to any six-base strands on the chip whose sequences are complementary to the unknown DNA segments sequences. Under laser light, the bound sequences fluoresce. Because the researcher (or computer program) knows which strands occupy which positions on the microchip, a scan of the chip reveals which six-base sequences make up the unknown sequence. Then, software aligns the identified fragments by their overlaps. This reconstructs the complement of the entire unknown sequence. Microarrays are increasingly common in research because they offer a fast, inexpensive way to generate sequence data. One day, they may also appear in doctors offices. If medical professionals can quickly determine a patients DNA sequence at key locations, they may be able to pinpoint the cause of a patients cancer or customize drug treatments to minimize side effects. Summary The Sanger method uses modified nucleotides to reveal the DNA sequence. Alternatively, DNA fragments of unknown sequence can stick to short DNA pieces of known sequence arranged on microchips.
7.5
C G A C
C C G T A T T A T C G A C G A T C C
Complementary sequence is derived from overlaps, revealing the original unknown sequence.
C C G T A T C G A T C C GG C A T A G C T A G G
MASTERING CoNCEPTS FIGURE 7.16 Sequencing on a Chip. A labeled DNA segment of unknown sequence binds to short, known DNA sequences immobilized on a small glass microchip. Identifying areas of overlap among the bound sequences reveals the unknown DNA sequence.
1. How does the Sanger method use modified nucleotides to produce truncated DNA fragments, and how do researchers use these fragments to deduce a DNA sequence? 2. What is a DNA microchip?
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7.6
DNA profiling detects genetic differences between individuals, with applications ranging from settling paternity claims to exonerating wrongly convicted criminals to identifying crime victims (see Can You Relate? Identifying Victims of the September 11, 2001, Attacks below). Rather than sequencing and comparing entire genomes, DNA profiling considers just the most variable parts. In one approach, researchers use single-base sites that tend to vary in a population. DNA samples are treated with bacterial enzymes (called restriction enzymes) that cut DNA at specific sequences. If two samples differ at a cutting site, the restriction enzyme produces DNA fragments of different sizes and numbers. Figure 7.17 illustrates how researchers use gel electrophoresis to reveal these underlying sequence differences.
DNA sample 2
DNA sample 3
Longer fragments
Power source
Shorter fragments
3 Labeled DNA probes highlight certain fragments of interest by binding to specific fragments of sampled DNA. This way, DNA from different individuals may have different banding patterns.
Sample 1
Sample 2
Sample 3
FIGURE 7.17 DNA Profiles from a Murder Case. (A) DNA samples from different sources produce different patterns of fragments when cut with the same restriction enzyme. (B) DNA from bloodstains on the defendants clothes matches the DNA profile of the victim but differs from the DNA profile of the defendant. This is evidence that the blood on the defendants clothes came from the victim, not the defendant.
b.
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Suppose that DNA extracted from a hair found on a murder victims body matches DNA from a suspects white blood cells at five restriction sites. What is the probability that the two matching DNA patterns come from the same personthe suspectrather than from two individuals who resemble each other by chance? To find out, investigators consult databases that compile the frequency of each allele in the population. If each of the five alleles occurs at a rate of 10% in the population, the chance of the match occurring by chance is 0.1 0.1 0.1 0.1 0.1, or 1 in 100,000. There is therefore a 99.999% chance that the samples came from the same person. Analysis of mitochondrial DNA is also sometimes useful. Mitochondrial DNA is typically only about 16,500 base pairs long, far shorter than nuclear DNA. But because each cell contains multiple mitochondria, each of which contains many DNA molecules, mitochondria can therefore often yield useful information even when nuclear DNA is badly degraded. Investigators extract mitochondrial DNA from hair, bones, and teeth, then use PCR to amplify the variable regions for sequencing. Because everyone inherits mitochondria only from his or her mother, this technique cannot discriminate be-
tween siblings or many other close family members. It is very useful, however, for verifying the relationship between woman and child. For example, children who were kidnapped during infancy can be matched to their biological mothers or grandmothers. The study of human evolution also has benefited from mitochondrial DNA. And, as you learned at this chapters beginning, mitochondrial DNA helped identify members of the Russian royal family. human evolution, p. 000 Summary DNA profiling reveals genetic differences between individuals by analyzing variable portions of the genome.
7.6
MASTERING CoNCEPTS
1. How are variable DNA sequences used in DNA profiling? 2. Why does DNA profiling require an understanding of probability? 3. Why does mitochondrial DNA provide different information from nuclear DNA?
