2 12 Molecular Genetics
wea25324_ch01_001-011.indd Page 5 10/19/10 12:03 PM user-f468 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile 1.2 Molecular Genetics Figure 1.5 Barbara McClintock. (Source: Bettmann Archive/Corbis.) Figure 1.6 Friedrich Miescher. (Source: National Library of Medicine.) himself). By the 1930s, other investigators found that the same rules applied to other eukaryotes (nucleus-containing organisms), including the mold Neurospora, the garden pea, maize (corn), and even human beings. These rules also apply to prokaryotes, organisms in which the genetic material is not confined to a nuclear compartment. 1.2 Physical Evidence for Recombination Barbara McClintock (Figure 1.5) and Harriet Creighton provided a direct physical demonstration of recombination in 1931. By examining maize chromosomes microscopically, they could detect recombinations between two easily identifiable features of a particular chromosome (a knob at one end and a long extension at the other). Furthermore, whenever this physical recombination occurred, they could also detect recombination genetically. Thus, they established a direct relationship between a region of a chromosome and a gene. Shortly after McClintock and Creighton performed this work on maize, Curt Stern observed the same phenomenon in Drosophila. So recombination could be detected both physically and genetically in animals as well as plants. McClintock later performed even more notable work when she discovered transposons, moveable genetic elements (Chapter 23), in maize. SUMMARY The chromosome theory of inheritance holds that genes are arranged in linear fashion on chromosomes. The reason that certain traits tend to be inherited together is that the genes governing these traits are on the same chromosome. However, recombination between two homologous chromosomes during meiosis can scramble the parental alleles to give nonparental combinations. The farther apart two genes are on a chromosome the more likely such recombination between them will be. 5 Molecular Genetics The studies just discussed tell us important things about the transmission of genes and even about how to map genes on chromosomes, but they do not tell us what genes are made of or how they work. This has been the province of molecular genetics, which also happens to have its roots in Mendel’s era. The Discovery of DNA In 1869, Friedrich Miescher (Figure 1.6) discovered in the cell nucleus a mixture of compounds that he called nuclein. The major component of nuclein is deoxyribonucleic acid (DNA). By the end of the nineteenth century, chemists had learned the general structure of DNA and of a related compound, ribonucleic acid (RNA). Both are long polymers— chains of small compounds called nucleotides. Each nucleotide is composed of a sugar, a phosphate group, and a base. The chain is formed by linking the sugars to one another through their phosphate groups. The Composition of Genes By the time the chromosome theory of inheritance was generally accepted, geneticists agreed that the chromosome must be composed of a polymer of some kind. This would agree with its role as a string of genes. But which polymer is it? Essentially, the choices were three: DNA, RNA, and protein. Protein was the other major component of Miescher’s nuclein; its chain is composed of links called amino acids. The amino acids in protein are joined by peptide bonds, so a single protein chain is called a polypeptide. Oswald Avery (Figure 1.7) and his colleagues demonstrated in 1944 that DNA is the right choice (Chapter 2). These investigators built on an experiment performed earlier by Frederick Griffith in which he transferred a genetic trait from one strain of bacteria to another. The trait was virulence, the ability to cause a lethal infection, wea25324_ch01_001-011.indd Page 6 6 10/19/10 12:03 PM user-f468 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 1 / A Brief History Figure 1.7 Oswald Avery. (Source: National Academy of Sciences.) (a) (b) Figure 1.8 (a) George Beadle; (b) E. L. Tatum. (Source: (a, b) AP/Wide World Photos.) and it could be transferred simply by mixing dead virulent cells with live avirulent (nonlethal) cells. It was very likely that the substance that caused the transformation from avirulence to virulence in the recipient cells was the gene for virulence, because the recipient cells passed this trait on to their progeny. What remained was to learn the chemical nature of the transforming agent in the dead virulent cells. Avery and his coworkers did this by applying a number of chemical and biochemical tests to the transforming agent, showing that it had the characteristics of DNA, not of RNA or protein. The Relationship Between Genes and Proteins The other major question in molecular genetics is this: How do genes work? To lay the groundwork for the