Coordinately Controlled Genes in Eukaryotes

Transcription

1 Figure Cell type specific transcription. Both liver cells and lens cells have the genes for making the proteins albumin and crystallin, but only liver cells make albumin (a blood protein) and only lens cells make crystallin (the main protein of the lens of the eye). The specific transcription factors made in a cell determine which genes are expressed. In this example, the genes for albumin and crystallin are shown at the top, each with an enhancer made up of three different control elements. Although the enhancers for the two genes both have a gray control element, each enhancer has a unique combination of elements. All the activator proteins required for high-level expression of the albumin gene are present in liver cells only (left), whereas the activators needed for expression of the crystallin gene are present in lens cells only (right). For simplicity, we consider only the role of specific transcription factors that are activators here, although repressors may also influence transcription in certain cell types. Enhancer for albumin gene DNA in both cells (activators not shown) Control elements Liver cell Enhancer for crystallin gene DNA in liver cell, showing available activators. The albumin gene is expressed, but the crystallin gene is not. Liver cell nucleus DNA in lens cell, showing available activators. The crystallin gene is expressed, but the albumin gene is not. Lens cell Available activators Albumin gene not expressed Albumin gene expressed Coordinately Controlled Genes in Eukaryotes How does the eukaryotic cell deal with a group of genes of related function that need to be turned on or off at the same time? Earlier in this chapter, you learned that in bacteria, such coordinately controlled genes are often clustered into an operon, which is regulated by a single promoter and transcribed into a single mrna molecule. Thus, the genes are expressed together, and the encoded proteins are produced concurrently. With a few exceptions, operons that work in this way have not been found in eukaryotic cells. Eukaryotic genes that are co-expressed, such as genes coding for the enzymes of a metabolic pathway, are typically scattered over different chromosomes. Here, coordinate gene expression depends on every gene of a dispersed group having a specific combination of control elements. Activator proteins in the nucleus that recognize the control elements bind to them, promoting simultaneous transcription of the genes, no matter where they are in the genome. Coordinate control of dispersed genes in a eukaryotic cell often occurs in response to chemical signals from outside the cell. A steroid hormone, for example, enters a cell and binds to a specific intracellular receptor protein, forming a hormone-receptor complex that serves as a transcription Genetics Crystallin gene Lens cell nucleus Crystallin gene not expressed Unit three Promoter Albumin gene Available activators VISUAL SKILLS Describe the enhancer for the albumin gene in each type of cell. How would the nucleotide sequence of this enhancer in the liver cell compare with that in the lens cell? 374 Promoter Crystallin gene expressed activator (see Figure 11.9). Every gene whose transcription is stimulated by a given steroid hormone, regardless of its chromosomal location, has a control element recognized by that hormone-receptor complex. This is how estrogen activates a group of genes that stimulate cell division in uterine cells, preparing the uterus for pregnancy. Many signaling molecules, such as nonsteroid hormones and growth factors, bind to receptors on a cell s surface and never actually enter the cell. Such molecules can control gene expression indirectly by triggering signal transduction pathways that activate particular transcription factors (see Figure 11.15). Coordinate regulation in such pathways is the same as for steroid hormones: Genes with the same sets of control elements are activated by the same chemical signals. Because this system for coordinating gene regulation is so widespread, biologists think that it probably arose early in evolutionary history. Nuclear Architecture and Gene Expression You saw in Figure 16.23b that each chromosome in the interphase nucleus occupies a distinct territory. The chromosomes are not completely isolated, however. Recently, chromosome conformation capture (3C) techniques have been developed that allow researchers to cross-link and identify regions of

2 Figure Chromosomal interactions in the interphase nucleus. Although each chromosome has its own territory (see Figure 16.23b), loops of chromatin may extend into other sites in the nucleus. Some of these sites are transcription factories that are occupied by multiple chromatin loops from the same chromosome (blue loops) or other chromosomes (red and green loops). 5 μm Chromosomes in the interphase nucleus (fluorescence micrograph) Chromatin loop Chromosome territory Transcription factory chromosomes that associate with each other during interphase. These studies reveal that loops of chromatin extend from individual chromosomal territories into specific sites in the nucleus (Figure 18.12). Different loops from the same chromosome and loops from other chromosomes may congregate in such sites, some of which are rich in RNA polymerases and other transcription-associated proteins. Like a recreation center that draws members from many different neighborhoods, these so-called transcription factories are thought to be areas specialized for a common function. The old view that the nuclear contents are like a bowl of amorphous chromosomal spaghetti has given way to a new model of a nucleus with a defined architecture and regulated movements of chromatin. Several lines of evidence suggest that genes that are not being expressed are located in the outer edges of the nucleus, while those that are being expressed are found in its interior region. Relocation of particular genes from their chromosomal territories to transcription factories in the interior may be part of the process of readying genes for transcription. How long an individual transcription factory may last has not yet been established. In 2014, the National Institutes of Health announced funding for a new 4D Nucleome program, which aims to investigate the many fascinating questions addressed by this exciting area of current research. Mechanisms of Post-Transcriptional Regulation Transcription alone does not constitute gene expression. The expression of a protein-coding gene is ultimately measured by the amount of functional protein a cell makes, and much happens between the synthesis of the RNA transcript and the activity of the protein in the cell. Many regulatory mechanisms operate at the various stages after transcription (see Figure 18.6). These mechanisms allow a cell to rapidly finetune gene expression in response to environmental changes without altering its transcription patterns. Here we discuss how cells can regulate gene expression once a gene has been transcribed. RNA Processing RNA processing in the nucleus and the export of mature RNA to the cytoplasm provide opportunities for regulating gene expression not available in prokaryotes. One example of regulation at the RNA-processing level is alternative RNA splicing, in which different mrna molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript. A simple example of alternative RNA splicing is shown in Figure for the troponin T gene, which encodes two different (though related) proteins. Other genes code for many more possible products. For instance, researchers have found a Drosophila gene with enough alternatively spliced exons to Figure Alternative RNA splicing of the troponin T gene. The primary transcript of this gene can be spliced in more than one way, generating different mrna molecules. Notice that one mrna molecule has ended up with exon 3 (green) and the other with exon 4 (purple). These two mrnas are translated into different but related muscle proteins. Exons DNA Primary RNA transcript mrna 1 Troponin T gene RNA splicing CHROMATIN MODIFICATION mrna DEGRADATION TRANSCRIPTION RNA PROCESSING TRANSLATION PROTEIN PROCESSING AND DEGRADATION OR chapter 18 Regulation of Gene Expression 375

3 generate about 19,000 membrane proteins that have different extracellular domains. At least 17,500 (94%) of the alternative mrnas are actually synthesized. Each developing nerve cell in the fly appears to synthesize a different form of the protein, which acts as unique identifier on the cell surface and helps prevent excessive overlap of nerve cells during development of the nervous system. It is clear that alternative RNA splicing can significantly expand the repertoire of a eukaryotic genome. In fact, alternative splicing was proposed as one explanation for the surprisingly low number of human genes counted when the human genome was sequenced. The number of human genes was found to be similar to that of a soil worm (nematode), a mustard plant, or a sea anemone. This discovery prompted questions about what, if not the number of genes, accounts for the more complex morphology (external form) of humans. It turns out that more than 90% of human protein-coding genes probably undergo alternative splicing. Thus, the extent of alternative splicing greatly multiplies the number of possible human proteins, which may be better correlated with complexity of form. Initiation of Translation and mrna Degradation Translation is another opportunity for regulating gene expression; it occurs most commonly at the initiation stage (see Figure 17.19). For some mrnas, the initiation of translation can be blocked by regulatory proteins that bind to specific sequences or structures within the untranslated region (UTR) at the 5 or 3 end, preventing the attachment of ribosomes. (Recall from Concept 17.3 that both the 5 cap and the poly-a tail of an mrna molecule are important for ribosome binding.) Alternatively, translation of all the mrnas in a cell may be regulated simultaneously. In a eukaryotic cell, such global control usually involves the activation or inactivation of one or more of the protein factors required to initiate translation. This mechanism plays a role in starting translation of mrnas that are stored in eggs. Just after fertilization, translation is triggered by the sudden activation of translation initiation factors. The response is a burst of synthesis of the proteins encoded by the stored mrnas. Some plants and algae store mrnas during periods of darkness; light then triggers the reactivation of the translational apparatus. The life span of mrna molecules in the cytoplasm is important in determining the pattern of protein synthesis in a cell. Bacterial mrna molecules typically are degraded by enzymes within a few minutes of their synthesis. This short life span of mrnas is one reason bacteria can change their patterns of protein synthesis so quickly in response to environmental changes. In contrast, mrnas in multicellular eukaryotes typically survive for hours, days, or even weeks. For instance, the mrnas for the hemoglobin polypeptides (α-globin and β-globin) in developing red blood cells are unusually stable, and these long-lived mrnas are translated repeatedly in red blood cells. Nucleotide sequences that affect how long an mrna remains intact are often found in the untranslated region at the 3 end of the molecule (see Figure 18.8). In one experiment, researchers transferred such a sequence from the short-lived mrna for a growth factor to the 3 end of a normally stable globin mrna. The globin mrna was quickly degraded. During the past few years, other mechanisms that degrade or block expression of mrna molecules have come to light. They involve a group of newly discovered RNA molecules that regulate gene expression at several levels, as we ll discuss shortly. Protein Processing and Degradation The final opportunities for controlling gene expression occur after translation. Often, eukaryotic polypeptides must be processed to yield functional protein molecules. For instance, cleavage of the initial insulin polypeptide (pro-insulin) forms the active hormone. In addition, many proteins undergo chemical modifications that make them functional. Regulatory proteins are commonly activated or inactivated by the reversible addition of phosphate groups (see Figure 11.10), and proteins destined for the surface of animal cells acquire sugars (see Figure 6.12). Cell-surface proteins and many others must also be transported to target destinations in the cell in order to function (see Figure 17.22). Regulation might occur at any of the steps involved in modifying or transporting a protein. Finally, the length of time each protein functions in the cell is strictly regulated by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived if the cell is to function appropriately (see Figure 12.16). To mark a protein for destruction, the cell commonly attaches molecules of a small protein called ubiquitin to the protein. Giant protein complexes called proteasomes then recognize the ubiquitintagged proteins and degrade them. Animation: Post-Transcriptional Control Mechanisms Concept Check In general, what are the effects of histone acetylation and DNA methylation on gene expression? 2. MAKE CONNECTIONS Speculate about whether the same enzyme could methylate both a histone and a DNA base. (See Concept 5.4.) 3. Compare the roles of general and specific transcription factors in regulating gene expression. 4. Once mrna encoding a particular protein reaches the cytoplasm, what are four mechanisms that can regulate the amount of the protein that is active in the cell? 5. WHAT IF? Suppose you compared the nucleotide sequences of the distal control elements in the enhancers of three genes that are expressed only in muscle cells. What would you expect to find? Why? For suggested answers, see Appendix A. 376 Unit three Genetics

