DNA The Chemical Nature

The Elegantly Stable Double Helix: Ice Man s DNA
DNA, with its gentle double-stranded spiral, is among the most elegant of all biological molecules. But the double helix is not just a beautiful structure; it also gives DNA incredible stability and permanence, as illustrated by the story of Ice Man.
On September 19, 1991, German tourists hiking in the Tyrolean Alps near the border between Austria and Italy spotted a corpse trapped in glacial ice. A copper ax, dagger, bow, and quiver with 14 arrows were found alongside the body. Not realizing its antiquity, local residents made several crude and unsuccessful attempts to free the body from the ice. After 4 days, a team of forensic experts arrived to recover the body and transport it to the University of Inns¬bruck. There the mummified corpse, known as Ice Man, was refrozen and subjected to scientific study.
Radiocarbon dating indicates that Ice Man is approxi¬mately 5000 years old. Recent evidence from the South Tyrol Museum of Archeology has led to the conclusion that Ice Man was shot in the chest with an arrow and died soon thereafter. The body became dehydrated in the cold high- altitude air, was covered with snow that turned into ice, and remained frozen for the next 5000 years.
Some experts challenged Ice Man’s origin, suggesting that he was a South American mummy who had been planted at the glacier site in an elaborate hoax. To establish his authenticity and ethnic origin, scientists removed eight samples of muscle, connective tissue, and bone from his left hip. Under sterile conditions, the investigators extracted DNA from the samples and used the polymerase chain reac¬tion to amplify a very small region of his mitochondrial DNA a million fold. They determined the base sequence of this amplified DNA and compared it with mitochondrial sequences from present-day humans.
This analysis revealed that Ice Man’s mitochondrial DNA sequences resemble those found in present-day Euro¬peans living north of the Alps and are quite different from those of sub-Saharan Africans, Siberians, and Native Ameri¬cans. Together, radiocarbon dating, the artifacts, and the DNA analysis all indicate that Ice Man was a Neolithic hunter who died while attempting to cross the Alps 5000 years ago. That some of Ice Man’s DNA persists and faith¬fully carries his genetic instructions even after the passage of 5000 years is testimony to the remarkable stability of the double helix. Even more ancient DNA has been isolated from the fossilized bones of Neanderthals that are at least 30,000 years old.
This chapter focuses on how DNA was identified as the source of genetic information and how this elegant mole¬cule encodes the genetic instructions. We begin by consider¬ing the basic requirements of the genetic material and the history of our understanding of DNA — how its relation to genes was uncovered and how its structure was determined. The history of DNA illustrates several important points about the nature of scientific research. As with so many important scientific advances, DNA’s structure and its role as the genetic material were not discovered by any single person but were gradually revealed over a period of almost 100 years, thanks to the work of many investigators. Our understanding of the relation between DNA and genes was enormously enhanced in 1953, when James Watson and Francis Crick proposed a three-dimensional structure for DNA that brilliantly illuminated its role in genetics. As illus¬trated by Watson and Crick’s discovery, major scientific advances are often achieved not through the collection of new data but through the interpretation of old data in new ways.
After reviewing the history of DNA, we will examine DNA structure. DNA structure is important in its own right, but the key genetic concept is the relation between the structure and the function of DNA how its structure allows it to serve as the genetic material.