answer to this question, we have to backtrack again, this time to 1902. That was the year Archibald Garrod noticed that the human disease alcaptonuria seemed to behave as a Mendelian recessive trait. It was likely, therefore, that the disease was caused by a defective, or mutant, gene. Moreover, the main symptom of the disease was the accumulation of a black pigment in the patient’s urine, which Garrod believed derived from the abnormal buildup of an intermediate compound in a biochemical pathway. By this time, biochemists had shown that all living things carry out countless chemical reactions and that these reactions are accelerated, or catalyzed, by proteins called enzymes. Many of these reactions take place in sequence, so that one chemical product becomes the starting material, or substrate, for the next reaction. Such sequences of reactions are called pathways, and the products or substrates within a pathway are called intermediates. Garrod postulated that an intermediate accumulated to abnormally high levels in alcaptonuria because the enzyme that would normally convert this intermediate to the next was defective. Putting this idea together with the finding that alcaptonuria behaved genetically as a Mendelian recessive trait, Garrod suggested that a defective gene gives rise to a defective enzyme. To put it another way: A gene is responsible for the production of an enzyme. Garrod’s conclusion was based in part on conjecture; he did not really know that a defective enzyme was involved in alcaptonuria. It was left for George Beadle and E. L. Tatum ( Figure 1.8 ) to prove the relationship between genes and enzymes. They did this using the mold Neurospora as their experimental system. Neurospora has an enormous advantage over the human being as the subject of genetic experiments. By using Neurospora, scientists are not limited to the mutations that nature provides, but can use mutagens to introduce mutations into genes and then observe the effects of these mutations on biochemical pathways. Beadle and Tatum found many instances where they could create Neurospora mutants and then pin the defect down to a single step in a biochemical pathway, and therefore to a single enzyme (see Chapter 3). They did this by adding the intermediate that would normally be made by the defective enzyme and showing that it restored normal growth. By circumventing the blockade, they discovered where it was. In these same cases, their genetic experiments showed that a single gene was involved. Therefore, a defective gene gives a defective (or absent) enzyme. In other words, a gene seemed to be responsible for making one enzyme. This was the one-gene/one-enzyme hypothesis. This hypothesis was actually not quite right for at least three reasons: (1) An enzyme can be composed of wea25324_ch01_001-011.indd Page 7 10/21/10 10:14 AM user-f494 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefiles 1.2 Molecular Genetics (a) 7 (b) Figure 1.11 (a) Matthew Meselson; (b) Franklin Stahl. (Sources: (a) Courtesy Dr. Matthew Meselson. (b) Cold Spring Harbor Laboratory Archives.) Activities of Genes Figure 1.9 James Watson (left) and Francis Crick. (Source: © A. Barrington Brown/Photo Researchers, Inc.) (a) (b) Figure 1.10 (a) Rosalind Franklin; (b) Maurice Wilkins. (Sources: (a) From The Double Helix by James D. Watson, 1968, Atheneum Press, NY. © Cold Spring Harbor Laboratory Archives. (b) Courtesy Professor M. H. F. Wilkins, Biophysics Dept., King’s College, London.) more than one polypeptide chain, whereas a gene has the information for making only one polypeptide chain. (2) Many genes contain the information for making polypeptides that are not enzymes. (3) As we will see, the end products of some genes are not polypeptides, but RNAs. A modern restatement of the hypothesis would be: Most genes contain the information for making one polypeptide. This hypothesis is correct for prokaryotes and lower eukaryotes, but must be qualifi ed for higher eukaryotes, such as humans, where a gene can give rise to different polypeptides through an alternative splicing mechanism we will discuss in Chapter 14. Let us now return to the question at hand: How do genes work? This is really more than one question because genes do more than one thing. First, they are replicated faithfully; second, they direct the production of RNAs and proteins; third, they accumulate mutations and so allow evolution. Let us look briefly at each of these activities. How Genes Are Replicated First of all, how is DNA replicated faithfully? To answer that question, we need to know the overall structure of the DNA molecule as it is found in the chromosome. James Watson and Francis Crick (Figure 1.9) provided the answer in 1953 by building models based on chemical and physical data that had been gathered in other laboratories, primarily x-ray