7.7
INVESTIGATING LIFE Genetic Messages from the Dead Tell Tales of Ancient Ecosystems
frozen land in Siberia, Alaska, and the Yukon. Could DNA trapped in frozen Siberian soil reveal an ancient ecosystems organisms? Danish researchers Eske Willerslev, Anders Hansen, and their colleagues decided to find out. To study the ancient remains, the scientists drove metal cylinders deep into the permafrost and removed long, thin rods (called cores) of ice, soil, and organic material (figure 7.18). Because new sediments accumulate over old ones, the deepest holes yield the oldest deposits. Radiometric dating (a technique that uses radioactive decay of isotopes as a type of clock), pollen analysis, and other techniques helped them estimate the age of each layer of material in the cores. radiometric dating, p. 000 Willerslev and company tried to extract DNA from eight small sediment samples ranging in age from modern to 1.5 to 2 million years old. Wherever DNA was present, they used PCR primers to amplify two sets of genes. One target sequence was part of the gene encoding the protein rubisco, which is essential for photosynthesis (see chapter 5). Because this gene occurs in chloroplasts, its presence indicates
Psychics claim to be able to communicate with the spirits of the dead. Although scientists do not assert the same spiritual connection, they can bring back the genetic remnants of species that lived and died long ago. When an organism dies, its DNA usually degrades rapidly. But in some special cases, the genetic material remains intact indefinitely. Millions of years ago, for example, sticky tree resin smothered and preserved insects, protecting the animals DNA in an amber tomb. (Blood-engorged mosquitoes preserved in amber were the fictional source of dinosaur DNA in Jurassic Park). Mineral crystals in bones and teeth can safeguard DNA for thousands of years too. Freezing also preserves DNA. An ideal source of diverse ancient DNA would therefore be a landscape that once teemed with life but that has since become permanently frozen. One such example is the land bridge, called Beringia, that once connected present-day northeastern Siberia to Alaska. During the last ice age, which ended about 10,000 years ago, much of Earths water was locked in polar ice caps and glaciers. As a result, global sea levels were low, exposing the Beringian land bridge. Although glaciers scoured most of North America during the ice age, Beringias dry climate kept it ice-free. Giant mammals such as mammoth, bear, bison, and large cats roamed the grassy Beringian landscape. Now that the ice age has ended, the land bridge is under the sea, and much of the rest of Beringia is permanently
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plant material in a sample. The other three target DNA sequences were fragments of genes found in mitochondria of vertebrate animals. (Vertebrates are animals with bony backbones, such as fishes, reptiles, and mammals). The PCR results showed that sediments from 300,000 to 400,000 years old contained plant DNA. When the scientists sequenced the DNA and compared it to genes cataloged in GenBank, a public database of nucleotide sequences, the scientists found that the ancient sediments harbored tremendous diversity: 11 classes, 23 orders, and 28 families representing mosses, herbs, shrubs, and trees. The oldest mitochondrial DNA was much younger, about 20,000 to 30,000 years old. Some came from grazers that still exist today, such as horse, lemming, hare, musk ox, and reindeer, but the researchers also found genes from extinct mammoth and bison. (Previous researchers studying fossils of these huge herbivores had deposited the gene sequences in GenBank.) The presence of genes from animals that vanished long ago suggests that the sediment DNA was authentic and not simply a modern contaminant. These gene fragments are important, but not for Jurassic Park-style reasons. After all, it is not possible to bring back the mammoth from just a tiny piece of its mitochondrial DNA. Rather, the study is significant because the long-buried sediments can help scientists reconstruct ancient ecosystems. The chloroplast DNA, for example, reveals that herbs (grasses and other nonwoody green plants) dominated the Beringian landscape 300,000 or so years ago but lost ground to shrubs over time (figure 7.19). The most dramatic decline of grasses occurred in the past 10,000 years, a time that coincided with the extinction of the mammoth and bison. Did one event
cause the other? Or did the end of the ice age cause both? What role did increasing human populations play? These questions remain unanswered for now. Willerslevs team hopes their work will inspire others to extract DNA from ancient sediments around the world. The resulting patchwork of gene fragments, pieced together, will reveal valuable information about the changes that shaped ancient ecosystemswithout the help of a psychic.
Willerslev, E., A. J. Hansen, J. Binladen, et al. May 2, 2003. Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science, vol. 300, pages 791795. 80 60 Percent 40 20 0 300-400 20-30 19 10.4 Thousands of years ago present
FIGURE 7.19 Communities Change. Chloroplast DNA isolated from sediment samples shows that grasses and other herbs have become much less common, while shrubs have become more common, over the past 400,000 or so years. Question: What are some alternative hypotheses for why the researchers failed to recover any DNA from sediments that were more than 300,000 to 400,000 years old? How would you test your hypotheses?
CHAPTER SUMMARy
7.1
7.2
Dna encodes the information necessary for a cells survival and specialization; it must be able to replicate for a cell to divide. Many experiments described DNA and showed it to be the genetic material. A. Griffith Discovered that Bacteria Can Transfer Genetic Information Griffith determined that an unknown substance transmits a disease-causing trait between two types of bacteria. B. Avery, MacLeod, and McCarty Showed that Genetic Information Is DNA With the help of protein- and DNA-destroying enzymes, scientists subsequently showed that Griffiths transforming principle is DNA. C. Hershey and Chase Confirmed the Genetic Role of DNA Using viruses that infect bacteria, Hershey and Chase confirmed that the genetic material is DNA and not protein.
