The union of genetics and cytology in the early 1900s was an exceedingly productive time. Heredity was based in genes, which were located on chromosomes. But what are genes made of? To some scientists, genes were almost unreal, a mental construct to explain real phenomena. We now know, of course, that genes are made of DNA. In fact, DNA was discovered decades before its significance was understood. The recognition of DNA as the genetic material, and the solution of its chemical and three-dimensional structure, brought genetics out of the realm of imagination and into the realm of chemistry. These discoveries sparked the fusion of chemistry and genetics to give us an entirely new scientific discipline: molecular genetics or, more generally, molecular biology. In this section, we outline some discoveries that led to our current understanding of DNA as the repository of biological information. We also describe how the information in DNA is translated into functional RNAs and proteins, and how this knowledge furthers our understanding of human health and disease.

Deoxyribonucleic acid (DNA), as we have noted, was identified long before its importance was recognized. The history begins with Friedrich Miescher, who carried out the first systematic chemical studies of cell nuclei in 1868. Miescher obtained white blood cells from pus that he collected from discarded surgical bandages. He carefully isolated the nuclei and then ruptured the nuclear membranes, releasing an acidic, phosphorus-containing substance that he called nuclein. Nuclein, a nucleic acid, was a new type of chemical polymer, different from all others previously identified. Around the turn of the century, Albrecht Kossel investigated the chemical structure of nucleic acids—both DNA and a similar molecule called ribonucleic acid (RNA)—and found that they contain nitrogenous bases, or nitrogen-containing basic compounds. Kossel identified five types of nitrogenous bases: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) (described in Chapters 3 and 6). By 1910, other investigators had determined that nitrogenous bases were but one component of a larger unit called a nucleotide, which consists of a phosphate group, a pentose sugar, and a nitrogenous base. DNA is composed of nucleotides that contain the bases A, G, C, or T, and RNA is composed of nucleotides with the bases A, G, C, or U.

By the 1920s, the chemical basis of heredity was thought to lie in chromosomes, but chromosomes are composed of both DNA and protein. Which one is the hereditary chemical? DNA was initially ruled out because it was believed to be too simple—just a repeating polymer of four different nucleotides. Surely such a monotonous molecule lacked the complexity to code for the working apparatus of a living cell! Attention turned to the other biopolymer, protein, for a chemical explanation of heredity. Only after biochemical studies conducted in the 1940s pointed to DNA as the genetic molecule was attention refocused on the structure and function of this molecule.

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DNA was shown to be the chemical of heredity in the 1940s, by Oswald T. Avery and his colleagues at the Rockefeller Institute in New York City. Their starting point was an observation made in 1928 by English microbiologist Frederick Griffith, who studied the pneumonia-causing bacterium Streptococcus pneumoniae. This pneumococcus exists as two types, virulent (disease-causing) and nonvirulent. Griffith noticed that virulent bacteria produced smooth colonies when grown on Petri plates, but colonies of a nonvirulent strain appeared rough. The difference in appearance lies in a polysaccharide capsule coat present only on the virulent strains. Griffith found that heat-killed virulent bacteria transformed live nonvirulent bacteria into live virulent bacteria (Figure 2-22a–d). The nonvirulent bacteria somehow acquired the smooth-colony trait from the heat-killed bacteria. The results suggested that the genetic material coding for capsules remained intact even after the virulent (smooth-colony) bacteria were killed, and this material could enter another cell and recombine with its genetic material.

Avery and his colleagues reproduced Griffith’s results, and they analyzed the heat-killed virulent bacterial extract for the chemical nature of the transforming factor. They selectively removed either DNA, RNA, or protein from the heat-killed virulent bacterial extract by treatment with DNase, RNase, or proteases (enzymes that specifically break down one of these cellular components, leaving the others intact). The DNase-treated extract lost the capacity to transform nonvirulent rough-colony cells into a virulent smooth-colony strain. The researchers then extracted DNA from virulent bacteria, purified it of contaminating proteins and RNA, and showed that this pure DNA was still capable of transforming nonvirulent bacteria into the virulent strain (Figure 2-22e). In 1944, Avery and colleagues reported their surprising conclusion that DNA was the carrier of genetic information. Another classic experiment, by Alfred Hershey and Martha Chase, supported this conclusion that DNA is the chemical of heredity (see the How We Know section at the end of this chapter).

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DNA and protein are chemically very different, and it was puzzling how a DNA sequence could code for a protein sequence. Regardless of the details, however, it now became easy to understand that mutations in a gene could lead to altered enzymes. In fact, even before the DNA structure was solved, the relationship between genes, mutations, and enzymes was well understood.