4 Concept 18.3 Noncoding RNAs play multiple roles in controlling gene expression Genome sequencing has revealed that protein-coding DNA accounts for only 1.5% of the human genome and a similarly small percentage of the genomes of many other multicellular eukaryotes. A very small fraction of the non-protein-coding DNA consists of genes for RNAs such as ribosomal RNA and transfer RNA. Until recently, scientists assumed that most of the remaining DNA was not transcribed, thinking that since it didn t specify proteins or the few known types of RNA, such DNA didn t contain meaningful genetic information in fact, it was called junk DNA. However, a flood of recent data has contradicted this idea. For example, a massive study of the entire human genome showed that roughly 75% of the genome is transcribed at some point in any given cell. Introns account for only a fraction of this transcribed, nontranslated RNA. These and other results suggest that a significant amount of the genome may be transcribed into non-protein-coding RNAs also called noncoding RNAs, or ncrnas including a variety of small RNAs. Researchers are uncovering more evidence of the biological roles of these ncrnas every day. Biologists are excited about these discoveries, which have revealed a large and diverse population of RNA molecules in the cell that play crucial roles in regulating gene expression but have gone largely unnoticed until fairly recently. Our long-standing view that because mrnas code for proteins, they are the most important RNAs functioning in the cell demands revision. This represents a major shift in the thinking of biologists, one that you are witnessing as students entering this field of study. Effects on mrnas by MicroRNAs and Small Interfering RNAs Regulation by both small and large ncrnas occurs at several points in the pathway of gene expression, including mrna translation and chromatin modification. We ll examine two types of small ncrnas, the importance of which was acknowledged when their discovery was the focus of the 2006 Nobel Prize in Physiology or Medicine, which was awarded for work completed only eight years earlier. Since 1993, a number of research studies have uncovered micrornas (mirnas) small, single-stranded RNA molecules capable of binding to complementary sequences in mrna molecules. A longer RNA precursor is processed by cellular enzymes into an mirna, a single-stranded RNA of about 22 nucleotides that forms a complex with one or more proteins (Figure 18.14). The mirna allows the complex to bind to any mrna molecule with at least 7 or 8 nucleotides of complementary sequence. The mirna-protein complex then degrades the target mrna or, less often, simply blocks its translation. There are approximately 1,500 genes for mirnas in the human genome, and biologists estimate that expression of at least one-half of all human genes may be regulated by mirnas, a remarkable figure given that the existence of mirnas was unknown 25 years ago. Another class of small RNAs, similar in size and function to mirnas, is called small interfering RNAs (sirnas). Both mirnas and sirnas can associate with the same proteins, producing similar results. In fact, if sirna precursor RNA molecules are injected into a cell, the cell s machinery can process them into sirnas that turn off expression of genes with related sequences, similarly to how mirnas function. The distinction between mirnas and sirnas is based on subtle differences in the structure of their precursors, which in both cases are RNA molecules that are mostly double-stranded. The blocking of gene expression by sirnas is referred to as RNA interference (RNAi), and it is used in the laboratory as a means of disabling specific genes to investigate their function. How did the RNAi pathway evolve? As you will learn in Concept 19.2, some viruses have double-stranded RNA genomes. Given that the cellular RNAi pathway can process double-stranded RNAs into homing devices that lead to destruction of related RNAs, some scientists think that this pathway may have evolved as a natural defense against 1 mrna 2 CHROMATIN MODIFICATION mrna DEGRADATION TRANSCRIPTION RNA PROCESSING TRANSLATION PROTEIN PROCESSING AND DEGRADATION The mirna binds to a target mrna with at least 7 complementary bases. mrna degraded Figure Regulation of gene expression by micrornas (mirnas). An mirna of about 22 nucleotides, formed by enzymatic processing of an RNA precursor, associates with one or more proteins in a complex that can degrade or block translation of target mrnas. mirna OR Translation blocked mirnaprotein complex If mirna and mrna bases are complementary all along their length, the mrna is degraded (left); if the match is less complete, translation is blocked (right). chapter 18 Regulation of Gene Expression 377

5 infection by such viruses. However, the fact that RNAi can also affect the expression of nonviral cellular genes may reflect a different evolutionary origin for the RNAi pathway. Moreover, many species, including mammals, apparently produce their own long, double-stranded RNA precursors to small RNAs such as sirnas. Once produced, these RNAs can interfere with gene expression at stages other than translation, as we ll discuss next. Chromatin Remodeling and Effects on Transcription by ncrnas In addition to regulating mrnas, some ncrnas act to bring about remodeling of chromatin structure. One example occurs during formation of heterochromatin at the centromere, as studied in a species of yeast. In the S phase of the cell cycle, the centromeric regions of DNA must be loosened for chromosomal replication and then re-condensed into heterochromatin in preparation for mitosis. In some yeasts, sirnas produced by the yeast cells themselves are required to re-form the heterochromatin at the centromeres. A model for how this happens is shown in Figure Exactly how the process starts is still under debate, but biologists agree on the general idea: The sirna system in yeast interacts with other ncrnas and with chromatin-modifying enzymes to condense the centromere chromatin into heterochromatin. Under most conditions in mammalian cells, sirnas are not known to occur, and the mechanism for centromere DNA condensation is not yet understood. However, it may turn out to involve other small ncrnas. A recently discovered class of small ncrnas is called piwi-interacting RNAs, or pirnas. These RNAs also induce formation of heterochromatin, blocking expression of some parasitic DNA elements in the genome known as transposons. (Transposons are discussed in Concepts 21.4 and 21.5.) Usually nucleotides in length, pirnas are processed from a longer, single-stranded RNA precursor. They play an indispensable role in the germ cells of many animal species, where they appear to help reestablish appropriate methylation patterns in the genome during gamete formation. Figure Condensation of chromatin at the centromere. In one type of yeast, sirnas and longer noncoding RNAs cooperate in the pathway that leads to re-formation of highly condensed heterochromatin at the centromere of each chromatid after DNA replication. Centromeric DNA RNA polymerase 1 RNA transcripts (red) are produced from centromeric DNA. RNA transcript 2 Each RNA transcript is used as a template by a yeast enzyme that synthesizes the complementary strand, forming double-stranded RNA. 3 The double-stranded RNA is processed into short, single-stranded sirnas that associate with proteins, forming sirna-protein complexes. 378 Unit three Genetics sirna-protein complex 4 The sirna-protein complexes bind the RNA transcripts being produced from the centromeric DNA and, in this way, are tethered to the centromere region. 5 Proteins in the sirna-protein complexes recruit enzymes (green) that chemically modify the histones within the chromatin and initiate chromatin condensation. Centromeric DNA Chromatinmodifying enzymes Interview with Haifan Lin: Discovering piwi-interacting RNAs and connections to testicular cancer Researchers have also found a relatively large number of long noncoding RNAs (lncrnas), ranging from 200 to hundreds of thousands of nucleotides in length, that are expressed at significant levels in specific cell types at particular times. One such lncrna is responsible for X chromosome inactivation, which, in most female mammals, prevents expression of genes located on one of the X chromosomes (see Figure 15.8). In this case, lncrnas transcripts of the XIST gene located on the Sister chromatids (two DNA molecules) 6 Ultimately, this process leads to formation of heterochromatin at the centromere. Heterochromatin at the centromere region

6 chromosome to be inactivated bind back to and coat that chromosome, and this binding leads to condensation of the entire chromosome into heterochromatin. The cases described above involve chromatin remodeling in large regions of the chromosome. Because chromatin structure affects transcription and thus gene expression, RNA-based regulation of chromatin structure is sure to play an important role in gene regulation. Additionally, some experimental evidence supports the idea of an alternate role for lncrnas in which they can act as a scaffold, bringing together DNA, proteins, and other RNAs into complexes. These associations may act either to condense chromatin or, in some cases, to help bring the enhancer of a gene together with mediator proteins and the gene s promoter, activating gene expression in a more direct fashion. The Evolutionary Significance of Small ncrnas Concept Check Compare mirnas and sirnas, including their functions. 2. WHAT IF? Suppose the mrna being degraded in Figure coded for a protein that promotes cell division in a multicellular organism. What would happen if a mutation disabled the gene for the mirna that triggers this degradation? 3. MAKE CONNECTIONS Inactivation of one of the X chromosomes in female mammals involves lncrna called XIST RNA, mentioned in this section and in Concept Describe transcription and binding of XIST RNA, then suggest a model for how it initiates Barr body formation. For suggested answers, see Appendix A. Concept 18.4 A program of differential gene expression leads to the different cell types in a multicellular organism In the embryonic development of multicellular organisms, a Evolution Small ncrnas can regulate gene expression fertilized egg (a zygote) gives rise to cells of many different types, at multiple steps and in many ways. While this section has each with a different structure and corresponding function. focused on ncrnas in eukaryotes, small ncrnas are also Typically, cells are organized into tissues, tissues into organs, used by bacteria as a defense system, called the CRISPR-Cas9 organs into organ systems, and organ systems into the whole system, against viruses that infect them. (You ll learn more organism. Thus, any developmental program must produce about this in Concept 19.2.) The use of ncrnas thus evolved cells of different types that form higher-level structures arranged long ago, but we don t yet know how bacterial ncrnas are in a particular way in three dimensions. The processes that related to those of eukaryotes. occur during development in plants and animals are detailed What about the evolutionary significance of small in Chapters 35 and 47, respectively. In this chapter, we focus eukaryotic ncrnas? In general, extra levels of gene on the program of regulation of gene expression that orchestrates development, using a few animal species as examples. regulation might allow evolution of a higher degree of complexity of form. The versatility of mirna regulation has therefore led some biologists to hypothesize that an A Genetic Program for Embryonic increase in the number of different mirnas specified by the genome of a given species has allowed morphological Development complexity to increase over evolutionary time. While this The photos in Figure illustrate the dramatic difference between a frog zygote (fertilized egg) and the tadpole it hypothesis is still being evaluated, it is logical to expand the discussion to include all small ncrnas. Exciting new techniques for rapidly sequencing genomes have allowed Figure From fertilized egg to animal: What a difference four days makes. biologists to begin asking how many genes for ncrnas are It takes just four days for cell division, differentiation, and morphogenesis to transform each of the fertilized present in the genome of any given species. A survey of frog eggs shown in (a) into a tadpole like the one in (b). different species supports the notion that sirnas evolved first, followed by mirnas and later pirnas, which are found only in animals. And while there are hundreds of types of mirnas, there appear to be 60,000 or so types of pirnas, allowing the potential for very sophisticated gene regulation by pirnas. Given the extensive functions of ncrnas, it is not surprising that many of the ncrnas characterized thus far play important roles in embryonic development the topic we turn to in the next section. Embryonic development is 1 mm 2 mm perhaps the ultimate example of precisely regulated gene expression. (a) Fertilized eggs of a frog (b) Newly hatched tadpole chapter 18 Regulation of Gene Expression 379

7 becomes. This remarkable transformation results from three interrelated processes: cell division, cell differentiation, and morphogenesis. Through a succession of mitotic cell divisions, the zygote gives rise to a large number of cells. Cell division alone, however, would merely produce a great ball of identical cells, nothing like a tadpole. During embryonic development, cells not only increase in number, but also undergo cell differentiation, the process by which cells become specialized in structure and function. Moreover, the different kinds of cells are not randomly distributed but are organized into tissues and organs in a particular threedimensional arrangement. The physical processes that give an organism its shape constitute morphogenesis, the development of the form of an organism and its structures. All three processes are rooted in cellular behavior. Even morphogenesis, the shaping of the organism, can be traced back to changes in the shape, motility, and other characteristics of the cells that make up various regions of the embryo. As you have seen, the activities of a cell depend on the genes it expresses and the proteins it produces. Almost all cells in an organism have the same genome; therefore, differential gene expression results from the genes being regulated differently in each cell type. In Figure 18.11, you saw a simplified view of how differential gene expression occurs in two cell types, a liver cell and a lens cell. Each of these fully differentiated cells has a particular mix of specific activators that turn on the collection of genes whose products are required in the cell. The fact that both cells arose through a series of mitoses from a common fertilized egg inevitably leads to a question: How do different sets of activators come to be present in the two cells? It turns out that materials placed into the egg by maternal cells set up a sequential program of gene regulation that is carried out as embryonic cells divide, and this program coordinates cell differentiation during embryonic development. To understand how this works, we will consider two basic developmental processes. First, we ll explore how cells that arise from early embryonic mitoses develop the differences that start each cell along its own differentiation pathway. Second, we ll see how cellular differentiation leads to one particular cell type, using muscle development as an example. Cytoplasmic Determinants and Inductive Signals What generates the first differences among cells in an early embryo? And what controls the differentiation of all the various cell types as development proceeds? By this point in the chapter, you can probably deduce the answer: The specific genes expressed in any particular cell of a developing organism determine its path. Two sources of information, used to varying extents in different species, tell a cell which genes to express at any given time during embryonic development. One important source of information early in development is the egg s cytoplasm, which contains both RNA and proteins encoded by the mother s DNA. The cytoplasm of an unfertilized egg is not homogeneous. Messenger RNA, proteins, other substances, and organelles are distributed unevenly in the unfertilized egg, and this unevenness has a profound impact on the development of the future embryo in many species. Maternal substances in the egg that influence the course of early development are called cytoplasmic determinants (Figure 18.17a). After fertilization, early mitotic divisions distribute the zygote s Figure Sources of developmental information for the early embryo. (a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Molecules of two different cytoplasmic determinants Nucleus Fertilization Zygote (fertilized egg) Mitotic cell division The unfertilized egg has molecules in its cytoplasm, encoded by the mother s genes, that influence development. Many of these cytoplasmic determinants, like the two shown here, are unevenly distributed in the egg. After fertilization and mitotic division, the cell nuclei of the embryo are exposed to different sets of cytoplasmic determinants and, as a result, express different genes. (b) Induction by nearby cells Signal transduction pathway Signal receptor Signaling molecule Early embryo (32 cells) NUCLEUS Two-celled embryo Cells at the bottom of the early embryo are releasing molecules that signal (induce) nearby cells to change their gene expression. 380 Unit three Genetics

