Bronze sculpture of the Watson–Crick model for double-helical DNA. ONLY 10 DNA Structure and Analysis CHAPTER CONCEPTS many complex processes that lead to an organism’s adult form. ■ Except in some viruses, DNA serves as the genetic Until 1944, it was not clear what chemical compo- material in all living organisms on Earth. nent of the chromosome makes up genes and constitutes ■ According to the Watson–Crick model, DNA exists in the genetic material. Because chromosomes were known the form of a right-handed double helix. to have both a nucleic acid and a protein component, both ■ The strands of the double helix are antiparallel and were candidates. In 1944, however, direct experimental evi- are held together by hydrogen bonding between dence emerged showing that the nucleic acid DNA serves complementary nitrogenous bases. as the informational basis for the process of heredity. ■ The structure of DNA provides the means of storing Once the importance of DNA to genetic processes was and expressing genetic information. realized, work was intensified with the hope of discerning ■ RNA has many similarities to DNAREVIEW but exists mostly as not only the structure of this molecule but also the relation- a single-stranded molecule. ship of its structure to its function. Between 1944 and 1953, many scientists sought information that might answer the ■ In some viruses, RNA serves as the genetic material. most significant and intriguing question in the history of ■ Many techniques have been developed that facilitate biology: How does DNA serve as the genetic basis for living the analysis of nucleic acids, most based on detection processes? Researchers believed the answer must depend of the complementarity of nitrogenous bases. strongly on the chemical structure of the DNA molecule, given the complex but orderly functions ascribed to it. p to this point in the text, we have described chro- These efforts were rewarded in 1953, when James mosomes as containing genes that control pheno- Watson and Francis Crick put forth their hypothesis for FORtypic traits transmitted through gametes to future the double-helical nature of DNA. The assumption that Uoffspring. Logically, genes must contain some sort of infor- the molecule’s functions would be easier to clarify once mation that, when passed to a new generation, influences its general structure was determined proved to be correct. the form and characteristics of each individual. We refer to In this chapter, we first review the evidence that DNA is that information as the genetic material. Logic also sug- the genetic material and then discuss the elucidation of its gests that this same information in some way directs the structure. We conclude the chapter with a discussion of 231 M10_KLUG8915_11_C10_pp231-260.indd 231 21/03/14 4:06 PM 232 10 DNA STRUCTURE AND ANALYSIS various analytical techniques useful during the investiga- DNA tion of the nucleic acids, DNA and RNA. Please note that some of the topics discussed in this chapter are explored in greater depth later in the text (see Transcription Special Topic Chapter 2—Emerging Roles of RNA). mRNA rRNA tRNA 10.1 The Genetic Material Must Exhibit Four Characteristics Ribosome For a molecule to serve as the genetic material, it must exhibit four crucial characteristics: replication, storage of Translation information, expression of information, and variation by mutation. Replication of the genetic material is one facet of the cell cycle and as such is a fundamental property of all living organisms. Once the genetic material of cells repli- Protein cates and is doubled in amount, it must then be partitioned FIGURE 10–1 Simplified diagram of information flow (the cen- equally—through mitosis—into daughter cells. The genetic tral dogma) from DNA to RNA to produceONLY the proteins within cells. material is also replicated during the formation of gametes, but is partitioned so that each cell gets only one-half of the original amount of genetic material—the process of meiosis translation, the chemical information in mRNA directs the (discussed in Chapter 2). Although the products of mitosis construction of a chain of amino acids, called a polypeptide, and meiosis are different, these processes are both part of which then folds into a protein. Collectively, these processes the more general phenomenon of cellular reproduction. serve as the foundation for the central dogma of molecular Storage of information requires the molecule to act as genetics: “DNA makes RNA, which makes proteins.” a repository of genetic information that may or may not be The genetic material is also the source of variabil- expressed by the cell in which it resides. It is clear that while ity among organisms, through the process of mutation. most cells contain a complete copy of the organism’s genome, If a mutation—a change in the chemical composition of at any point in time they express only a part of this genetic DNA—occurs, the alteration is reflected during transcrip- potential. For example, in bacteria