diffraction data collected by Rosalind Franklin and Maurice Wilkins (Figure 1.10). Watson and Crick proposed that DNA is a double helix—two DNA strands wound around each other. More important, the bases of each strand are on the inside of the helix, and a base on one strand pairs with one on the other in a very specific way. DNA has only four different bases: adenine, guanine, cytosine, and thymine, which we abbreviate A, G, C, and T. Wherever we find an A in one strand, we always find a T in the other; wherever we find a G in one strand, we always find a C in the other. In a sense, then, the two strands are complementary. If we know the base sequence of one, we automatically know the sequence of the other. This complementarity is what allows DNA to be replicated faithfully. The two strands come apart, and enzymes build new partners for them using the old strands as templates and following the Watson–Crick base-pairing rules (A with T, G with C). This is called semiconservative replication because one strand of the parental double helix is conserved in each of the daughter double helices. In 1958, Matthew Meselson and Franklin Stahl (Figure 1.11) wea25324_ch01_001-011.indd Page 8 8 10/19/10 12:03 PM user-f468 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 1 / A Brief History (a) (b) Figure 1.12 (a) François Jacob; (b) Sydney Brenner. (Source: (a, b) Cold Spring Harbor Laboratory Archives.) proved that DNA replication in bacteria follows the semiconservative pathway (see Chapter 20). Figure 1.13 Gobind Khorana (left) and Marshall Nirenberg. How Genes Direct the Production of Polypeptides Gene expression is the process by which a cell makes a gene product (an RNA or a polypeptide). Two steps, called transcription and translation, are required to make a polypeptide from the instructions in a DNA gene. In the transcription step, an enzyme called RNA polymerase makes a copy of one of the DNA strands; this copy is not DNA, but its close cousin RNA. In the translation step, this RNA (messenger RNA, or mRNA) carries the genetic instructions to the cell’s protein factories, called ribosomes. The ribosomes “read” the genetic code in the mRNA and put together a protein according to its instructions. Actually, the ribosomes already contain molecules of RNA, called ribosomal RNA (rRNA). Francis Crick originally thought that this RNA residing in the ribosomes carried the message from the gene. According to this theory, each ribosome would be capable of making only one kind of protein—the one encoded in its rRNA. François Jacob and Sydney Brenner (Figure 1.12) had another idea: The ribosomes are nonspecific translation machines that can make an unlimited number of different proteins, according to the instructions in the mRNAs that visit the ribosomes. Experiment has shown that this idea is correct (Chapter 3). What is the nature of this genetic code? Marshall Nirenberg and Gobind Khorana (Figure 1.13), working independently with different approaches, cracked the code in the early 1960s (Chapter 18). They found that 3 bases constitute a code word, called a codon, that stands for one amino acid. Out of the 64 possible 3-base codons, 61 specify amino acids; the other three are stop signals. The ribosomes scan a messenger RNA 3 bases at a time and bring in the corresponding amino acids to link to the growing polypeptide chain. When they reach a stop signal, they release the completed polypeptide. (Source: Corbis/Bettmann Archive.) How Genes Accumulate Mutations Genes change in a number of ways. The simplest is a change of one base to another. For example, if a certain codon in a gene is GAG (for the amino acid called glutamate), a change to GTG converts it to a codon for another amino acid, valine. The protein that results from this mutated gene will have a valine where it ought to have a glutamate. This may be one change out of hundreds of amino acids, but it can have profound effects. In fact, this specific change has occurred in the gene for one of the human blood proteins and is responsible for the genetic disorder we call sickle cell disease. Genes can suffer more profound changes, such as deletions or insertions of large pieces of DNA. Segments of DNA can even move from one locus to another. The more drastic the change, the more likely that the gene or genes involved will be totally inactivated. Gene Cloning Since the 1970s, geneticists have learned to isolate genes, place them in new organisms, and reproduce them by a set of techniques collectively known as gene cloning. Cloned genes not only give molecular biologists plenty of raw materials for their studies, they also can be induced to yield their protein products. Some of these, such as human insulin or blood clotting factors, can be very useful. Cloned genes can also be transplanted to plants and animals, including humans.