8 cytoplasm into separate cells. The nuclei of these cells may thus be exposed to different cytoplasmic determinants, depending on which portions of the zygotic cytoplasm a cell received. The combination of cytoplasmic determinants in a cell helps determine its developmental fate by regulating expression of the cell s genes during the course of cell differentiation. The other major source of developmental information, which becomes increasingly important as the number of embryonic cells increases, is the environment around a particular cell. Most influential are the signals conveyed to an embryonic cell from other embryonic cells in the vicinity, including contact with cell-surface molecules on neighboring cells and the binding of growth factors secreted by neighboring cells (see Concept 11.1). Such signals cause changes in the target cells, a process called induction (Figure 18.17b). The molecules passing along these signals within the target cell are cell-surface receptors and other signaling pathway proteins. In general, the signaling molecules send a cell down a specific developmental path by causing changes in its gene expression that eventually result in observable cellular changes. Thus, interactions between embryonic cells help induce differentiation into the many specialized cell types making up a new organism. Sequential Regulation of Gene Expression During Cellular Differentiation The earliest changes that set a cell on its path to specialization are subtle ones, showing up only at the molecular level. Before biologists knew much about the molecular changes occurring in embryos, they coined the term determination to refer to the point at which an embryonic cell is irreversibly committed to becoming a particular cell type. Once it has undergone determination, an embryonic cell can be experimentally placed in another location in the embryo and it will still differentiate into the cell type that is its normal fate. Differentiation, then, is the process by which a cell attains its determined fate. As the tissues and organs of an embryo develop and their cells differentiate, the cells become more noticeably different in structure and function. Today we understand determination in terms of molecular changes. The outcome of determination, observable cell differentiation, is marked by the expression of genes for tissue-specific proteins. These proteins are found only in a specific cell type and give the cell its characteristic structure and function. The first evidence of differentiation is the appearance of mrnas for these proteins. Eventually, differentiation is observable with a microscope as changes in cellular structure. On the molecular level, different sets of genes are sequentially expressed in a regulated manner as new cells arise from division of their precursors. A number of the steps in gene expression may be regulated during differentiation, transcription being the most common. In the fully differentiated cell, transcription remains the principal regulatory point for maintaining appropriate gene expression. Differentiated cells are specialists at making tissue-specific proteins. For example, as a result of transcriptional regulation, liver cells specialize in making albumin, and lens cells specialize in making crystallin (see Figure 18.11). Skeletal muscle cells in vertebrates are another instructive example. Each of these cells is a long fiber containing many nuclei within a single plasma membrane. Skeletal muscle cells have high concentrations of muscle-specific versions of the contractile proteins myosin and actin, as well as membrane receptor proteins that detect signals from nerve cells. Muscle cells develop from embryonic precursor cells that have the potential to develop into a number of cell types, including cartilage cells and fat cells, but particular conditions commit them to becoming muscle cells. Although the committed cells appear unchanged under the microscope, determination has occurred, and they are now myoblasts. Eventually, myoblasts start to churn out large amounts of muscle-specific proteins and fuse to form mature, elongated, multinucleate skeletal muscle cells. Researchers have worked out what happens at the molecular level during muscle cell determination. To do so, they grew embryonic precursor cells in culture and analyzed them using molecular techniques you will learn about in Concepts 20.1 and In a series of experiments, they isolated different genes, caused each to be expressed in a separate embryonic precursor cell, and then looked for differentiation into myoblasts and muscle cells. In this way, they identified several so-called master regulatory genes whose protein products commit the cells to becoming skeletal muscle. Thus, in the case of muscle cells, the molecular basis of determination is the expression of one or more of these master regulatory genes. To understand more about how determination occurs in muscle cell differentiation, let s focus on the master regulatory gene called myod. The myod gene deserves its designation as a master regulatory gene. Researchers have shown that the MyoD protein it encodes is capable of changing some kinds of fully differentiated nonmuscle cells, such as fat cells and liver cells, into muscle cells. Why doesn t MyoD work on all kinds of cells? One likely explanation is that activation of musclespecific genes is not solely dependent on MyoD but requires a particular combination of regulatory proteins, some of which are lacking in cells that do not respond to MyoD. The determination and differentiation of other kinds of tissues may play out in a similar fashion. A growing body of experimental evidence supports the idea that master regulatory proteins like MyoD might actually function by opening the chromatin in particular regions. This allows access to transcription machinery for activation of the next set of cell-type-specific genes. What is the molecular basis for muscle cell differentiation? The MyoD protein is a transcription factor (see Figure 18.9) that binds to specific control elements in the enhancers of various target genes and stimulates their expression (Figure 18.18). Some target genes for MyoD encode still other muscle-specific transcription factors. MyoD also stimulates expression of the chapter 18 Regulation of Gene Expression 381

9 Figure Determination and differentiation of muscle cells. Skeletal muscle cells arise from embryonic cells as a result of changes in gene expression. (In this depiction, the process of gene activation is greatly simplified.) Nucleus Master regulatory gene myod Other muscle-specific genes 1 Determination. Signals from other cells lead to activation of a master regulatory gene called myod, and the cell makes MyoD protein, a specific transcription factor that acts as an activator. The cell, now called a myoblast, is irreversibly committed to becoming a skeletal muscle cell. Embryonic precursor cell Myoblast (determined) DNA OFF mrna MyoD protein (transcription factor) OFF OFF 2 Differentiation. MyoD protein stimulates the myod gene further and activates genes encoding other muscle-specific transcription factors, which in turn activate genes for muscle proteins. MyoD also turns on genes that block the cell cycle, thus stopping cell division. The nondividing myoblasts fuse to become mature multinucleate muscle cells, also called muscle fibers. Part of a muscle fiber (fully differentiated cell) mrna MyoD mrna mrna mrna A different transcription factor Myosin, other muscle proteins, and cell cycleblocking proteins WHAT IF? What would happen if a mutation in the myod gene resulted in the production of an altered MyoD protein that could not activate the myod gene? myod gene itself, an example of positive feedback that perpetuates MyoD s effect in maintaining the cell s differentiated state. Presumably, all the genes activated by MyoD have enhancer control elements recognized by MyoD and are thus coordinately controlled. Finally, the secondary transcription factors activate the genes for proteins such as myosin and actin that confer the unique properties of skeletal muscle cells. We have now seen how different programs of gene expression that are activated in the fertilized egg can result in differentiated cells and tissues. But for the tissues to function effectively in the organism as a whole, the organism s body plan its overall three-dimensional arrangement must be established and superimposed on the differentiation process. Next we ll investigate the molecular basis for the establishment of the body plan, using the well-studied fruit fly Drosophila melanogaster as an example. Pattern Formation: Setting Up the Body Plan Cytoplasmic determinants and inductive signals both contribute to the development of a spatial organization in which the tissues and organs of an organism are all in their characteristic places. This process is referred to as pattern formation. Just as the locations of the front, back, and sides of a new building are determined before construction begins, pattern formation in animals begins in the early embryo, when the major axes of an animal are established. In a bilaterally symmetrical animal, the relative positions of head and tail, right and left sides, and back and front the three major body axes are set up before the organs appear. The molecular cues that control pattern formation, collectively called positional information, are provided by cytoplasmic determinants and inductive signals (see Figure 18.17). These cues tell a cell its location relative to the body axes and to neighboring cells and determine how the cell and its descendants will respond to future molecular signals. During the first half of the 20th century, classical embryologists made detailed anatomical observations of embryonic development in a number of species and performed experiments in which they manipulated embryonic tissues. Although this research laid the groundwork for understanding the mechanisms of development, it did not reveal the 382 Unit three Genetics

10 specific molecules that guide development or determine how patterns are established. In the 1940s, scientists began using the genetic approach the study of mutants to investigate Drosophila development. That approach has had spectacular success. These studies have established that genes control development and have led to an understanding of the key roles that specific molecules play in defining position and directing differentiation. By combining anatomical, genetic, and biochemical approaches to the study of Drosophila development, researchers have discovered developmental principles common to many other species, including humans. The Life Cycle of Drosophila Fruit flies and other arthropods have a modular construction, an ordered series of segments. These segments make up the body s three major parts: the head, the thorax (the mid-body, from which the wings and legs extend), and the abdomen (Figure 18.19a). Like other bilaterally symmetrical animals, Drosophila has an anterior-posterior (head-to-tail) axis, a dorsalventral (back-to-belly) axis, and a right-left axis. In Drosophila, cytoplasmic determinants that are localized in the unfertilized egg provide positional information for the placement of anterior-posterior and dorsal-ventral axes even before fertilization. We ll focus here on the molecules involved in establishing the anterior-posterior axis. The Drosophila egg develops in one of the female s ovaries, adjacent to ovarian cells called nurse cells and surrounded by so-called follicle cells (Figure 18.19b, top). These support cells supply the egg with nutrients, mrnas, and other substances needed for development and make the egg shell. After fertilization and laying of the egg, embryonic development results in the formation of a segmented larva, which goes through three larval stages. Then, in a process much like that by which a caterpillar becomes a butterfly, the fly larva forms a pupa in which it metamorphoses into the adult fly pictured in Figure 18.19a. Genetic Analysis of Early Development: Scientific Inquiry Edward B. Lewis was a visionary American biologist who, in the 1940s, first showed the value of the genetic approach to studying embryonic development in Drosophila. Lewis studied bizarre mutant flies with developmental defects that led to extra wings or legs in the wrong place (Figure 18.20). He located the mutations on the fly s genetic map, thus connecting the developmental abnormalities to specific genes. This research supplied the first concrete evidence that genes somehow direct the developmental processes studied by embryologists. The genes Lewis discovered, called homeotic genes, are regulatory genes that control pattern formation in the late embryo, larva, and adult. Figure Key events in the Drosophila life cycle. Anterior Dorsal Left Ventral BODY AXES Right Posterior Head Thorax Abdomen (a) Adult. The adult fly is segmented, and multiple segments make up each of the three main body parts head, thorax, and abdomen. The body axes are shown by arrows. 1 Developing egg within one ovarian follicle (among many in an ovary). The egg (yellow) is surrounded by support cells (follicle cells). 2 Mature, unfertilized egg. The developing egg enlarges as nutrients and mrnas are supplied to it by other support cells (nurse cells), which shrink. Eventually, the mature egg fills the egg shell that is secreted by the follicle cells. 3 Fertilized egg. The egg is fertilized within the mother and then laid. 4 Segmented embryo. The egg develops into a segmented embryo. 5 Larva. The embryo develops into a larva, which has three stages. The third stage forms a pupa (not shown), within which the larva metamorphoses into the adult shown in (a). (b) Development from egg to larva. Nurse cell Depleted nurse cells Body segments Follicle cell Fertilization Laying of egg Embryonic development Hatching Nucleus Egg Egg shell 0.1 mm Figure Abnormal pattern formation in Drosophila. Mutations in homeotic genes cause abnormal placement of structures in an animal, such as the legs extending from the mutant fly s head in place of antenna. (Colorized SEMs) Eye Normal antenna Wild type Leg instead of antenna Mutant 0.5 mm chapter 18 Regulation of Gene Expression 383