Characteristics of Genetic Material
Life is characterized by tremendous diversity, but the coding instructions of all living organisms are written in the same genetic language that of nucleic acids. Surprisingly, the idea that genes are made of nucleic acids was not widely accepted until after 1950. This late recognition of the role of nucleic acids in genetics resulted principally from a lack of knowledge about the structure of deoxyribonucleic acid (DNA). Until the structure of DNA was fully elucidated, it wasn’t clear how DNA could store and transmit genetic information. Even before nucleic acids were identified as the genetic material, biologists recognized that, whatever the nature of genetic material, it must possess three important characteristics.
1. Genetic material must contain complex information.
First and foremost, the genetic material must be capable of storing large amounts of information—instructions for all the traits and functions of an organism. This information must have the capacity to vary, because different species and even individual members of a species differ in their genetic makeup. At the same time, the genetic material must be stable, because most alterations to the genetic instructions (mutations) are likely to be detrimental.
2. Genetic material must replicate faithfully.
A second necessary feature is that genetic material must have the capacity to be copied accurately. Every organism begins life as a single cell, which must undergo billions of cell divisions to produce a complex, multicellular creature like ourselves. At each cell division, the genetic instructions must be transmitted to descendent cells with great accuracy. When organisms reproduce and pass genes to their progeny, the coding instructions must be copied with fidelity.
3. Genetic material must encode phenotype.
The genetic material (the genotype) must have the capacity to “code for” (determine) traits (the phenotype). The product of a gene is often a protein; so there must be a mechanism for genetic instructions to be translated into the amino acid sequence of a protein.
Concepts
The genetic material must be capable of carrying large amounts information, replicating faithfully, and translating its coding instructions into phenotypes.
The Molecular Basis of Heredity
Although our understanding of how DNA encodes genetic information is relatively recent, the study of DNA structure stretches back 100 years.
Early Studies of DNA
In 1868, Johann Friedrich Miescher grad¬uated from medical school in Switzerland. Influenced by an uncle who believed that the key to understanding dis¬ease lay in the chemistry of tissues, Miescher traveled to Tubingen, Germany, to study under Ernst Felix Hoppe- Seyler, an early leader in the emerging field of biochem¬istry. Under Hoppe-Seyler’s direction, Miescher turned his attention to the chemistry of pus, a substance of clear medical importance. Pus contains white blood cells with large nuclei; Miescher developed a method of isolating these nuclei. The minute amounts of nuclear material that he obtained were insufficient for a thorough chemical analysis, but he did establish that it contained a novel sub¬stance that was slightly acidic and high in phosphorus. This material, which consisted of DNA and protein, Miescher called nuclein. The substance was later renamed nucleic acid by one of his students.
By 1887, researchers had concluded that the physical basis of heredity lies in the nucleus. Chromatin was shown to consist of nucleic acid and proteins, but which of these substances is actually the genetic information was not clear. In the late 1800s, further work on the chemistry of DNA was carried out by Albrecht Kossel, who determined that DNA contains four nitrogenous bases: adenine, cytosine, guanine, and thymine (abbreviated A, C, G, and T).
In the early twentieth century, the Rockefeller Institute in New York City became a center for nucleic acid research. Phoebus Aaron Levene joined the Institute in 1905 and spent the next 40 years studying the chemistry of DNA. He discovered that DNA consists of a large number of linked, repeating units, each containing a sugar, a phosphate, and a base (together forming a nucleotide).
As additional studies of the chemistry of DNA were completed in the 1940s and 1950s, this notion of DNA as a simple, invariant molecule began to change. Erwin Chargaff and his colleagues carefully measured the amounts of the four bases in DNA from a variety of organisms and found that DNA from different organisms varies greatly in base composition. This finding disproved the tetranucleotide the¬ory. They discovered that, within each species, there is some regularity in the ratios of the bases: the total amount of ade¬nine is always equal to the amount of thymine (A = T), and the amount of guanine is always equal to the amount of cytosine (G = C). These findings became known as Chargaff’s rules.

(Concepts)
Details of the structure of DNA were worked out by a number of scientists. At first, DNA was interpreted as being too regular in structure to carry genetic information but, by the 1940s, DNA from different organisms was shown to vary in its base composition.


The discovery of the transforming principle
The first clue that DNA was the carrier of hereditary information came with the demonstration that DNA was responsible for a phenomenon called transformation. The phenomenon was first observed in 1928 by Fred Griffith, an English physician whose special interest was the bacterium that causes pneumonia, Streptococcus pneumonia. Griffith had succeeded in isolating several different strains of S. pneumo¬nia (type I, II, III, and so forth). In the virulent (disease- causing) forms of a strain, each bacterium is surrounded by a polysaccharide coat, which makes the bacterial colony appear smooth when grown on an agar plate; these forms are referred to as S, for smooth. Griffith found that these virulent forms occasionally mutated to non-virulent forms, which lack a polysaccharide coat and produce a rough ¬appearing colony on an agar plate; these forms are referred to as R, for rough.
Griffith was interested in the origins of the different strains of S. pneumonia and why some types were virulent, whereas others were not. He observed that small amounts of living type IIIS bacteria injected into mice caused the mice to develop pneumonia and die; on autopsy, he found large amounts of type IIIS bacteria in the blood of the mice ( . 10.2a). When Griffith injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood. Griffith knew that boil¬ing killed all the bacteria and destroyed their virulence; when he injected large amounts of heat-killed type IIIS bac¬teria into mice, the mice lived and no type IIIS bacteria were recovered from their blood.
The results of these experiments were not unusual. However, Griffith got a surprise when he infected his mice with a small amount of living type IIR bacteria, along with a large amount of heat-killed type IIIS bacteria. Because both the type IIR bacteria and the heat-killed type IIIS bac-teria were non virulent, he expected these mice to live. Sur¬prisingly, 5 days after the injections, the mice became infected with pneumonia and died. When Griffith examined blood from the hearts of these mice, he observed live type IIIS bacteria. Furthermore, these bacteria retained their type IIIS characteristics through several gen¬erations; so the infectivity was heritable.