many genes “turn on” in tion and translation, affecting the specific protein. If a response to specific environmental cues and “turn off ” when mutation is present in a gamete, it may be passed to future conditions change. In vertebrates, skin cells may display generations and, with time, become distributed in the pop- active melanin genes but never activate their hemoglobin ulation. Genetic variation, which also includes alterations genes; in contrast, digestive cells activate many genes specific of chromosome number and rearrangements within and to their function but do not activate their melanin genes. between chromosomes (as discussed in Chapter 8), pro- Inherent in the concept of storageREVIEW is the need for the vides the raw material for the process of evolution. genetic material to be able to encode the vast variety of gene products found among the countless forms of life on our planet. The chemical language of the genetic material must have the capability of storing such diverse informa- tion and transmitting it to progeny cells and organisms. 10.2 Until 1944, Observations Favored Expression of the stored genetic information is a com- Protein as the Genetic Material plex process that is the underlying basis for the concept of information flow within the cell (Figure 10–1). The initial The idea that genetic material is physically transmitted from event in this flow of information is the transcription of parent to offspring has been accepted for as long as the concept DNA, inFOR which three main types of RNA molecules are syn- of inheritance has existed. Beginning in the late nineteenth thesized: messenger RNA (mRNA), transfer RNA (tRNA), century, research into the structure of biomolecules progressed and ribosomal RNA (rRNA). Of these, mRNAs are trans- considerably, setting the stage for describing the genetic mate- lated into proteins, by means of a process mediated by the rial in chemical terms. Although proteins and nucleic acid tRNA and rRNA. Each mRNA is the product of a specific were both considered major candidates for the role of genetic gene and leads to the synthesis of a different protein. In material, until the 1940s many geneticists favored proteins. M10_KLUG8915_11_C10_pp231-260.indd 232 4/9/14 11:30 AM 10.3 EVIDENCE FAVORING DNA AS THE GENETIC MATERIAL WAS FIRST OBTAINED DURING THE STUDY OF BACTERIA AND BACTERIOPHagES 233 This is not surprising, since proteins were known to be both strains of the bacterium Diplococcus pneumoniae.* Some diverse and abundant in cells, and much more was known were virulent, that is, infectious, strains that cause pneu- about protein than about nucleic acid chemistry. monia in certain vertebrates (notably humans and mice), DNA was first studied in 1869 by a Swiss chemist, whereas others were avirulent, or noninfectious strains, Friedrich Miescher. He isolated cell nuclei and derived which do not cause illness. an acidic substance, now known to contain DNA, that he The difference in virulence depends on the presence called nuclein. As investigations of DNA progressed, how- of a polysaccharide capsule; virulent strains have this cap- ever, showing it to be present in chromosomes, the sub- sule, whereas avirulent strains do not. The nonencapsulated stance seemed to lack the chemical diversity necessary to bacteria are readily engulfed and destroyed by phagocytic store extensive genetic information. cells in the host animal’s circulatory system. Virulent bac- This conclusion was based largely on Phoebus A. Levene’s teria, which possess the polysaccharide coat, are not easily observations in 1910 that DNA contained approximately engulfed; they multiply and cause pneumonia. equal amounts of four similar molecules called nucleotides. The presence or absence of the capsule causes a visible Levene postulated incorrectly that identical groups of these difference between colonies of virulent and avirulent four components were repeated over and over, which was the strains. Encapsulated bacteria form smooth, shiny-surfaced basis of his tetranucleotide hypothesis for DNA structure. colonies (S) when grown on an agar culture plate; non- Attention was thus directed away from DNA, thereby favor- encapsulated strains produce rough colonies (R). Thus, ing proteins. However, in the 1940s, Erwin Chargaff showed virulent and avirulent strains are easily distinguished by that Levene’s proposal was incorrect when he demonstrated standard microbiological culture techniques. that most organisms do not contain precisely equal propor- Each strain of DiplococcusONLY may be one of dozens of dif- tions of the four nucleotides. We shall see later that the struc- ferent types called serotypes that differ in the precise chemical ture of DNA accounts for Chargaff’s observations. structure of the polysaccharide constituent of the thick, slimy capsule. Serotypes are identified by immunological techniques and are usually designated by Roman numerals. In the United States, types I and II are the most common in causing pneu- monia.