11 Further insight into pattern formation during early embryonic development did not come for another 30 years, when two researchers in Germany, Christiane Nüsslein- Volhard and Eric Wieschaus, set out to identify all the genes that affect segment formation in Drosophila. The project was daunting for three reasons. The first was the sheer number of Drosophila genes, now known to total about 14,000. The genes affecting segmentation might be just a few needles in a haystack or might be so numerous and varied that the scientists would be unable to make sense of them. Second, mutations affecting a process as fundamental as segmentation would surely be embryonic lethals, mutations with phenotypes causing death at the embryonic or larval stage. Because organisms with embryonic lethal mutations never reproduce, they cannot be bred for study. The researchers dealt with this problem by looking for recessive mutations, which can be propagated in heterozygous flies that act as genetic carriers. Third, cytoplasmic determinants in the egg were known to play a role in axis formation, so the researchers knew they would have to study the mother s genes as well as those of the embryo. It is the mother s genes that we will discuss further as we focus on how the anteriorposterior body axis is set up in the developing egg. Nüsslein-Volhard and Wieschaus began their search for segmentation genes by exposing flies to a mutagenic chemical that affected the flies gametes. They mated the mutagenized flies and then scanned their descendants for dead embryos or larvae with abnormal segmentation or other defects. For example, to find genes that might set up the anterior-posterior axis, they looked for embryos or larvae with abnormal ends, such as two heads or two tails, predicting that such abnormalities would arise from mutations in maternal genes required for correctly setting up the offspring s head or tail end. Using this approach, Nüsslein-Volhard and Wieschaus eventually identified about 1,200 genes essential for pattern formation during embryonic development. Of these, about 120 were essential for normal segmentation. Over several years, the researchers were able to group these segmentation genes by general function, to find them on the fly s chromosomes, and to isolate many of them for further study in the lab. The result was a detailed molecular understanding of the early steps in pattern formation in Drosophila. When the results of Nüsslein-Volhard and Wieschaus were combined with Lewis s earlier work, a coherent picture of Drosophila development emerged. In recognition of their discoveries, the three researchers were awarded a Nobel Prize in Next, let s consider a specific example of the genes that Nüsslein-Volhard, Wieschaus, and co-workers found. Drosophila body. These substances are encoded by genes of the mother, fittingly called maternal effect genes. A gene classified as a maternal effect gene is one that, when mutant in the mother, results in a mutant phenotype in the offspring, regardless of the offspring s own genotype. In fruit fly development, the mrna or protein products of maternal effect genes are placed in the egg while it is still in the mother s ovary. When the mother has a mutation in such a gene, she makes a defective gene product (or none at all), and her eggs are defective; when these eggs are fertilized, they fail to develop properly. Because they control the orientation (polarity) of the egg and consequently that of the fly, these maternal effect genes are also called egg-polarity genes. One group of these genes sets up the anterior-posterior axis of the embryo, while a second group establishes the dorsal-ventral axis. Like mutations in segmentation genes, mutations in maternal effect genes are generally embryonic lethals. Bicoid: A Morphogen That Determines Head Structures To see how maternal effect genes determine the body axes of the offspring, we will focus on one such gene, called bicoid, a term meaning two-tailed. An embryo or larva whose mother has two mutant bicoid alleles lacks the front half of its body and has posterior structures at both ends (Figure 18.21). This phenotype suggested to Nüsslein-Volhard and her colleagues that the product of the mother s bicoid gene is essential for setting up the anterior end of the fly and might be concentrated at the future anterior end of the embryo. This hypothesis is an example of the morphogen gradient hypothesis first proposed Figure Effect of the bicoid gene on Drosophila development. A wild-type fruit fly larva has a head, three thoracic (T) segments, eight abdominal (A) segments, and a tail. A larva whose mother has two mutant alleles of the bicoid gene has two tails and lacks all anterior structures (LMs). Head T1 T2 Tail T3 A1 A2 A3 A4 Wild-type larva A5 A8 A6 A7 Tail 250 μm Tail Axis Establishment As we mentioned earlier, cytoplasmic determinants in the egg are the substances that initially establish the axes of the A8 A7 A6 A7 Mutant larva (bicoid ) A8 384 Unit three Genetics

12 by embryologists a century ago, in which gradients of substances called morphogens establish an embryo s axes and other features of its form. DNA technology and other modern biochemical methods enabled the researchers to test whether the bicoid product, a protein called Bicoid, is in fact a morphogen that determines the anterior end of the fly. The first question they asked was whether the mrna and protein products of this gene are located in the egg in a position consistent with the hypothesis. They found that bicoid mrna is highly concentrated at the extreme anterior end of the mature egg (Figure 18.22). After the egg is fertilized, the mrna is translated into protein. The Bicoid protein then diffuses from the anterior end toward the posterior, resulting in a gradient of protein within the early embryo, with the highest concentration at the anterior end. These results are consistent with the hypothesis that Bicoid protein specifies the fly s anterior end. To test the hypothesis more specifically, scientists injected pure bicoid mrna into various regions of early embryos. The protein that resulted from its translation caused anterior structures to form at the injection sites. The bicoid research was groundbreaking for several reasons. First, it led to the identification of a specific protein required for some of the earliest steps in pattern formation. It thus helped us understand how different regions of the egg can give rise to cells that go down different developmental pathways. Second, it increased our understanding of the mother s critical role in the initial phases of embryonic development. Finally, the principle that a gradient of morphogens can determine polarity and position has proved to be a key developmental concept for a number of species, just as early embryologists had hypothesized. Maternal mrnas are crucial during development of many species. In Drosophila, gradients of specific proteins encoded by maternal mrnas not only determine the posterior and anterior ends but also establish the dorsal-ventral axis. As the fly embryo grows, it reaches a point when the embryonic program of gene expression takes over, and the maternal mrnas must be destroyed. (This process involves mirnas in Drosophila and other species.) Later, positional information encoded by the embryo s genes, operating on an ever finer scale, establishes a specific number of correctly oriented segments and triggers the formation of each segment s characteristic structures. When the genes operating in this final step are abnormal, the pattern of the adult is abnormal, as you saw in Figure Interview with Nancy Hopkins: Studying the genetic basis of development in zebrafish Evolutionary Developmental Biology ( Evo-Devo ) Evolution The fly with legs emerging from its head in Figure is the result of a single mutation in one gene, a homeotic gene. The gene does not encode any antenna Figure Inquiry Could Bicoid be a morphogen that determines the anterior end of a fruit fly? Experiment Using a genetic approach to study Drosophila development, Christiane Nüsslein-Volhard and colleagues at two research institutions in Germany analyzed expression of the bicoid gene. The researchers hypothesized that bicoid normally codes for a morphogen that specifies the head (anterior) end of the embryo. To begin to test this hypothesis, they used molecular techniques to determine whether the mrna and protein encoded by this gene were found in the anterior end of the fertilized egg and early embryo of wild-type flies. Results Bicoid mrna (dark blue in the light micrographs and drawings) was confined to the anterior end of the unfertilized egg. Later in development, Bicoid protein (dark orange) was seen to be concentrated in cells at the anterior end of the embryo. Bicoid mrna in mature unfertilized egg Fertilization, translation of bicoid mrna Anterior end Bicoid protein in early embryo 100 μm Conclusion The location of bicoid mrna and the diffuse gradient of Bicoid protein seen later are consistent with the hypothesis that Bicoid protein is a morphogen specifying formation of head-specific structures. Further Reading C. Nüsslein-Volhard et al., Determination of anteroposterior polarity in Drosophila, Science 238: (1987); W. Driever and C. Nüsslein-Volhard, A gradient of Bicoid protein in Drosophila embryos, Cell 54:83 93 (1988); T. Berleth et al., The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo, EMBO Journal 7: (1988). WHAT IF? The researchers needed further evidence, so they injected bicoid mrna into the anterior end of an egg from a female with a mutation disabling the bicoid gene. Given that the hypothesis was supported, what must their results have been? Animation: Role of bicoid Gene in Drosophila Development protein, however. Instead, it encodes a transcription factor that regulates other genes, and its malfunction leads to misplaced structures, such as legs instead of antennae. The observation that a change in gene regulation during development could lead to such a fantastic change in body form prompted some scientists to consider whether these types of mutations could contribute to evolution by generating novel body shapes. Ultimately, this line of inquiry gave rise to the field of evolutionary developmental biology, so-called evo-devo, which will be further discussed in Concept In this section, we have seen how a carefully orchestrated program of sequential gene regulation controls the chapter 18 Regulation of Gene Expression 385

13 transformation of a fertilized egg into a multicellular organism. The program is carefully balanced between turning on the genes for differentiation in the right place and turning off other genes. Even when an organism is fully developed, gene expression is regulated in a similarly fine-tuned manner. In the final section of the chapter, we ll consider how fine this tuning is by looking at how specific changes in expression of just a few genes can lead to the development of cancer. Concept Check MAKE CONNECTIONS As you learned in Chapter 12, mitosis gives rise to two daughter cells that are genetically identical to the parent cell. Yet you, the product of many mitotic divisions, are not composed of identical, zygote-like cells. Why? 2. MAKE CONNECTIONS Explain how the signaling molecules released by an embryonic cell can induce changes in a neighboring cell without entering the cell. (See Figures and ) 3. How do fruit fly maternal effect genes determine the polarity of the egg and the embryo? 4. WHAT IF? In Figure 18.17b, the lower cell is synthesizing signaling molecules, whereas the upper cell is expressing receptors for these molecules. In terms of gene regulation and cytoplasmic determinants, explain how these cells came to synthesize different molecules. For suggested answers, see Appendix A. Concept 18.5 Cancer results from genetic changes that affect cell cycle control In Concept 12.3, we considered cancer as a type of disease in which cells escape from the control mechanisms that normally limit their growth. Now that we have discussed the molecular basis of gene expression and its regulation, we are ready to look at cancer more closely. The gene regulation systems that go wrong during cancer turn out to be the very same systems that play important roles in embryonic development, the immune response, and many other biological processes. Thus, research into the molecular basis of cancer has both benefited from and informed many other fields of biology. Types of Genes Associated with Cancer The genes that normally regulate cell growth and division during the cell cycle include genes for growth factors, their receptors, and the intracellular molecules of signaling pathways. (To review cell signaling, see Concept 11.2; for regulation of the cell cycle, see Concept 12.3.) Mutations that alter any of these genes in somatic cells can lead to cancer. The agent of such change can be random spontaneous mutation. However, it is also likely that many cancer-causing mutations result from environmental influences, such as chemical carcinogens, X-rays and other high-energy radiation, and some viruses. Cancer research led to the discovery of cancer-causing genes called oncogenes (from the Greek onco, tumor) in certain types of viruses. Subsequently, close counterparts of viral oncogenes were found in the genomes of humans and other animals. The normal versions of the cellular genes, called proto-oncogenes, code for proteins that stimulate normal cell growth and division. How might a proto-oncogene a gene that has an essential function in normal cells become an oncogene, a cancercausing gene? In general, an oncogene arises from a genetic change that leads to an increase either in the amount of the proto-oncogene s protein product or in the intrinsic activity of each protein molecule. The genetic changes that convert proto-oncogenes to oncogenes fall into three main categories: movement of DNA within the genome, amplification of a proto-oncogene, and point mutations in a control element or in the proto-oncogene itself (Figure 18.23). Figure Genetic changes that can turn proto-oncogenes into oncogenes. Proto-oncogene Proto-oncogene Proto-oncogene Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene Point mutation within a control element Point mutation within the gene New promoter Oncogene Oncogene Oncogene Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Hyperactive or degradationresistant protein 386 Unit three Genetics