Griffith’s results had several possible interpretations, all of which he considered. First, it could have been the case that he had not sufficiently sterilized the type IIIS bacteria and thus a few live bacteria remained in the culture. Any live bacteria injected into the mice would have multiplied and caused pneumonia. Griffith knew that this possibility was unlikely, because he had used only heat-killed type IIIS bac¬teria in the control experiment, and they never produced pneumonia in the mice.
A second interpretation was that the live, type IIR bac¬teria had mutated to the virulent S form. Such a mutation would cause pneumonia in the mice, but it would produce type IIS bacteria, not the type IIIS that Griffith found in the dead mice. Many mutations would be required for type II bacteria to mutate to type III bacteria, and the chance of all the mutations occurring simultaneously was impossibly low.
Griffith finally concluded that the type IIR bacteria had somehow been transformed, acquiring the genetic virulence of the dead type IIIS bacteria. This transformation had pro¬duced a permanent, genetic change in the bacteria; though Griffith didn’t understand the nature of transformation, he theorized that some substance in the polysaccharide coat of the dead bacteria might be responsible. He called this sub¬stance the transforming principle.

Identification of the transforming principle
At the time of Griffith’s report, Oswald Avery was a microbiologist at the Rockefeller Institute. At first Avery was skeptical but, after other microbiologists successfully repeated Griffith’s experiments using other bacteria and showed that transformation took place, Avery set out to identify the nature of the transforming substance.
After 10 years of research, Avery, Colin MacLeod, and Maclyn McCarty succeeded in isolating and purifying the transforming substance. They showed that it had a chemical composition closely matching that of DNA and quite differ¬ent from that of proteins. Enzymes such as trypsin and chymotrypsin, known to break down proteins, had no effect on the transforming substance. Ribonuclease, an enzyme that destroys RNA, also had no effect. Enzymes capable of destroying DNA, however, eliminated the biological activity of the transforming substance. Avery, MacLeod, and McCarty showed that purified transforming substance precipitated at about the same rate as purified DNA and that it absorbed ultraviolet light at the same wave¬lengths as does DNA. These results, published in 1944, provided compelling evidence that the transforming princi¬ple and therefore genetic information resides in DNA. Many biologists still refused to accept the idea, however, still preferring the hypothesis that the genetic material is protein.








Concepts
The process of transformation indicates that some substance—the transforming principle is capable of genetically altering bacteria. Avery, MacLeod, and McCarty demonstrated that the transforming principle is DNA, providing the first evidence that DNA is the genetic material.
The Hershey-Chase experiment
A second piece of evi¬dence implicating DNA as the genetic material resulted from a study of the T2 virus conducted by Alfred Hershey and Martha T2 is a bacteriophage (phage) that infects the bacterium Escherichia coli. A phage reproduces by attaching to the outer wall of a bacterial cell and injecting its DNA into the cell, where it replicates and directs the cell to synthesize phage protein. The phage DNA becomes encapsulated within the proteins, producing progeny phages that lyse (break open) the cell and escape.
At the time of the Hershey-Chase study (their paper was published in 1952), biologists did not understand exactly how phages reproduce. What they did know was that the T2 phage consists of approximately 50% protein and 50% nucleic acid, which a phage infects a cell by first attaching to the cell wall, and that progeny phages are ulti¬mately produced within the cell. Because the progeny carried the same traits as the infecting phage, genetic mater¬ial from the infecting phage must be transmitted to the progeny, but how this occurs was unknown.