A. Biochemists and Physicists Discovered DNAs Structure Chargaff discovered that A and T, and G and C, occur in equal proportions in DNA. Wilkins and Franklin provided X-ray diffraction data. Watson and Crick combined these clues to propose the double-helix structure of DNA. DNA consists of building blocks called nucleotides. The rungs of the DNA double helix consist of hydrogen-bonded complementary base pairs (A with T, and C with G). The rails are antiparallel chains of alternating deoxyribose and phosphate. B. DNA Contains the Information Needed for Lifes Functions An organisms genome is all of the DNA in its cells. In eukaryotic cells, the genome is divided into chromosomes. Genes are sequences of DNA that encode a cells proteins.
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DNA replication is semiconservative because each double-stranded molecule contains one strand from the original DNA. A. Replication Requires Many Enzymes To replicate, DNA unwinds, and the hydrogen bonds between strands break. DNA polymerase adds DNA nucleotides to a short RNA primer. Ligase seals the sugarphosphate backbone after the RNA primer is replaced with DNA. Replication proceeds only in a 5 to 3 direction, so the process is discontinuous in short stretches on one strand. Enzymes repair mistakes in replication or damaged DNA. B. Mutations May Occur During Replication Mutations are changes in a cells DNA sequence; they give rise to new alleles (gene variants). DNA repair disorders raise cancer risk.
7.4
A. Sanger Used Fragments to Determine a DNA Sequence The Sanger method uses modified nucleotides to generate DNA fragments of various lengths. Sorting the fragments by size reveals the DNA sequence. B. DNA Microarrays Speed Sequencing DNA chips contain all possible combinations of short nucleotide sequences. DNA of unknown sequence sticks to some of the chip-bound DNA, then computers reconstruct the original sequence.
7.6
Individuals vary genetically in single bases and short repeated DNA sequences. DNA profiling detects these differences. Investigators can use known frequencies of alleles in the population to calculate the probability that two DNA samples match. Analysis of mitochondrial DNA can verify maternal relationships.
7.7
In the polymerase chain reaction (PCR), DNA separates into two strands, then a primer provides DNA polymerase with a starting point for replication. Repeated cycles of heating and cooling allow for rapid amplification of the target DNA sequence. PCR finds many applications in research, forensics, medicine, agriculture, and other fields.
Investigating Life: Genetic Messages from the Dead Tell Tales of Ancient Ecosystems
Researchers have extracted DNA from plants and animals buried long ago in frozen sediments. The DNA evidence reveals changes in the landscape over hundreds of thousands of years.
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8. Why did the DNA from the heat-killed S-type cells transform the R-type cells? a. Because it was mutated b. Because DNA determines what proteins are made in a cell c. Because the R-type cells did not have DNA d. Because DNA is less complex than proteins 9. What is a DNA microarray used for? a. Determining the number of base pairs in a persons chromosome b. Determining the sequence of a segment of DNA c. Creating genetic mutations d. Identifying a DNA profile
10. In 1920, a woman claiming to be a surviving member of the royal family of Tsar Nicholas II of Russia appeared in Europe. What modern method of DNA profiling would you use to determine if this person was a member of the Romanov family? a. Analysis of variable single-base sites b. Analysis of the sequence of the womans entire genome c. Analysis of mitochondrial DNA sequences d. Comparison of allele frequencies to population databases
THINKING AS A SCIENTIST
1. Choose an experiment mentioned in the chapter and analyze how it follows the scientific method. 2. Give an example from the chapter of different types of experiments used to address the same hypothesis. Why might this be necessary? 3. The experiments that revealed DNA structure and function used a variety of organisms. How can such diverse organisms demonstrate the same genetic principles? 4. How would the results of the Hershey-Chase experiment have differed if protein was the genetic material? 5. A person with deficient DNA repair may have an increased cancer risk and chromosomes that cannot heal breaks. The person is, nevertheless, alive. How
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long would an individual lacking DNA polymerase be likely to survive? 6. To diagnose encephalitis (brain inflammation) caused by West Nile virus infection, a researcher needs a million copies of a viral gene. She decides to use PCR on a sample of cerebrospinal fluid, which bathes the persons infected brain. If one cycle of PCR takes 2 minutes, how long will it take the researcher to obtain her million-fold amplification if she starts with a single copy of the viral gene? 7. As the climate warms, permafrost in Siberia and other northern regions could melt. How might global warming affect future DNA-based studies of ancient ecosystems whose remains are trapped in the frozen soil?
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