14 Cancer cells are frequently found to contain chromosomes that have broken and rejoined incorrectly, translocating fragments from one chromosome to another (see Figure 15.14). Having learned how gene expression is regulated, you can now see the possible consequences of such translocations. If a translocated proto-oncogene ends up near an especially active promoter (or other control element), its transcription may increase, making it an oncogene. The second main type of genetic change, amplification, increases the number of copies of the proto-oncogene in the cell through repeated gene duplication (discussed in Concept 21.5). The third possibility is a point mutation either in the promoter or an enhancer that controls a proto-oncogene, causing an increase in its expression, or in the coding sequence of the proto-oncogene, changing the gene s product to a protein that is more active or more resistant to degradation than the normal protein. These mechanisms can lead to abnormal stimulation of the cell cycle and put the cell on the path to becoming a cancer cell. In addition to genes whose products normally promote cell division, cells contain genes whose normal products inhibit cell division. Such genes are called tumor-suppressor genes since the proteins they encode help prevent uncontrolled cell growth. Any mutation that decreases the normal activity of a tumor-suppressor protein may contribute to the onset of cancer, in effect stimulating growth through the absence of suppression. The protein products of tumor-suppressor genes have various functions. Some repair damaged DNA, a function that Figure Normal and mutant cell cycle stimulating pathway. (a) The normal pathway is triggered by 1 a growth factor that binds to 2 its receptor in the plasma membrane. The signal is relayed to 3 a G protein called Ras. Like all G proteins, Ras is active when GTP is bound to it. Ras passes the signal to 4 a series of protein kinases. The last kinase activates 5 a transcription factor (activator) that turns on one or more genes for 6 a protein that stimulates the cell cycle. (b) If a mutation makes Ras or any other pathway component abnormally active, excessive cell division and cancer may result. 1 Growth factor prevents the cell from accumulating cancer-causing mutations. Other tumor-suppressor proteins control the adhesion of cells to each other or to the extracellular matrix; proper cell anchorage is crucial in normal tissues and is often absent in cancers. Still other tumor-suppressor proteins are components of cellsignaling pathways that inhibit the cell cycle. Interference with Normal Cell-Signaling Pathways The proteins encoded by many proto-oncogenes and tumorsuppressor genes are components of cell-signaling pathways. Let s take a closer look at how such proteins function in normal cells and what goes wrong with their function in cancer cells. We will focus on the products of two key genes, the ras proto-oncogene and the p53 tumor-suppressor gene. Mutations in ras occur in about 30% of human cancers, and mutations in p53 in more than 50%. The Ras protein, encoded by the ras gene (named for rat sarcoma, a connective tissue cancer), is a G protein that relays a signal from a growth factor receptor on the plasma membrane to a cascade of protein kinases (see Figures 11.8 and 11.10). The cellular response at the end of the pathway is the synthesis of a protein that stimulates the cell cycle (Figure 18.24a). Normally, such a pathway will not operate unless triggered by the appropriate growth factor. But certain mutations in the ras gene can lead to production of a hyperactive Ras protein that triggers the kinase cascade even in 3 G protein P P P P P P 2 Receptor Ras GTP NUCLEUS 5 Transcription factor (activator) 6 Protein that stimulates the cell cycle 4 Protein kinases Normal cell division (a) Normal cell cycle stimulating pathway. MUTATION Ras GTP NUCLEUS Transcription factor (activator) Hyperactive Ras protein (product of oncogene) issues signals on its own, whether or not growth factor is bound to the receptor. Protein expressed all the time (overexpression) Increased cell division (b) Mutant cell cycle stimulating pathway. chapter 18 Regulation of Gene Expression 387

15 the absence of growth factor, resulting in increased cell division (Figure 18.24b). In fact, hyperactive versions or excess amounts of any of the pathway s components can have the same outcome: excessive cell division. Figure 18.25a shows a pathway in which an intracellular signal leads to the synthesis of a protein that suppresses the cell cycle. In this case, the signal is damage to the cell s DNA, perhaps as the result of exposure to ultraviolet light. Operation of this signaling pathway blocks the cell cycle until the damage has been repaired. Otherwise, the damage might contribute to tumor formation by causing mutations or chromosomal abnormalities. Thus, the genes for the components of the pathway act as tumor-suppressor genes. The p53 gene, named for the 53,000-dalton molecular weight of its protein product, is a tumor-suppressor gene. The protein it encodes is a specific transcription factor that promotes the synthesis of cell cycle inhibiting proteins. That is why a mutation that knocks out the p53 gene, like a mutation that leads to a hyperactive Ras protein, can lead to excessive cell growth and cancer (Figure 18.25b). The p53 gene has been called the guardian angel of the genome. Once the gene is activated for example, by DNA damage the p53 protein functions as an activator for several other genes. Often it activates a gene called p21, whose product halts the cell cycle by binding to cyclindependent kinases, allowing time for the cell to repair the DNA. Researchers recently showed that p53 also activates Figure Normal and mutant cell cycle inhibiting pathway. (a) In the normal pathway, 1 DNA damage is an intracellular signal that is passed via 2 protein kinases, leading to activation of 3 p53. Activated p53 promotes 4 transcription of the gene for 5 a protein that inhibits the cell cycle. The resulting suppression of cell division ensures that the damaged DNA is not replicated. If the DNA damage is irreparable, then the p53 signal leads to programmed cell death (apoptosis). (b) Mutations causing deficiencies in any pathway component can contribute to the development of cancer.? Explain whether a cancercausing mutation in a tumorsuppressor gene, such as p53, is more likely to be a recessive or a dominant mutation. 388 Unit three Genetics expression of a group of mirnas, which in turn inhibit the cell cycle. In addition, the p53 protein can turn on genes directly involved in DNA repair. Finally, when DNA damage is irreparable, p53 activates suicide genes, whose protein products bring about programmed cell death (apoptosis; see Figure 11.20). Thus, p53 acts in several ways to prevent a cell from passing on mutations due to DNA damage. If mutations do accumulate and the cell survives through many divisions as is more likely if the p53 tumor-suppressor gene is defective or missing cancer may ensue. The many functions of p53 suggest a complex picture of regulation in normal cells, one that we do not yet fully understand. A recent study may underscore the protective role of p53 while illuminating a long-standing research question: Why is cancer so rare among elephants? The incidence of cancer among elephants in zoo-based studies has been estimated at about 3%, compared to closer to 30% for humans. Genome sequencing revealed that elephants have 20 copies of the p53 gene, compared to one copy in humans, other mammals, and even manatees, elephants closest living relatives. There are undoubtedly other underlying reasons, but the correlation between low cancer rate and extra copies of the p53 gene bears further investigation. For the present, the diagrams in Figure and Figure are an accurate view of how mutations can contribute to cancer, but we still don t know exactly how a particular cell becomes a cancer cell. As we discover previously unknown 2 Protein kinases NUCLEUS 5 Protein that inhibits the cell cycle Damaged DNA is not replicated. UV light 1 DNA damage in genome 3 Active form of p53 4 Transcription No cell division (a) Normal cell cycle inhibiting pathway UV light DNA damage in genome Defective or missing MUTATION transcription factor, such as p53, cannot activate transcription. (b) Mutant cell cycle inhibiting pathway Inhibitory protein absent Cell cycle is not inhibited. Increased cell division

16 aspects of gene regulation, it is informative to study their role in the onset of cancer. Such studies have shown, for instance, that DNA methylation and histone modification patterns differ in normal and cancer cells and that mirnas probably participate in cancer development. While we ve learned a lot about cancer by studying cell-signaling pathways, there is still much more to learn. The Multistep Model of Cancer Development More than one somatic mutation is generally needed to produce all the changes characteristic of a full-fledged cancer cell. This may help explain why the incidence of cancer increases greatly with age. If cancer results from an accumulation of mutations and if mutations occur throughout life, then the longer we live, the more likely we are to develop cancer. The model of a multistep path to cancer is well supported by studies of one of the best-understood types of human cancer: colorectal cancer, which affects the colon and/or rectum. About 140,000 new cases of colorectal cancer are diagnosed each year in the United States, and the disease causes 50,000 deaths per year. Like most cancers, colorectal cancer develops gradually (Figure 18.26). The first sign is often a polyp, a small, benign growth in the colon lining. The cells of the polyp look normal, although they divide unusually frequently. The tumor grows and may eventually become malignant, invading other tissues. The development of a malignant tumor is paralleled by a gradual accumulation of mutations that convert proto-oncogenes to oncogenes and knock out tumor-suppressor genes. A ras oncogene and a mutated p53 tumor-suppressor gene are often involved. About half a dozen changes must occur at the DNA level for a cell to become fully cancerous. These changes usually include the appearance of at least one active oncogene and the mutation or loss of several tumor-suppressor genes. Furthermore, since mutant tumor-suppressor alleles are usually recessive, in most cases mutations must knock out both alleles in a cell s genome to block tumor suppression. (Most oncogenes, on the other hand, behave as dominant alleles.) Since we understand the progression of this type of cancer, routine screenings (colonoscopies, for example) are recommended to identify and remove any suspicious polyps. The colorectal cancer mortality rate has been declining for the past 20 years due to increased screening and improved treatments. Treatments for other cancers have improved as well. Advances in the sequencing of DNA and mrna allow medical researchers to compare the genes expressed by different types of tumors and by the same type in different people. These comparisons have led to personalized treatments based on the molecular characteristics of a person s tumor. Breast cancer is the second most common form of cancer in the United States, and the first among women. Each year, this cancer strikes over 230,000 women (and some men) in the United States and kills 40,000 (450,000 worldwide). A major problem with understanding breast cancer is its heterogeneity: Tumors differ in significant ways. Identifying differences between types of breast cancer is expected to improve treatment and decrease the mortality rate. In 2012, The Cancer Genome Atlas Network, sponsored by the National Institutes of Health, published the results of a multi-team effort that used a genomics approach to profile subtypes of breast cancer based on their molecular signatures. Four major types of breast cancer were identified (Figure 18.27). It is now routine to screen for the presence of particular signaling receptors in any breast cancer tumors, and individuals with breast cancer, along with their physicians, can now make more informed decisions about their treatments. Figure A multistep model for the development of colorectal cancer. This type of cancer is one of the best understood. Changes in a tumor parallel a series of genetic changes, including mutations affecting several tumor-suppressor genes (such as p53) and the ras proto-oncogene. Mutations of tumor-suppressor genes often entail loss (deletion) of the gene. APC stands for adenomatous polyposis coli, and SMAD4 is a gene involved in signaling that results in apoptosis. Colon Colon wall 1 Loss of tumorsuppressor gene ras oncogene 2 Activation of APC (or other) 4 Loss of tumor-suppressor gene p53 Normal colon epithelial cells Small benign growth (polyp) 3 Loss of tumor-suppressor gene SMAD4 Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma) chapter 18 Regulation of Gene Expression 389

17 Figure Make Connections Genomics, Cell Signaling, and Cancer Modern medicine that melds genome-wide molecular studies with cell-signaling research is transforming the treatment of many diseases, such as breast cancer. Using microarray analysis (see Figure 20.13) and other techniques, researchers measured the relative levels of mrna transcripts for every gene in hundreds of breast cancer tumor samples. They identified four major subtypes of breast cancer that differ in their expression of three signal receptors involved in regulating cell growth and division: Estrogen receptor alpha (ERα) Progesterone receptor (PR) HER2, a type of receptor called a receptor tyrosine kinase (see Figure 11.8) (ERα and PR are steroid receptors; see Figure 11.9.) The absence or excess expression of these receptors can cause aberrant cell signaling, leading in some cases to inappropriate cell division, which may contribute to cancer (see Figure 18.24). A research scientist examines DNA sequencing data from breast cancer samples. Milk duct Normal Breast Cells in a Milk Duct In a normal breast cell, the three signal receptors are at normal levels (indicated by +): ERα+ PR+ HER2+ Estrogen receptor alpha (ERα) Progesterone receptor (PR) Duct interior Mammary gland lobule Epithelial milk-secreting cell HER2 receptor Support cell Extracellular matrix 390 Unit three Genetics

18 Breast Cancer Subtypes Each breast cancer subtype is characterized by the overexpression (indicated by ++ or +++) or absence ( ) of three signal receptors: ERα, PR, and HER2. Breast cancer treatments are becoming more effective because they can be tailored to the specific cancer subtype. Luminal A Luminal B Basal-like ERα ERα+++ PR++ HER2 40% of breast cancers Best prognosis ERα++ PR++ HER2 (shown here); some HER % of breast cancers Poorer prognosis than luminal A subtype Both luminal subtypes overexpress ERα (luminal A more than luminal B) and PR, and usually lack expression of HER2. Both can be treated with drugs that target ERα and inactivate it, the most well-known drug being tamoxifen. These subtypes can also be treated with drugs that inhibit estrogen synthesis. PR ERα PR HER % of breast cancers More aggressive; poorer prognosis than other subtypes The basal-like subtype is "triple negative" it does not express ERα, PR, or HER2. It often has a mutation in the tumor-suppressor gene BRCA1 (see Concept 18.5). Treatments that target ER, PR, or HER2 are not effective, but new treatments are being developed. Currently, patients are treated with cytotoxic chemotherapy, which selectively kills fast-growing cells. HER2 HER2 ERα PR HER % of breast cancers Poorer prognosis than luminal A subtype The HER2 subtype overexpresses HER2. Because it does not express either ERα or PR at normal levels, the cells are unresponsive to therapies that target those two receptors. However, patients with the HER2 subtype can be treated with Herceptin, an antibody protein that inactivates HER2 (see Concept 12.3). Signaling molecule HER2 receptor Dimer ATP ADP P P P P P P Response (cell division) 1 Signaling molecules 2 Binding of 3 Formation of a 4 Each monomer 5 The signal is transduced through (such as a growth factor) bind to HER2 receptor monomers (single receptor proteins). signaling molecules causes two receptor monomers to associate closely with each other, forming a dimer. dimer activates each monomer. adds phosphate from ATP to the other monomer, triggering a signal transduction pathway. the cell, which leads to a cellular response in this case, turning on genes that trigger cell division. HER2 cells have up to 100 times as many HER2 receptors as normal cells, so they undergo uncontrolled cell division. Treatment with Herceptin for the HER2 subtype Herceptin molecule HER2 receptors MAKE CONNECTIONS When researchers compared gene expression in normal breast cells and cells from breast cancers, they found that the genes showing the most significant differences in expression encoded signal receptors, as shown here. Given what you learned in Chapters 11, 12, and this chapter, explain why this result is not surprising. 1 The drug Herceptin binds to 2 In certain patients with the HER2 HER2 receptors in place of the usual signaling molecules. subtype, signaling is blocked and excessive cell division does not occur. chapter 18 Regulation of Gene Expression 391