Hershey and Chase designed a series of experiments to determine whether the phage protein or the phage DNA was transmitted in phage reproduction. To follow the fate of protein and DNA, they used radioactive forms (isotopes) of phosphorus and sulfur. A radioactive isotope can be used as a tracer to identify the location of a specific molecule, because any molecule containing the isotope will be radioactive and therefore easily detected. DNA contains phosphorus but not sulfur; so Hershey and Chase used 32P to follow phage DNA during reproduction. Protein contains sulfur but not phosphorus; so they used 35S to follow the protein.
First, Hershey and Chase grew E. coli in a medium containing 32P and infected the bacteria with T2 so that all the new phages would have DNA labeled with 32P. They grew a second batch of E. coli in a medium containing 35S and infected these bacteria with T2 so that all these new phages would have protein labeled with 35S. Hershey and Chase then infected separate batches of unlabeled E. coli with the 35S- and 32P-labeled phages. After allowing time for the phages to infect the cells, they placed the E. coli cells in a blender and sheared off the now-empty protein coats (ghosts) from the cell walls. They separated out the protein coats and cultured the infected bacterial cells. Eventually, the cells burst and new phage particles emerged.

When phages labeled with 35S infected the bacteria, most of the radioactivity separated with the protein ghosts and little remained in the cells. Furthermore, when new phages emerged from the cell, they contained almost no radioactivity. This result indicated that, although the protein component of a phage was necessary for infection, it didn’t enter the cell and was not transmitted to progeny phages.

In contrast, when Hershey and Chase infected bacteria with 32P-labeled phages and removed the protein ghosts, the bacteria were still radioactive. Most significantly, after the cells lysed and new progeny phages emerged, many of these phages emitted radioactivity from 32P, demonstrating that DNA from the infecting phages had been passed on to the progeny. These results confirmed that DNA, not protein, is the genetic material of phages.
[Concepts]
Using radioactive isotopes, Hershey and Chase traced the movement of DNA and protein during phage infection. They demonstrated that DNA, not protein, enters the bacterial cell during phage reproduction and that only DNA is passed on to progeny phages.
Watson and Crick s Discovery of the Three-Dimensional Structure of DNA
The experiments on the nature of the genetic material set the stage for one of the most important advances in the history of biology the discovery of the three-dimensional structure of DNA by James Watson and Francis Crick in 1953.
Watson had studied bacteriophage for his Ph.D.; he was familiar with Avery’s work and thus understood the tremen¬dous importance of DNA to genetics. Shortly after receiving his Ph.D., Watson went to the Cavendish Laboratory at Cambridge University in England, where a number of researchers were studying the three-dimensional structure of large molecules. Among these researchers was Francis Crick, who was still working on his Ph.D. Watson and Crick immediately became friends and colleagues.
Much of the basic chemistry of DNA had already been determined by Miescher, Kossel, Levene, Chargaff, and others, who had established that DNA consisted of nucleotides, and that each nucleotide contained a sugar, base, and phosphate group. However, how the nucleotides fit together in the three¬ dimensional structure of the molecule was not at all clear.
In 1947, William Ashbury began studying the three¬ dimensional structure of DNA by using a technique called X-ray diffraction, but his diffraction pic¬tures did not provide enough resolution to reveal the struc¬ture. A research group at King s College in London, led by Maurice Wilkins and Rosalind Franklin, also was studying the structure of DNA by using X-ray diffraction and obtained strikingly better pictures of the molecule. Wilkins and Franklin, however, were unable to develop a complete structure of the molecule; their progress was impeded by personal discord that existed between them.
Watson and Crick investigated the structure of DNA, not by collecting new data but by using all available infor¬mation about the chemistry of DNA to construct molecular models. By applying the laws of structural chemistry, they were able to limit the number of possible structures that DNA could assume. Watson and Crick tested various structures by building models made of wire and metal plates. With their models, they were able to see whether a structure was compatible with chemical princi¬ples and with the X-ray images.
The key to solving the structure came when Watson recognized that an adenine base could bond with a thymine base and that a guanine base could bond with a cytosine base; these pairings accounted for the base ratios that Chargaff had discovered earlier. The model developed by Watson and Crick showed that DNA consists of two strands of nucleotides wound around each other to form a right- handed helix, with the sugars and phosphates on the outside and the bases in the interior. They published an electrifying description of their model in Nature in 1953.