In 1902, the physician Archibald Garrod studied patients with alkaptonuria, a disease of little consequence for the patients, except that they excreted urine that turned black. Mendel’s work had recently been rediscovered, and by noticing how alkaptonuria was inherited, Garrod realized that this disorder behaved as a recessive trait. It was already known that the synthesis and breakdown of biomolecules occur in multistep pathways, each step requiring a different enzyme—a protein catalyst that facilitates the reaction. Garrod hypothesized that alkaptonuria was caused by a mutation that inactivated a gene required for the production of one enzyme in a metabolic pathway. Without this functional enzyme, the pathway was blocked, resulting in the buildup of an intermediate compound, which was excreted in the urine and turned black. Garrod’s reasoning drew the connection between a mutation in a gene and a mutation in an enzyme.

Formal proof that genes encode enzymes came from a series of elegant experiments in the 1940s by George Beadle and Edward L. Tatum. They introduced a new microorganism into the study of genetics: the bread mold, Neurospora crassa (see the Model Organisms Appendix). This haploid organism can grow on a simple, defined medium, called minimal medium. Minimal medium contains sugar, nitrogen, inorganic salts, and biotin, and the cell must make all the rest of the biochemicals that it needs to live from these simple starting compounds. Beadle and Tatum irradiated Neurospora spores to intentionally produce mutations, then germinated individual spores on a complete medium (i.e., one made with cell extracts that have all the necessary amino acids, nucleotides, and vitamins) to obtain genetically pure colonies and their spores. These spores were then tested for their ability to germinate on the minimal medium. An inability to grow on minimal medium indicates a mutation in one of the metabolic pathways required for growth. These mutants are called auxotrophs. Spores of different auxotrophs were then analyzed for growth on a range of minimal media supplemented with selected compounds, to identify the defective metabolic pathways and the steps affected. An example of one such study is illustrated in Figure 2-23, for auxotrophs of arginine metabolism.

Beadle and Tatum had a collection of Neurospora mutants that were auxotrophic for the amino acid arginine. The arginine synthetic pathway was known to include the intermediate compounds ornithine and citrulline, so they tested their arginine auxotrophs for growth on minimal media containing ornithine, citrulline, or arginine. The arginine auxotrophs fell into three classes, depending on which intermediate(s) they required for growth (see Figure 2-23). Beadle and Tatum also mapped the mutant genes and found that mutants in a particular class of auxotrophs mapped to the same chromosomal location. They concluded that each class of mutant was caused by a single defective gene. Their findings also held true for genes in other metabolic pathways.

On the basis of these experiments, Beadle and Tatum proposed the one gene, one enzyme hypothesis, which stated that each gene codes for one enzyme. We now know that some enzymes are composed of multiple subunits encoded by different genes; furthermore, not all proteins are enzymes. So, the hypothesis was later revised to one gene, one polypeptide. A polypeptide is a chain of amino acids, and a functional protein can be composed of a single polypeptide or multiple polypeptide subunits. For a large number of genes, one gene, one polypeptide holds true. But as we will see throughout this textbook, even this hypothesis is not entirely accurate. Some genes code for functional RNAs rather than for protein. And through a process called alternative splicing (see Chapter 16), some genes code for more than one type of polypeptide.

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Watson and Crick’s determination of DNA structure was a turning point in understanding how information flows in biological systems. Their model of DNA structure, which they reasoned from data collected by other scientists—most notably Rosalind Franklin—consists of two strands of DNA wound about one another in a spiral, double helix. Each strand is composed of a long string of the four nucleotides containing the bases adenine (A), guanine (G), cytosine (C), and thymine (T). The nucleotides in one strand pair with those in the other. Because A pairs only with T, and G pairs only with C, the sequence of each strand contains information about the sequence of the other, and the two strands are said to be complementary. The A–T and G–C pairs are referred to as base pairs. The detailed structure of DNA and the nucleotide bases, how the nucleotides base-pair in a specific way, and the critical contributions Rosalind Franklin made to Watson and Crick’s structure are described in Chapter 6.

The double-helical DNA structure immediately suggested a mechanism for the transmission of genetic information. The essential feature of the model is the complementarity of the two DNA strands. As Watson and Crick realized well before confirmatory data became available, DNA could logically be replicated by separating the two strands and using each as a template to synthesize a new, complementary strand, thereby generating two new DNA duplexes that are identical to each other and to the original double-stranded DNA.