19 Inherited Predisposition and Environmental Factors Contributing to Cancer The fact that multiple genetic changes are required to produce a cancer cell helps explain the observation that cancers can run in families. An individual inheriting an oncogene or a mutant allele of a tumor-suppressor gene is one step closer to accumulating the necessary mutations for cancer to develop than is an individual without any such mutations. Geneticists are working to identify inherited cancer alleles so that predisposition to certain cancers can be detected early in life. About 15% of colorectal cancers, for example, involve inherited mutations. Many of these affect the tumor-suppressor gene called adenomatous polyposis coli, or APC (see Figure 18.26). This gene has multiple functions in the cell, including regulation of cell migration and adhesion. Even in patients with no family history of the disease, the APC gene is mutated in 60% of colorectal cancers. In these individuals, new mutations must occur in both APC alleles before the gene s function is lost. Since only 15% of colorectal cancers are associated with known inherited mutations, researchers continue to try to identify markers that could predict the risk of developing this type of cancer. Given the prevalence and significance of breast cancer, it is not surprising that it was one of the first cancers for which the role of inheritance was investigated. It turns out that for 5 10% of patients with breast cancer, there is evidence of a strong inherited predisposition. Geneticist Mary-Claire King began working on this problem in the mid-1970s. After 16 years of research, she convincingly demonstrated that mutations in one gene BRCA1 were associated with increased susceptibility to breast cancer, a finding that flew in the face of medical opinion at the time. (BRCA stands for breast cancer.) Mutations in that gene or a gene called BRCA2 are found in at least half of inherited breast cancers, and tests using DNA sequencing can detect these mutations. A woman who inherits one mutant BRCA1 allele has a 60% probability of developing breast cancer before the age of 50, compared with only a 2% probability for an individual homozygous for the normal allele. BRCA1 and BRCA2 are considered tumor-suppressor genes because their wild-type alleles protect against breast cancer and their mutant alleles are recessive. (Note that mutations in BRCA1 are commonly found in the genomes of cells from basal-like breast cancers; see Figure ) The BRCA1 and BRCA2 proteins both appear to function in the cell s DNA damage repair pathway. More is known about BRCA2, which, along with another protein, helps repair breaks that occur in both strands of DNA; this repair function is crucial for maintaining undamaged DNA. Interview with Mary-Claire King: Discovering breast cancer genes Because DNA breakage can contribute to cancer, it makes sense that the risk of cancer can be lowered by minimizing exposure to DNA-damaging agents, such as the ultraviolet radiation in sunlight and chemicals found in cigarette smoke. Novel genomics-based analyses of specific cancers, such as the approach described in Figure 18.27, are contributing to both early diagnosis and development of treatments that interfere with expression of key genes in tumors. Ultimately, such approaches are expected to lower the death rate from cancer. The Role of Viruses in Cancer The study of genes associated with cancer, inherited or not, increases our basic understanding of how disruption of normal gene regulation can result in this disease. In addition to the mutations and other genetic alterations described in this section, a number of tumor viruses can cause cancer in various animals, including humans. In fact, one of the earliest breakthroughs in understanding cancer came in 1911, when Peyton Rous, an American pathologist, discovered a virus that causes cancer in chickens. The Epstein-Barr virus, which causes infectious mononucleosis, has been linked to several types of cancer in humans, notably Burkitt s lymphoma. Papillomaviruses are associated with cancer of the cervix, and a virus called HTLV-1 causes a type of adult leukemia. Viruses play a role in about 15% of the cases of human cancer. Viruses may at first seem very different from mutations as a cause of cancer. However, we now know that viruses can interfere with gene regulation in several ways if they integrate their genetic material into the DNA of a cell. Viral integration may donate an oncogene to the cell, disrupt a tumor-suppressor gene, or convert a proto-oncogene to an oncogene. Some viruses produce proteins that inactivate p53 and other tumor-suppressor proteins, making the cell more prone to becoming cancerous. Viruses are powerful biological agents; you ll learn more about them in Chapter 19. Animation: Causes of Cancer Concept Check Cancer-promoting mutations are likely to have different effects on the activity of proteins encoded by proto-oncogenes than they do on proteins encoded by tumor-suppressor genes. Explain. 2. Under what circumstances is cancer considered to have a hereditary component? 3. MAKE CONNECTIONS The p53 protein can activate genes involved in apoptosis. Review Concept 11.5, and discuss how mutations in genes coding for proteins that function in apoptosis could contribute to cancer. For suggested answers, see Appendix A. 392 Unit three Genetics

20 18 Chapter Review Go to MasteringBiology for Videos, Animations, Vocab Self-Quiz, Practice Tests, and more in the Study Area. Concept 18.2 Summary of Key Concepts Eukaryotic gene expression is regulated at many stages (pp ) Concept 18.1 Bacteria often respond to environmental change by regulating transcription (pp ) VOCAB SELF-QUIZ goo.gl/6u55ks Cells control metabolism by regulating enzyme activity or the expression of genes coding for enzymes. In bacteria, genes are often clustered into operons, with one promoter serving several adjacent genes. An operator site on the DNA switches the operon on or off, resulting in coordinate regulation of the genes. Chromatin modification Genes in highly compacted chromatin are generally not transcribed. Histone acetylation loosens chromatin structure, enhancing transcription. DNA methylation generally reduces transcription. Operon CHROMATIN MODIFICATION Promoter Genes A B TRANSCRIPTION C Operator RNA polymerase RNA PROCESSING A B mrna DEGRADATION C Polypeptides Both repressible and inducible operons are examples of negative gene regulation. In either type of operon, binding of a specific repressor protein to the operator shuts off transcription. (The repressor is encoded by a separate regulatory gene.) In a repressible operon, the repressor is active when bound to a corepressor, usually the end product of an anabolic pathway. Repressible operon: Genes expressed Transcription Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Coordinate regulation: Enhancer for Enhancer for liver-specific genes lens-specific genes Genes not expressed TRANSLATION RNA processing Alternative RNA splicing: Primary RNA transcript mrna OR PROTEIN PROCESSING AND DEGRADATION Translation mrna degradation Each mrna has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs.? Promoter Initiation of translation can be controlled via regulation of initiation factors. Protein processing and degradation Protein processing and degradation are subject to regulation. Describe what must happen in a cell for a gene specific to that cell type to be transcribed. Genes Operator Inactive repressor: no corepressor present Active repressor: corepressor bound Corepressor In an inducible operon, binding of an inducer to an innately active repressor inactivates the repressor and turns on transcription. Inducible enzymes usually function in catabolic pathways. Concept 18.3 Noncoding RNAs play multiple roles in controlling gene expression (pp ) CHROMATIN MODIFICATION Inducible operon: Genes not expressed Promoter Operator Genes expressed Translation RNA PROCESSING Genes Active repressor: no inducer present mirna or sirna can block the translation of specific mrnas. Inducer mrna DEGRADATION Inactive repressor: inducer bound Some operons are also subject to positive gene regulation via a stimulatory activator protein, such as camp receptor protein (CRP), which, when activated by cyclic AMP, binds to a site within the promoter and stimulates transcription.? TRANSCRIPTION Chromatin modification Small and/or large noncoding RNAs can promote heterochromatin formation in certain regions, which can block transcription. Compare and contrast the roles of a corepressor and an inducer in negative regulation of an operon. TRANSLATION PROTEIN PROCESSING AND DEGRADATION mrna degradation mirna or sirna can target specific mrnas for destruction.? Why are mirnas called noncoding RNAs? Explain how they participate in gene regulation. chapter 18 Regulation of Gene Expression 393

21 Concept 18.4 A program of differential gene expression leads to the different cell types in a multicellular organism (pp )? Embryonic cells become committed to a certain fate (determination), and undergo differentiation, becoming specialized in structure and function for their determined fate. Cells differ in structure and function not because they contain different genomes but because they express different genes. Morphogenesis encompasses the processes that give shape to the organism and its various structures. Localized cytoplasmic determinants in the unfertilized egg are distributed differentially to daughter cells, where they regulate the expression of those cells developmental fate. In the process called induction, signaling molecules from embryonic cells cause transcriptional changes in nearby target cells. Differentiation is heralded by the appearance of tissue-specific proteins, which enable differentiated cells to carry out their specialized roles. In animals, pattern formation, the development of a spatial organization of tissues and organs, begins in the early embryo. Positional information, the molecular cues that control pattern formation, tells a cell its location relative to the body s axes and to other cells. In Drosophila, gradients of morphogens encoded by maternal effect genes determine the body axes. For example, the gradient of Bicoid protein determines the anterior-posterior axis. Describe the two main processes that cause embryonic cells to head down different pathways to their final fates. Concept 18.5 Cancer results from genetic changes that affect cell cycle control (pp ) The products of proto-oncogenes and tumor-suppressor genes control cell division. A DNA change that makes a proto-oncogene excessively active converts it to an oncogene, which may promote excessive cell division and cancer. A tumor-suppressor gene encodes a protein that inhibits abnormal cell division. A mutation that reduces the activity of its protein product may lead to excessive cell division and cancer. Many proto-oncogenes and tumor-suppressor genes encode components of growth-stimulating and growth-inhibiting signaling pathways, respectively, and mutations in these genes can interfere with normal cell-signaling pathways. A hyperactive version of a protein in a stimulatory pathway, such as Ras (a G protein), functions as an oncogene protein. A defective version of a protein in an inhibitory pathway, such as p53 (a transcription activator), fails to function as a tumor suppressor. Protein overexpressed Cell cycle overstimulated EFFECTS OF MUTATIONS Increased cell division Protein absent Cell cycle not inhibited In the multistep model of cancer development, normal cells are converted to cancer cells by the accumulation of mutations affecting proto-oncogenes and tumor-suppressor genes. Technical advances in DNA and mrna sequencing are enabling cancer treatments that are more individually based.? Genomics-based studies have resulted in researchers proposing four subtypes of breast cancer, based on expression of genes by tumor cells. Individuals who inherit a mutant allele of a proto-oncogene or tumor-suppressor gene have a predisposition to develop a particular cancer. Certain viruses promote cancer by integration of viral DNA into a cell s genome. Compare the usual functions of proteins encoded by proto-oncogenes with those of proteins encoded by tumor-suppressor genes. Test Your Understanding Level 1: Knowledge/Comprehension 1. If a particular operon encodes enzymes for making an essential amino acid and is regulated like the trp operon, then (A) the amino acid inactivates the repressor. (B) the repressor is active in the absence of the amino acid. (C) the amino acid acts as a corepressor. (D) the amino acid turns on transcription of the operon. 2. Muscle cells differ from nerve cells mainly because they (A) express different genes. (B) contain different genes. (C) use different genetic codes. (D) have unique ribosomes. 3. The functioning of enhancers is an example of (A) a eukaryotic equivalent of prokaryotic promoter functioning. (B) transcriptional control of gene expression. (C) the stimulation of translation by initiation factors. (D) post-translational control that activates certain proteins. 4. Cell differentiation always involves (A) transcription of the myod gene. (B) the movement of cells. (C) the production of tissue-specific proteins. (D) the selective loss of certain genes from the genome. 5. Which of the following is an example of post-transcriptional control of gene expression? (A) the addition of methyl groups to cytosine bases of DNA (B) the binding of transcription factors to a promoter (C) the removal of introns and alternative splicing of exons (D) gene amplification contributing to cancer Level 2: Application/Analysis PRACTICE TEST goo.gl/cuygkd 6. What would occur if the repressor of an inducible operon were mutated so it could not bind the operator? (A) irreversible binding of the repressor to the promoter (B) reduced transcription of the operon s genes (C) buildup of a substrate for the pathway controlled by the operon (D) continuous transcription of the operon s genes 7. Absence of bicoid mrna from a Drosophila egg leads to the absence of anterior larval body parts and mirror-image duplication of posterior parts. This is evidence that the product of the bicoid gene (A) normally leads to formation of head structures. (B) normally leads to formation of tail structures. (C) is transcribed in the early embryo. (D) is a protein present in all head structures. 8. Which of the following statements about the DNA in one of your brain cells is true? (A) Most of the DNA codes for protein. (B) The majority of genes are likely to be transcribed. (C) It is the same as the DNA in one of your liver cells. (D) Each gene lies immediately adjacent to an enhancer. 394 Unit three Genetics