At the same time, Wilkins and Franklin published their X-ray diffraction data, which demonstrated experimentally the theory that DNA was helical in structure.
Many have called the solving of DNA’s structure the most important biological discovery of the twentieth cen¬tury. For their discovery, Watson and Crick, along with Maurice Wilkins, were awarded a Nobel Prize in 1962. (Rosalind Franklin had died of cancer in 1957 and, thus, could not be considered a candidate for the shared prize.)


Concepts)
By collecting existing information about the chemistry of DNA and building molecular models, Watson and Crick were able to discover the three-dimensional structure of the DNA molecule.

RNA As Genetic Material
In most organisms, DNA carries the genetic information. However, a few viruses utilize RNA, not DNA, as their genetic material. This fact was demonstrated in 1956 by Heinz Fraenkel-Conrat and Bea Singer, who worked with tobacco mosaic virus (TMV), a virus that infects and causes disease in tobacco plants. The tobacco mosaic virus pos¬sesses a single molecule of RNA surrounded by a helically arranged cylinder of protein molecules. Fraenkal-Conrat found that, after separating the RNA and protein of TMV, he could remix them and obtain intact, infectious viral particles.
With Singer, Fraenkal-Conrat then created hybrid viruses by mixing RNA and protein from different strains of TMV (< . 10.8). When these hybrid viruses infected tobacco leaves, new viral particles were produced. The new viral progeny were identical to the strain from which the RNA had been isolated and did not exhibit the characteris¬tics of the strain that donated the protein. These results showed that RNA carries the genetic information in TMV.
Also in 1956, Alfred Gierer and Gerhard Schramm demonstrated that RNA isolated from TMV is sufficient to infect tobacco plants and direct the production of new TMV particles, confirming that RNA carries genetic instructions.
Concepts
RNA serves as the genetic material in some viruses.

The Structure of DNA
DNA, though relatively simple in structure, has an elegance and beauty unsurpassed by other large molecules. It is use¬ful to consider the structure of DNA at three levels of increasing complexity, known as the primary, secondary, and tertiary structures of DNA. The primary structure of DNA refers to its nucleotide structure and how the nucleotides are joined together. The secondary structure refers to DNA’s stable three-dimensional configuration, the helical structure worked out by Watson and Crick.

The Primary Structure of DNA
The primary structure of DNA consists of a string of nucleotides joined together by phosphodiester linkages.
Nucleotides DNA is typically a very long molecule and is therefore termed a macromolecule. For example, within each human chromosome is a single DNA molecule that, if stretched out straight, would be several centimeters in length. In spite of its large size, DNA has a relatively simple structure: it is a polymer, a chain made up of many repeating units linked together. As already mentioned, the repeating units of DNA are nucleotides, each comprising three parts: (1) a sugar, (2) a phosphate, and (3) a nitrogen-containing base.
The sugars of nucleic acids called pentose sugars have five carbon atoms, numbered 1 , 2 , 3 , and so forth. Four of the carbon atoms are joined by an oxygen atom to form a five-sided ring; the fifth (5 ) carbon atom projects upward from the ring. Hydrogen atoms or hydroxyl groups (OH) are attached to each carbon atom.




The sugars of DNA and RNA are slightly different in structure. RNA’s ribose sugar has a hydroxyl group at¬tached to the 2 -carbon atom, whereas DNA’s sugar, called deoxyribose, has a hydrogen atom at this position and contains one oxygen atom fewer overall. This difference gives rise to the names ribonucleic acid (RNA) and de¬oxyribonucleic acid (DNA). This minor chemical differ¬ence is recognized by all the cellular enzymes that interact with DNA or RNA, thus yielding specific functions for each nucleic acid. Further, the additional oxygen atom in the RNA nucleotide makes it more reactive and less chem¬ically stable than DNA. For this reason, DNA is better suited to serve as the long-term repository of genetic information.
The second component of a nucleotide is its nitroge¬nous base, which may be of two types a purine or a pyrimidine. Each purine consists of a six-sided ring attached to a five-sided ring, whereas each pyrimidine consists of a six-sided ring only. DNA and RNA both contain two purines, adenine and guanine (A and G), which differ in the positions of their double bonds and in the groups attached to the six-sided ring. There are three pyrimidines found in nucleic acids: cytosine (C), thymine (T), and uracil (U). Cytosine is present in both DNA and RNA; however, thymine is restricted to DNA, and uracil is found only in RNA.