With discovery of the DNA structure, genetics could now be described in chemical terms. Both DNA and proteins are linear polymers, so the sequence of nucleotides in DNA must somehow be converted to a sequence of amino acids. But DNA is located in the nucleus, whereas proteins are synthesized in the cytoplasm. Therefore, there must be an intermediary molecule to shuttle information between the two locations. RNA was believed to play a role in this, and the similarities between DNA and RNA made it a simple matter to understand how an RNA molecule could be made from a DNA template. Crick proposed that biological information flows in the direction DNA→RNA→protein and that DNA acts as a template for its own synthesis (DNA→DNA) (Figure 2-24). Crick’s proposal is known as the central dogma of information flow.

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Over the years, it has become obvious that the nice and tidy linear flow of information in Crick’s central dogma is not really all that simple after all. Several different paths of information flow are now known to exist. Among these different pathways are the ability of some enzymes to synthesize DNA from RNA (RNA→DNA) and the ability of some viruses to use RNA as a template to make more RNA (RNA→RNA). But the most profound change in what we know about information flow is the finding that the cell makes a huge amount of RNA that is not translated into protein, and it is not just tRNA and rRNA (whose functions we describe below). Indeed, much of the mammalian genome is transcribed into RNA that does not code for protein. We discuss this topic briefly at the end of this section.

RNA was widely expected to be the molecule that mediates the transfer of information from DNA in the nucleus to the site of protein synthesis in the cytoplasm. However, no one imagined that three different types of RNA would be required for the process.

Ribosomal RNA In the early 1950s, Paul Zamecnik and his colleagues identified the site of protein synthesis as particles in the cytoplasm called ribosomes. Ribosomes are large structures composed of both protein and RNA. The RNA component is called ribosomal RNA (rRNA). In bacteria and eukaryotes, ribosomes consist of a large subunit and a small subunit.

Messenger RNA The combined findings that ribosomes are the site of protein synthesis and that rRNA is the most abundant RNA (>80%) in the cell led most researchers to believe that rRNA was the carrier of information from DNA to protein. However, some features of rRNA are incompatible with its function as an information carrier. For example, rRNA is an integral part of the ribosome, so there would have to be specific ribosomes to make each specific protein. Further, the nucleotide composition of rRNAs from different organisms was relatively constant, whereas the nucleotide composition of chromosomal DNA varied considerably from one organism to the next.

Studies by Sydney Brenner, Jacques Monod, and Matthew Meselson in the early 1960s, using Escherichia coli (see the Model Organisms Appendix), suggested that another type of RNA carries the message from DNA to protein. They discovered a class of RNA that targets preexisting ribosomes, and the nucleotide composition of this RNA was more similar to chromosomal DNA than was rRNA. These properties are exactly those expected for a true messenger between DNA and protein. The investigators called this RNA messenger RNA (mRNA) and concluded that ribosomes are protein-synthesizing factories that use mRNA as a template to direct construction of the protein sequence.

RNA synthesis is carried out by the enzyme RNA polymerase, which synthesizes RNA by reading one strand of the duplex DNA, pairing RNA bases to the bases in the DNA strand, to synthesize a single-stranded RNA molecule that has a sequence directed by the DNA sequence (Figure 2-25). This process of making single-stranded RNA copies of a DNA strand is known as transcription.

Transfer RNA The discovery of mRNA was a crucial piece of the information puzzle. But a problem remained: how is a sequence of nucleotides in mRNA converted to a sequence of amino acids in protein? Furthermore, DNA and RNA each consist of only four different nucleotides, whereas proteins have 20 different amino acids. Hence, one must assume the existence of a code that uses combinations of nucleotides to specify amino acids. Combinations of two nucleotides yield only 16 permutations (4²). Combinations of three nucleotides yield 64 permutations (4³), more than enough to specify a code for 20 amino acids.

In 1955, Crick hypothesized the existence of an adaptor molecule, perhaps a small RNA, that could read three nucleotides and also carry amino acids. It was not long after Crick’s adaptor hypothesis (see Chapter 17) that Paul Zamecnik and Mahlon Hoagland discovered a small RNA to which amino acids could attach. This small RNA, later called transfer RNA (tRNA), was the adaptor between nucleic acid and protein.