22 9. Within a cell, the amount of protein made using a given mrna molecule depends partly on (A) the degree of DNA methylation. (B) the rate at which the mrna is degraded. (C) the number of introns present in the mrna. (D) the types of ribosomes present in the cytoplasm. 10. Proto-oncogenes can change into oncogenes that cause cancer. Which of the following best explains the presence of these potential time bombs in eukaryotic cells? (A) Proto-oncogenes first arose from viral infections. (B) Proto-oncogenes are mutant versions of normal genes. (C) Proto-oncogenes are genetic junk. (D) Proto-oncogenes normally help regulate cell division. Level 3: Synthesis/Evaluation 11. DRAW IT The diagram below shows five genes, including their enhancers, from the genome of a certain species. Imagine that yellow, blue, green, black, red, and purple activator proteins exist that can bind to the appropriately color-coded control elements in the enhancers of these genes. Enhancer Promoter Gene 1 an androgen in these cancer cells. Describe one or more experiments to test this hypothesis. (See Figure 11.9 to review the action of these steroid hormones.) 14. SCIENCE, TECHNOLOGY, AND SOCIETY Trace amounts of dioxin were present in Agent Orange, a defoliant sprayed on vegetation during the Vietnam War. Animal tests suggest that dioxin can cause birth defects, cancer, liver and thymus damage, and immune system suppression, sometimes leading to death. But the animal tests are equivocal; a hamster is not affected by a dose that can kill a guinea pig. Dioxin acts like a steroid hormone, entering a cell and binding to a cytoplasmic receptor that then binds the cell s DNA. (a) Discuss how this mechanism might help explain the variety of dioxin s effects on different body systems and in different animals. (b) Discuss how you might determine whether a type of illness is related to dioxin exposure. Next, discuss how you might determine whether a particular individual became ill as a result of exposure to dioxin. Which would be more difficult to demonstrate? Why? 15. WRITE ABOUT A THEME: INTERACTIONS In a short essay ( words), discuss how the processes shown in Figure 18.2 are examples of feedback mechanisms regulating biological systems in bacterial cells. 16. SYNTHESIZE YOUR KNOWLEDGE Gene 2 Gene 3 Gene 4 Gene 5 (a) Draw an X above enhancer elements (of all the genes) that would have activators bound in a cell in which only gene 5 is transcribed. Identify which colored activators would be present. (b) Draw a dot above all enhancer elements that would have activators bound in a cell in which the green, blue, and yellow activators are present. Identify which gene(s) would be transcribed. (c) Imagine that genes 1, 2, and 4 code for nerve-specific proteins, and genes 3 and 5 are skin specific. Identify which activators would have to be present in each cell type to ensure transcription of the appropriate genes. 12. EVOLUTION CONNECTION DNA sequences can act as tape measures of evolution (see Concept 5.6). Scientists analyzing the human genome sequence were surprised to find that some of the regions of the human genome that are most highly conserved (similar to comparable regions in other species) don t code for proteins. Propose a possible explanation for this observation. 13. SCIENTIFIC INQUIRY Prostate cells usually require testosterone and other androgens to survive. But some prostate cancer cells thrive despite treatments that eliminate androgens. One hypothesis is that estrogen, often considered a female hormone, may be activating genes normally controlled by The flashlight fish has an organ under its eye that emits light, which serves to startle predators and attract prey, and allows the fish to communicate with other fish. Some species can rotate the organ inside and then out, so the light appears to flash on and off. The light is actually emitted by bacteria (of the genus Vibrio) that live in the organ in a mutualistic relationship with the fish. (The bacteria receive nutrients from the fish.) The bacteria must multiply until they reach a certain density in the organ (a quorum ; see Concept 11.1), at which point they all begin emitting light at the same time. There is a group of six or so genes, called lux genes, whose gene products are necessary for light formation. Given that these bacterial genes are regulated together, propose a hypothesis for how the genes are organized and regulated. For selected answers, see Appendix A. For additional practice questions, check out the Dynamic Study Modules in MasteringBiology. You can use them to study on your smartphone, tablet, or computer anytime, anywhere! chapter 18 Regulation of Gene Expression 395

23 Superset Viruses 19 Figure 19.1 Are the viruses (reddish-purple) budding from these cells alive? Key Concepts 19.1 A virus consists of a nucleic acid surrounded by a protein coat 19.2 Viruses replicate only in host cells 19.3 Viruses and prions are formidable pathogens in animals and plants A Borrowed Life The illustration in Figure 19.1 shows a remarkable event: Human immune cells (purple) infected by human immunodeficiency viruses (HIV) are releasing more HIV viruses. These viruses (red, surrounded by a protein-studded purple membrane from the immune cell) will infect other cells. (The SEM below shows one infected cell.) By injecting its genetic information into a cell, a virus hijacks the cell, recruiting cellular machinery to manufacture many new viruses and promote further infection. Left untreated, HIV causes acquired immunodeficiency syndrome (AIDS) by destroying vital immune system cells. Compared with eukaryotic and even prokaryotic cells, viruses are much smaller and simpler in structure. Lacking the structures and metabolic machinery found in a cell, a virus is an infectious particle consisting of little more than genes packaged in a protein coat. Are viruses living or nonliving? Early on, they were considered biological chemicals; the Latin root for virus means poison. Viruses are capable of causing a wide variety of diseases, so researchers in the late 1800s saw a parallel with bacteria and proposed that viruses were the simplest of living forms. However, viruses cannot reproduce or carry out metabolic activities outside of a host cell. Most biologists studying viruses today would probably agree that they are not alive but exist in a shady area between life-forms and chemicals. The simple phrase used recently by two researchers describes them aptly enough: Viruses lead a kind of borrowed life. A human immune cell infected with HIV. New viruses (red) are budding from the plasma membrane (colorized SEM). When you see this blue icon, log in to MasteringBiology and go to the Study Area for digital resources. Get Ready for This Chapter 396

24 To a large extent, molecular biology was born in the laboratories of biologists studying viruses that infect bacteria. Experiments with these viruses provided evidence that genes are made of nucleic acids, and they were critical in working out the molecular mechanisms of the fundamental processes of DNA replication, transcription, and translation. In this chapter, we will explore the biology of viruses, beginning with their structure and then describing how they replicate. Next, we will discuss the role of viruses as diseasecausing agents, or pathogens, and conclude by considering some even simpler infectious agents called prions. Figure 19.2 Inquiry What causes tobacco mosaic disease? Experiment In the late 1800s, Martinus Beijerinck, of the Technical School in Delft, the Netherlands, investigated the properties of the agent that causes tobacco mosaic disease (then called spot disease). Concept 19.1 A virus consists of a nucleic acid surrounded by a protein coat Scientists were able to detect viruses indirectly long before they were actually able to see them. The story of how viruses were discovered begins near the end of the 19th century. 1 Extracted sap from tobacco plant with tobacco mosaic disease 2 Passed sap through a porcelain filter known to trap bacteria 3 Rubbed filtered sap on healthy tobacco plants The Discovery of Viruses: Scientific Inquiry Tobacco mosaic disease stunts the growth of tobacco plants and gives their leaves a mottled, or mosaic, coloration. In 1883, Adolf Mayer, a German scientist, discovered that he could transmit the disease from plant to plant by rubbing sap extracted from diseased leaves onto healthy plants. After an unsuccessful search for an infectious microbe in the sap, Mayer suggested that the disease was caused by unusually small bacteria that were invisible under a microscope. This hypothesis was tested a decade later by Dmitri Ivanowsky, a Russian biologist who passed sap from infected tobacco leaves through a filter designed to remove bacteria. After filtration, the sap still produced mosaic disease. But Ivanowsky clung to the hypothesis that bacteria caused tobacco mosaic disease. Perhaps, he reasoned, the bacteria were small enough to pass through the filter or made a toxin that could do so. The second possibility was ruled out when the Dutch botanist Martinus Beijerinck carried out a classic series of experiments that showed that the infectious agent in the filtered sap could replicate (Figure 19.2). In fact, the pathogen replicated only within the host it infected. In further experiments, Beijerinck showed that unlike bacteria used in the lab at that time, the mysterious agent of mosaic disease could not be cultivated on nutrient media in test tubes or petri dishes. Beijerinck imagined a replicating particle much smaller and simpler than a bacterium, and he is generally credited with being the first scientist to voice the concept of a virus. His suspicions were confirmed in 1935 when the American scientist Wendell Stanley crystallized the infectious particle, now known as tobacco mosaic virus (TMV). Subsequently, TMV and many other viruses were actually seen with the help of the electron microscope. 4 Healthy plants became infected Results When the filtered sap was rubbed on healthy plants, they became infected. Their sap, extracted and filtered, could then act as a source of infection for another group of plants. Each successive group of plants developed the disease to the same extent as earlier groups. Conclusion The infectious agent was apparently not a bacterium because it could pass through a bacterium-trapping filter. The pathogen must have been replicating in the plants because its ability to cause disease was undiluted after several transfers from plant to plant. Data from M. J. Beijerinck, Concerning a contagium vivum fluidum as cause of the spot disease of tobacco leaves, Verhandelingen der Koninkyke akademie Wettenschappen te Amsterdam 65:3 21 (1898). Translation published in English as Phytopathological Classics Number 7 (1942), American Phytopathological Society Press, St. Paul, MN. WHAT IF? If Beijerinck had observed that the infection of each group was weaker than that of the previous group and that ultimately the sap could no longer cause disease, what might he have concluded? Structure of Viruses The tiniest viruses are only 20 nm in diameter smaller than a ribosome. Millions could easily fit on a pinhead. Even the largest known virus, which has a diameter of 1,500 nanometers (1.5 µm), is barely visible under the light microscope. Stanley s discovery that some viruses could be crystallized was exciting and puzzling news. Not even the simplest of cells can aggregate into regular crystals. But if viruses are not cells, then what are they? Examining the structure of a virus more closely reveals that it is an infectious particle consisting of nucleic acid enclosed in a protein coat and, for some viruses, surrounded by a membranous envelope. chapter 19 Viruses 397