The three pyrimidines differ in the groups or atoms attached to the carbon atoms of the ring and in the number of double bonds in the ring. In a nucleotide, the nitrogenous base always forms a cova-lent bond with the 1 -carbon atom of the sugar. A deoxyribose (or ribose) sugar and a base together are referred to as a nucleoside.
The third component of a nucleotide is the phosphate group, which consists of a phosphorus atom bonded to four oxygen atoms. Phosphate groups are found in every nucleotide and frequently carry a negative charge, which makes DNA acidic. The phosphate is always bonded to the 5 -carbon atom of the sugar in a nucleotide.

The DNA nucleotides are properly known as deoxyri- bonucleotides or deoxyribonucleoside 5 -monophos- phates. Because there are four types of bases, there are four different kinds of DNA nucleotides. The equivalent RNA nucleotides are termed ribonu¬cleotides or ribonucleoside 5 -monophosphates. RNA molecules sometimes contain additional rare bases, which are modified forms of the four common bases.
Concepts
The primary structure of DNA consists of a string of nucleotides. Each nucleotide consists of a five- carbon sugar, a phosphate, and a base. There are two types of DNA bases: purines (adenine and guanine) and pyrimidines (thymine and cytosine).

Polynucleotide strands
DNA is made up of many nucleotides connected by covalent bonds, which join the 5 -phosphate group of one nucleotide to the 3 -carbon atom of the next nucleotide ( . 10.13). These bonds, called phosphodiester linkages, are relatively strong cova¬lent bonds; a series of nucleotides linked in this way consti¬tutes a polynucleotide strand. The backbone of the polynucleotide strand is composed of alternating sugars and phosphates; the bases project away from the long axis of the strand. The negative charges of the phosphate groups are frequently neutralized by the association of positive charges on proteins, metals, or other molecules.
An important characteristic of the polynucleotide strand is its direction, or polarity. At one end of the strand a phosphate group is attached only to the 5 -carbon atom of the sugar in the nucleotide. This end of the strand is there¬fore referred to as the 5 end. The other end of the strand, referred to as the 3 end, has an OH group attached to the 3 -carbon atom of the sugar. RNA nucleotides also are connected by phosphodiester linkages to form similar polynucleotide strands.

The nature of the hydrogen bond imposes a limita¬tion on the types of bases that can pair. Adenine normally pairs only with thymine through two hydrogen bonds, and cytosine normally pairs only with guanine through three hydrogen bonds. Because three hydrogen bonds form between C and G and only two hydrogen bonds form between A and T, C - G pairing is stronger than A - T pairing. The specificity of the base pairing means that wherever there is an A on one strand, there must be a T in the corresponding position on the other strand, and wherever there is a G on one strand, a C must be on the other. The two polynucleotide strands of a DNA molecule are therefore not identical but are complementary.
The second force that holds the two DNA strands together is the interaction between the stacked base pairs. These stacking interactions contribute to the stability of the DNA molecule and do not require that any particular base follow another. Thus, the base sequence of the DNA molecule is free to vary, allowing DNA to carry genetic information.
Concepts
DNA consists of two polynucleotide strands. The sugar-phosphate groups of each polynucleotide strand are on the outside of the molecule, and the bases are in the interior. Hydrogen bonding joins the bases of the two strands: guanine pairs with cytosine, and adenine pairs with thymine. The two polynucleotide strands of a DNA molecule are complementary and antiparallel.

Secondary Structures of DNA
Different secondary structures as we have seen, DNA normally consists of two polynucleotide strands that are antiparallel and complementary (exceptions are single¬ stranded DNA molecules in a few viruses). The precise three-dimensional shape of the molecule can vary, however, depending on the conditions in which the DNA is placed and, in some cases, on the base sequence itself.
The three-dimensional structure of DNA that Watson and Crick described is termed the B-DNA structure. This structure exists when plenty of water surrounds the molecule and there is no unusual base sequence in the DNA — conditions that are likely to be present in cells. The B-DNA structure is the most stable configuration for a random sequence of nucleotides under physiological conditions, and most evidence suggests that it is the predominate structure in the cell.
B-DNA is an alpha helix, meaning that it has a right¬ handed, or clockwise, spiral. It possesses approximately 10 base pairs (bp) per 360-degree rotation of the helix; so B-DNA consists of an alpha helix with approximately 10 bases per turn. (a) Diagrammatic representation showing that the bases are 0.34 nanometer (nm) apart, that each rotation encompasses 3.4 nm, and that the diameter of the helix is 2 nm.