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The discovery of tRNA, combined with the idea of a three-letter code, suggested how the DNA sequence could be converted to an amino acid sequence. Three bases in the tRNA form base pairs with a triplet sequence in the mRNA. When two amino acid–linked tRNAs align side-by-side on the mRNA by base-pairing to adjacent triplets, the amino acids attached to the tRNAs can be joined together. By continuing this process over the length of an mRNA strand, amino acids carried to the mRNA by tRNAs become connected together in a linear order specified by the mRNA sequence. These connections occur as the mRNA-tRNA complexes thread through the ribosome. The overall process of protein synthesis, involving three different types of RNA molecules, is known as translation (Figure 2-26). (Translation is covered in detail in Chapter 18.)

Functional RNAs All RNAs, whether they code for protein or not, are transcribed from DNA genes. Messenger RNA is needed only transiently, to instruct the synthesis of proteins. But the end products of tRNA and rRNA genes are the RNA molecules themselves. These functional RNAs fold into specific three-dimensional shapes and constitute the majority of the RNA in a cell.

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There is an abundance of other functional RNAs besides tRNA and rRNA, some of which have known functions. For example, some small nuclear RNAs (<150 nucleotides) associate with protein to form ribonucleoprotein particles that process the introns from mRNAs (see Chapters 16 and 22). Other types of small RNA, the microRNAs (miRNA) and the short interfering RNAs (siRNA), have important gene regulatory functions. MicroRNAs anneal to particular mRNAs, usually causing their degradation and thereby effectively turning off, or silencing, the gene (see Chapter 22). Perhaps the most mysterious of the non-protein-coding RNAs are the “long noncoding RNAs.” These RNAs (>200 nucleotides) are not translated and have no known function, yet they are more abundant than translated mRNA. There is accumulating evidence that at least some members of this abundant class of RNA may be needed for proper cell function. Identification and understanding of the function of these new RNAs is a fast-paced field, and new types of RNA are almost certain to be discovered in the near future.

There are yet other types of information flow, besides the use of RNAs, that fall outside the classic central dogma. Notable among these is the epigenetic control of gene regulation, based in specific chemical modifications of particular nucleotides and of the proteins that package DNA (in structures called nucleosomes). Combinations of these chemical changes can program the transcriptional control of a cell and can be inherited in cell divisions during an organism’s development (see Chapter 10). This epigenetic inheritance falls outside the domain of DNA sequence. There are also many other types of protein modifications that transduce the flow of information within the cell and from one cell to another. Suffice it to say that the new dogma is that there is no simple “central dogma.” The flow of information is so vital to life and evolution that it takes many forms, some hard to recognize, and scientists have no shortage of work ahead of them to elucidate these important mechanisms.

Most cellular functions are carried out by proteins. The precise sequence of amino acids in each protein molecule and the specific rules governing the timing and quantity of its production are programmed into an organism’s DNA. When changes in the DNA sequence occur, cellular function can be altered. Mutations in DNA can be beneficial or harmful to an organism, or can have no effect at all. For example, if the mutation does not change the sequence of a protein or how the protein is regulated, the mutation has no effect and is said to be silent. Evolution depends on mutations that are beneficial, and these usually alter the sequence or regulation of a protein in a way that enhances its function or confers a new, beneficial function that increases the viability of the organism. However, most mutations that change a protein sequence are harmful, because they lead to altered proteins with decreased function or new, detrimental function, and give rise to various diseases. When these DNA mutations occur in germ-line cells (cells that give rise to gametes), the disease can be inherited. There are many examples of inherited diseases, some of which have altered the course of history. One such disease is hemophilia.

Hemophilia afflicted the interrelated royal families of England, Russia, Spain, and Prussia in the 1800s. At the root of this malady is an inability of the blood to clot, resulting in excessive bleeding from even the slightest injury. The disease typically results in death at an early age. Tracing hemophilia through the royal families of Europe indicates that it originated with Queen Victoria (Figure 2-27a). It is interesting that none of the current family members are carriers, presumably the result of natural selection against this trait.

Hemophilia is about 10,000 times more common in males than in females. This is because the blood-clotting factor involved in 90% of cases of this disease is factor VIII, encoded by a gene on the X chromosome. A mutant recessive allele of the factor VIII gene is responsible for hemophilia A, the most common form of the disease. Males have only one copy of the X chromosome, and the recessive allele, when inherited, is always expressed. Females have two X chromosomes, and if one X contains a wild-type allele, it masks the expression of the mutant recessive allele. A female with only one copy of the recessive allele is called a carrier, because she is phenotypically normal but may pass on this allele to her offspring (Figure 2-27b).

Many other inherited diseases have been mapped to their particular genes. One of the first to be identified was the gene involved in Huntington disease (Figure 2-28). The gene, HTT, is located on chromosome 4. The disease is associated with a region of the HTT gene that can have a variable number of repeats of the triplet nucleotide sequence CAG (encoding the amino acid glutamine). The HTT gene in healthy individuals has about 27 or fewer of these repeats, but when the number exceeds 36, it is often associated with disease. The likelihood of having Huntington disease increases with the number of trinucleotide repeats in the HTT gene. The function of the protein encoded by HTT is unknown, but the disease results in the degeneration of neurons in areas of the brain that affect motor coordination, memory, and cognitive function.

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The number of triplet repeats in HTT can increase during gamete production, resulting in earlier onset and increased severity of the disease over successive generations. This is thought to occur by template slippage (the same segment of DNA replicated more than once) during DNA synthesis due to the repetitive nature of the sequence. Other diseases caused by “triplet expansion” of this type have now been identified. These include Kennedy disease, spinocerebellar ataxia, and Machado-Joseph disease, all caused by an increase in CAG repeats. The CGG repeat is associated with fragile X syndrome, a neurological disorder; expansion of the CTG repeat is associated with myotonic dystrophy, a muscular wasting disease. In these triplet repeat mutations, it is not the sequence of the repeat that matters; rather, it is the disruptive effect of the iterative amino acid they encode within the sequence of the expressed protein that causes the disease.

Cystic fibrosis is another genetic disease that has been identified at the molecular level. The gene (CFTR) is on chromosome 7 and encodes a chloride channel protein, the cystic fibrosis transmembrane conductance regulator (M_r 168,173). The protein contains five domains: two domains that span the cytoplasmic membrane for chloride transport; two domains that bind and use ATP, the energy that fuels transport of the chloride ions; and a regulatory domain (Figure 2-29). The most common mutation (occurring in about 60% of cases) is CFTRΔF508, in which three nucleotides are deleted (denoted by Δ), resulting in the deletion of phenylalanine (denoted by F) at position 508 in the amino acid sequence. This amino acid residue is located in the first of the ATP-binding domains, and its deletion prevents proper folding of the protein. Many other mutations in CFTR have also been discovered. CFTR mutations are most prevalent in Caucasians from Northern Europe.

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The ΔF508 mutation in CFTR is autosomal recessive, and therefore an individual must inherit two copies of the mutant allele to develop cystic fibrosis, one from each parent. Without functional CFTR chloride channels, individuals with cystic fibrosis develop abnormally high sweat and mucus production, and a major complication is the buildup of mucus in the lungs. Patients experience breathing difficulties and often have pneumonia. Individuals with cystic fibrosis have typically had an average life span of about 30 years, but as new treatments are developed, survival is increasing greatly.

Although many mutations are detrimental, other mutations can be beneficial. For example, the protein CCR5 is a coreceptor for HIV, the AIDS virus. There is much speculation about a 32 amino acid deletion mutation of CCR5 (due to the CCR5Δ32 mutation), which is widely dispersed among people of European descent (an occurrence of 5% to 14% in these groups), although much rarer among Asians and Africans. Researchers speculate that this mutation may have conferred resistance to the bubonic plague or smallpox, thereby becoming enriched in the population, by natural selection, in endemic areas. Although the allele has a negative effect on T-cell (a type of immune cell) function, it seems to provide protection against HIV infection, as well as smallpox.

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Another example of a mutation that confers some benefit is the one that causes sickle-cell anemia (Highlight 2-1), a mutation of hemoglobin. When the mutation is inherited from both parents, the result is misshapen red blood cells that can get stuck in capillaries and impede blood flow, with possibly fatal results. However, people who are heterozygous for this mutation have enhanced resistance to malaria. Geographic areas where this mutation is prevalent in the population correlate with locations that are plagued by malaria.

Discovery of DNA as the hereditary material, and the understanding of how it is transcribed and translated into RNA and protein, is a most fascinating story in science. Darwin’s theory of the origin of species through evolution by natural selection was compelling, but the mechanism that drove the variation on which natural selection could act remained a mystery in his lifetime. Yet, the key to understanding this mystery had already been discovered by Mendel. Mutations create the natural variation needed for the forces of natural selection to mold new species. It seems almost ludicrous that Mendel and Darwin were alive at the same time and separately uncovered secrets that together explained the diversity of planetary life. Lack of a robust means of communication kept these two vital pieces of information segregated for decades—an improbable situation today, given the rapid pace of global communication. Although most mutations are deleterious, the rare mutation that carries a beneficial change eventually enters the population through natural selection over the expanse of evolutionary time. Natural selection still drives change and the evolution of new species today.

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