Space-filling model of B-DNA showing major and minor grooves, each base pair is twisted 36 degrees relative to the adjacent bases. The base pairs are 0.34 nanome¬ter (nm) apart; so each complete rotation of the molecule encompasses 3.4 nm. The diameter of the helix is 2 nm, and the bases are perpendicular to the long axis of the DNA molecule. A space-filling model shows that B-DNA has a relatively slim and elongated structure. Spiraling of the nucleotide strands creates major and minor grooves in the helix, features that are important for the binding of some DNA-binding proteins that regulate the expression of genetic information. Some char¬acteristics of the B-DNA structure, along with characteris¬tics of other secondary structures that exist under certain conditions or with unusual base sequences.
Another secondary structure that DNA can assume is the A-DNA structure, which exists when less water is pre¬sent. Like B-DNA, A-DNA is an alpha (right-handed) helix, but it is shorter and wider than B-DNA and its bases are tilted away from the main axis of the molecule. There is little evidence that A-DNA exists under physiological conditions.
A radically different secondary structure called Z-DNA forms a left-handed helix. In this form, the sugar - phosphate backbones zigzag back and forth, giv¬ing rise to the name Z-DNA (for zigzag). Z-DNA structures can arise under physiological conditions when particular base sequences are present, such as stretches of alternating C and G sequences. Parts of some active genes form Z-DNA, suggesting that Z-DNA may play a role in regulat¬ing gene transcription. Other secondary structures may exist under special conditions or with special base sequences. Structures other than B-DNA exist rarely, if ever, within cells.

(Concepts)
DNA can assume different secondary structures depending on the conditions in which it is placed and on its base sequence. B-DNA is thought to be the most common configuration in the cell. Local variation in DNA arises as a result of environmental factors and base sequence.

Genetic Implications of DNA Structure
After Oswald Avery and his colleagues demonstrated that the transforming principle is DNA, it was clear that the genotype resides within the chemical structure of DNA. Watson and Crick’s great contribution was their elucida¬tion of the genotype’s chemical structure, making it possi¬ble for geneticists to begin to examine genes directly, in¬stead of looking only at the phenotypic consequences of gene action. Determining the structure of DNA permitted the birth of molecular genetics the study of the chemi¬cal and molecular nature of genetic information.
Watson and Crick’s structure did more than just cre¬ate the potential for molecular genetic studies; it was an immediate source of insight into key genetic processes. At the beginning of this chapter, three fundamental proper¬ties of the genetic material were identified. First, it must be capable of carrying large amounts of information; so it must vary in structure. Watson and Crick’s model sug¬gested that genetic instructions are encoded in the base sequence, the only variable part of the molecule. The sequence of the four bases adenine, guanine, cytosine, and thymine along the helix encodes the information that ultimately determines the phenotype. Watson and Crick were not sure how the base sequence of DNA deter¬mined the phenotype, but their structure clearly indicated that the genetic instructions were encoded in the bases.
A second necessary property of genetic material is its ability to replicate faithfully. The complementary polynu¬cleotide strands of DNA make this replication possible. Watson and Crick wrote, “It has not escaped our attention that the specific base pairing we have postulated immedi-ately suggests a possible copying mechanism for the genetic material.” They proposed that, in replication, the two strand, they can pair and form a hairpin (. 10.17a). A hairpin consists of a region of paired bases (the stem) and sometimes includes intervening unpaired bases (the loop). When the comple¬mentary sequences are contiguous, the hairpin has a stem but no loop (. 10.17b). Hairpins frequently control aspects of information transfer. RNA molecules may contain numerous hairpins, allowing them to fold up into complex structures.
In double-stranded DNA, sequences that are inverted replicas of each other are called inverted repeats. The fol¬lowing double-stranded sequence is an example of inverted repeats:
5 -AAAG . . . CTTT-3
3 -TTTC . . . GAAA-5
Notice that the sequences on the two strands are the same when read from 5 to 3 but, because the polarities of the two strands are opposite, their sequences are reversed from left to right. An inverted repeat that is complementary to itself, such as:
5 - ATCGAT - 3
3 - TAGCTA - 5
is also a palindrome, defined as a word or sentence that reads the same forward and backward, such as “rotator.” Inverted repeats are palindromes because the sequences on the two strands are the same but in reverse orientation. When an inverted repeat forms a perfect palindrome, the double-stranded sequence reads the same forward and backward.
Another secondary structured, called a cruciform, can be made from an inverted repeat when a hairpin forms within each of the two single-stranded sequences.
Concepts
In DNA and RNA, base pairing between nucleotides on the same strand produces special secondary structures such as hairpins and cruciform.
DNA Methylation
The primary structure of DNA can be modified in various ways. These modifications are important in the expression of the genetic material, as we will see in the chapters to come. One such modification is DNA methylation, in which methyl groups (-CH3) are added (by specific en¬zymes) to certain positions on the nucleotide bases.
In eukaryotic DNA, cytosine bases are often methy¬lated to form 5-methylcytosine. The ex¬tent of cytosine methylation varies; in most animal cells, about 5% of the cytosine bases are methylated, but more than 50% of the cytosine bases in some plants are methy¬lated. On the other hand, no methylation of cytosine has been detected in yeast cells, and only very low levels of methylation (about 1 methylated cytosine base per 12,500 nucleotides) are found in Drosophila. Why eukaryotic or¬ganisms differ so widely in their degree of methylation is not clear.
Methylation is most frequent on cytosine nucleotides that sit next to guanine nucleotides on the same strand:
. . . GC . . .
. . . CG . . .
In eukaryotic cells, methylation is often related to gene expression. Sequences that are methylated typically show low levels of transcription while sequences lacking methyla- tion are actively being transcribed. Methylation can also affect the three-dimensional structure of the DNA molecule.
Concepts
Methyl groups may be added to certain bases in DNA, depending on their positions in the molecule. Both prokaryotic and eukaryotic DNA can be methylated. In eukaryotes, cytosine bases are most often methylated to form 5- methylcytosine, and methylation is often related to gene expression.

Bends in DNA
Some specific base sequences — such as a series of four or more adenine - thymine base pairs — cause the DNA dou¬ble helix to bend. Bending affects how the DNA binds to certain proteins and may be important in controlling the transcription of some genes.
The DNA helix can also be made to bend by the binding of proteins to specific DNA sequences. The SRY protein, which is encoded by a Y-linked gene and is responsible for sex determination in mam¬mals, binds to certain DNA sequences (along the minor groove) and activates nearby genes that encode male traits. When the SRY protein grips the DNA, it bends the molecule about 80 degrees. This distortion of the DNA helix apparently facilitates the binding of other proteins that activate the transcription of genes that en¬code male characteristics.
This chapter has shifted the focus of our study to molecu¬lar genetics. The first nine chapters of this book examined various aspects of transmission genetics. In these chapters, the focus was on the individual: which phenotype was produced by an individual genotype, how the genes of an individual were transmitted to the next generation, and what types of offspring were produced when two individ¬uals were crossed. In molecular genetics, our focus now shifts to genes: how they are encoded in DNA, how they are replicated, and how they are expressed.
Much of what follows in this book will depend on your knowledge of DNA. An understanding of all the major processes of information transfer — replication, transcription, and translation — requires an understand¬ing of nucleic acid structure; discussions of recombinant DNA, mutation, gene expression, cancer genetics, and even population genetics are based on the assumption that you understand the basic structure and function of DNA. Thus the information in this chapter provides a critical foundation for much of the remainder of the book.
In this chapter, the history of how DNA’s structure and function were unraveled has been strongly empha¬sized, because the DNA story illustrates how pivotal scientific discoveries are often made. No one scientist dis¬covered the structure of DNA; rather, numerous persons, over a long period of time, made important contributions to our understanding of its structure. Watson and Crick’s proposal for DNA’s double-helical structure stands out as a singularly important contribution, because it combined many known facts about the structure into a new model that allowed important inferences about the fundamental nature of genes. The DNA story also illustrates the impor¬tant lesson that science is a human enterprise, influenced by personalities, relations, and motivation.