25 Viral Genomes We usually think of genes as being made of double-stranded DNA, but many viruses defy this convention. Their genomes may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA, depending on the type of virus. A virus is called a DNA virus or an RNA virus based on the kind of nucleic acid that makes up its genome. In either case, the genome is usually organized as a single linear or circular molecule of nucleic acid, although the genomes of some viruses consist of multiple molecules of nucleic acid. The smallest viruses known have only three genes in their genome, while the largest have several hundred to 2,000. For comparison, bacterial genomes contain about 200 to a few thousand genes. Capsids and Envelopes The protein shell enclosing the viral genome is called a capsid. Depending on the type of virus, the capsid may be rod-shaped, polyhedral, or more complex in shape. Capsids are built from a large number of protein subunits called capsomeres, but the number of different kinds of proteins in a capsid is usually small. Tobacco mosaic virus has a rigid, rod-shaped capsid made from over 1,000 molecules of a single type of protein arranged in a helix; rod-shaped viruses are commonly called helical viruses for this reason (Figure 19.3a). Adenoviruses, which infect the respiratory tracts of animals, have 252 identical protein molecules arranged in a polyhedral capsid with 20 triangular facets an icosahedron; thus, these and other similarly shaped viruses are referred to as icosahedral viruses (Figure 19.3b). Some viruses have accessory structures that help them infect their hosts. For instance, a membranous envelope surrounds the capsids of influenza viruses and many other viruses found in animals (Figure 19.3c). These viral envelopes, which are derived from the membranes of the host cell, contain host cell phospholipids and membrane proteins. They also contain proteins and glycoproteins of viral origin. (Glycoproteins are proteins with carbohydrates covalently attached.) Some viruses carry a few viral enzyme molecules within their capsids. Many of the most complex capsids are found among the viruses that infect bacteria, called bacteriophages, Figure 19.3 Viral structure. Viruses are made up of nucleic acid (DNA or RNA) enclosed in a protein coat (the capsid) and sometimes further wrapped in a membranous envelope. The individual protein subunits making up the capsid are called capsomeres. Although diverse in size and shape, viruses have many common structural features. (All micrographs are colorized TEMs.) RNA Capsomere DNA RNA-protein complex Protein Viral polymerase Head DNA Capsomere of capsid RNA Tail sheath Tail fiber Protein spike Membranous envelope Glycoprotein spikes nm nm (diameter) nm (diameter) nm 40 nm 40 nm 40 nm 40 nm (a) Tobacco mosaic virus has a helical capsid surrounding a single helical RNA molecule, with the overall shape of a rigid rod. (b) Adenoviruses have an icosahedral capsid with a protein spike at each vertex (corner). The capsid, made up of capsomeres, contains double-stranded DNA. (c) Influenza viruses have an outer envelope, studded with glycoprotein spikes, that contains eight double-helical RNA-protein complexes, each associated with a viral polymerase. (d) Bacteriophage T4, like other T-even phages, has a complex capsid consisting of an icosahedral head and a tail apparatus. T4's double-stranded DNA is enclosed in the head. 398 Unit three Genetics

26 or simply phages. The first phages studied included seven that infect Escherichia coli. These seven phages were named type 1 (T1), type 2 (T2), and so forth, in the order of their discovery. The three T-even phages (T2, T4, and T6) turned out to be very similar in structure. Their capsids have elongated icosahedral heads enclosing their DNA. Attached to the head is a protein tail piece with fibers by which the phages attach to a bacterial cell (Figure 19.3d). In the next section, we ll examine how these few viral parts function together with cellular components to produce large numbers of viral progeny. Concept Check VISUAL SKILLS Compare the structures of tobacco mosaic virus (TMV) and influenza virus (see Figure 19.3). 2. MAKE CONNECTIONS Bacteriophages were used to provide evidence that DNA carries genetic information (see Figure 16.4). Briefly describe the experiment carried out by Hershey and Chase, including in your description why the researchers chose to use phages. For suggested answers, see Appendix A. Concept 19.2 Viruses replicate only in host cells Viruses lack metabolic enzymes and equipment for making proteins, such as ribosomes. They are obligate intracellular parasites; in other words, they can replicate only within a host cell. It is fair to say that viruses in isolation are merely packaged sets of genes in transit from one host cell to another. Each particular virus can infect cells of only a limited number of host species, called the host range of the virus. This host specificity results from the evolution of recognition systems by the virus. Viruses usually identify host cells by a lock-and-key fit between viral surface proteins and specific receptor molecules on the outside of cells. According to one model, such receptor molecules originally carried out functions that benefited the host cell but were co-opted later by viruses as portals of entry. Some viruses have broad host ranges. For example, West Nile virus and equine encephalitis virus are distinctly different viruses that can each infect mosquitoes, birds, horses, and humans. Other viruses have host ranges so narrow that they infect only a single species. Measles virus, for instance, can infect only humans. Further more, viral infection of multicellular eukaryotes is usually limited to particular tissues. Human cold viruses infect only the cells lining the upper respiratory tract, and the HIV seen in Figure 19.1 binds to receptors present only on certain types of immune cells. General Features of Viral Replicative Cycles A viral infection begins when a virus binds to a host cell and the viral genome makes its way inside (Figure 19.4). The mechanism of genome entry depends on the type of virus and the type of host cell. For example, T-even phages use their elaborate tail apparatus to inject DNA into a bacterium (see Figure 19.3d). Other viruses are taken up by endocytosis or, in the case of enveloped viruses, by fusion of the viral envelope with the host s plasma membrane. Once the viral genome is inside, the proteins it encodes can commandeer the host, reprogramming the cell to copy the viral genome and manufacture viral proteins. The host provides the nucleotides for making viral nucleic acids, as well as enzymes, ribosomes, trnas, amino acids, ATP, and other components needed for making the viral proteins. Many DNA viruses use the DNA polymerases of the host cell to synthesize new genomes along the templates provided by the viral DNA. In contrast, to replicate their genomes, RNA viruses use virally encoded RNA polymerases that can use RNA as a template. (Uninfected cells generally make no enzymes for carrying out this process.) Figure 19.4 A simplified viral replicative cycle. A virus is an intracellular parasite that uses the equipment and small molecules of its host cell to replicate. In this simplest of viral cycles, the parasite is a DNA virus with a capsid consisting of a single type of protein. 1 The virus enters the cell and is uncoated, releasing viral DNA and capsid proteins. DNA 2 Host enzymes replicate the viral genome. Viral DNA Capsid HOST CELL Viral DNA VIRUS mrna Capsid proteins 3 Meanwhile, host enzymes transcribe the viral genome into viral mrna, which host ribosomes use to make more capsid proteins. 4 Viral genomes and capsid proteins self-assemble into new virus particles, which exit the cell. MAKE CONNECTIONS Label each of the straight gray arrows with one word representing the name of the process that is occurring. Review Figure Animation: Simplified Viral Replicative Cycle chapter 19 Viruses 399

27 After the viral nucleic acid molecules and capsomeres are produced, they spontaneously self-assemble into new viruses. In fact, researchers can separate the RNA and capsomeres of TMV and then reassemble complete viruses simply by mixing the components together under the right conditions. The simplest type of viral replicative cycle ends with the exit of hundreds or thousands of viruses from the infected host cell, a process that often damages or destroys the cell. Such cellular damage and death, as well as the body s responses to this destruction, cause many of the symptoms associated with viral infections. The viral progeny that exit a cell have the potential to infect additional cells, spreading the viral infection. There are many variations on the simplified viral replicative cycle we have just described. We will now take a look at some of these variations in bacterial viruses (phages) and animal viruses; later in the chapter, we will consider plant viruses. Replicative Cycles of Phages Phages are the best understood of all viruses, although some of them are also among the most complex. Research on phages led to the discovery that some double-stranded DNA viruses can replicate by two alternative mechanisms: the lytic cycle and the lysogenic cycle. Figure 19.5 The lytic cycle of phage T4, a virulent phage. Phage T4 has almost 300 genes, which are transcribed and translated using the host cell s machinery. One of the first phage genes translated after the viral DNA enters the host cell codes for an enzyme that degrades the host cell s DNA (step 2 ); the phage DNA is protected from breakdown because it contains a modified form of cytosine that is not recognized by the phage enzyme. The entire lytic cycle, from the phage s first contact with the cell surface to cell lysis, takes only minutes at 37 C. The Lytic Cycle A phage replicative cycle that culminates in death of the host cell is known as a lytic cycle. The term refers to the last stage of infection, during which the bacterium lyses (breaks open) and releases the phages that were produced within the cell. Each of these phages can then infect a healthy cell, and a few successive lytic cycles can destroy an entire bacterial population in just a few hours. A phage that replicates only by a lytic cycle is a virulent phage. Figure 19.5 illustrates the major steps in the lytic cycle of T4, a typical virulent phage. The Lysogenic Cycle Instead of lysing their host cells, many phages coexist with them in a state called lysogeny. In contrast to the lytic cycle, which kills the host cell, the lysogenic cycle allows replication of the phage genome without destroying the host. Phages capable of using both modes of replicating within a bacterium are called temperate phages. A temperate phage called lambda, written with the Greek letter l, has been widely used in biological research. Phage l resembles T4, but its tail has only one short tail fiber. Infection of an E. coli cell by phage l begins when the phage binds to the surface of the cell and injects its linear DNA 1 Attachment. The T4 phage uses its tail fibers to bind to specific surface proteins on an E. coli cell that act as receptors. 5 Release. The phage directs production of an enzyme that damages the bacterial cell wall, allowing fluid to enter. The cell swells and finally bursts, releasing 100 to 200 phage particles. 2 Entry of phage DNA and degradation of host DNA. The sheath of the tail contracts, injecting the phage DNA into the cell and leaving an empty capsid outside. The cell s DNA is hydrolyzed. Phage assembly Head Tail Animation: Phage Lytic Cycle 400 Unit three Genetics Tail fibers 4 Self-assembly. Three separate sets of proteins self-assemble to form phage heads, tails, and tail fibers. The phage genome is packaged inside the capsid as the head forms. 3 Synthesis of viral genomes and proteins. The phage DNA directs production of phage proteins and copies of the phage genome by host and viral enzymes, using components within the cell.

28 genome (Figure 19.6). Within the host, the l DNA molecule forms a circle. What happens next depends on the replicative mode: lytic cycle or lysogenic cycle. During a lytic cycle, the viral genes immediately turn the host cell into a l-producing factory, and the cell soon lyses and releases its virus progeny. During a lysogenic cycle, however, the l DNA molecule is incorporated into a specific site on the E. coli chromosome by viral proteins that break both circular DNA molecules and join them to each other. When integrated into the bacterial chromosome in this way, the viral DNA is known as a prophage. One prophage gene codes for a protein that prevents transcription of most of the other prophage genes. Thus, the phage genome is mostly silent within the bacterium. Every time the E. coli cell prepares to divide, it replicates the phage DNA along with its own chromosome such that each daughter cell inherits a prophage. A single infected cell can quickly give rise to a large population of bacteria carrying the virus in prophage form. This mechanism enables viruses to propagate without killing the host cells on which they depend. The term lysogenic signifies that prophages are capable of generating active phages that lyse their host cells. This occurs when the l genome (or that of another temperate phage) is induced to exit the bacterial chromosome and initiate a lytic cycle. An environmental signal, such as a certain chemical or high-energy radiation, usually triggers the switchover from the lysogenic to the lytic mode. In addition to the gene for the viral protein that prevents transcription, a few other prophage genes may be expressed during lysogeny. Expression of these genes may alter the host s phenotype, a phenomenon that can have important medical significance. For example, the three species of bacteria that cause the human diseases diphtheria, botulism, and scarlet fever would not be so harmful to humans without certain prophage genes that cause the host bacteria to make toxins. And the difference between the E. coli strain in our intestines and the O157:H7 strain that has caused several deaths by food poisoning appears to be the presence of toxin genes of prophages in the O157:H7 strain. Bacterial Defenses Against Phages After reading about the lytic cycle, you may have wondered why phages haven t exterminated all bacteria. Lysogeny is one major reason why bacteria have been spared from extinction caused by phages. Bacteria also have their own defenses against phages. First, natural selection favors bacterial mutants with surface proteins that are no longer recognized as receptors by a particular type of phage. Second, when phage DNA does enter a bacterium, the DNA Figure 19.6 The lytic and lysogenic cycles of phage L, a temperate phage. After entering the bacterial cell and circularizing, the l DNA can immediately initiate the production of a large number of progeny phages (lytic cycle) or integrate into the bacterial chromosome (lysogenic cycle). In most cases, phage l follows the lytic pathway, which is similar to that detailed in Figure However, once a lysogenic cycle begins, the prophage may be carried in the host cell s chromosome for many generations. Phage l has one main tail fiber, which is short. Phage DNA The phage attaches to a host cell and injects its DNA. Daughter cell with prophage Phage Tail fiber Phage DNA circularizes. Many cell divisions produce a large population of bacteria infected with the prophage. Bacterial chromosome Occasionally, a prophage exits the bacterial chromosome, initiating a lytic cycle. Lytic cycle Lysogenic cycle The cell lyses, releasing phages. Certain factors determine whether lytic cycle is induced OR lysogenic cycle is entered Prophage The bacterium reproduces normally, copying the prophage and transmitting it to daughter cells. New phage DNA and proteins are synthesized and self-assemble into phages. Phage DNA integrates into the bacterial chromosome, becoming a prophage. Animation: Phage Lysogenic and Lytic Cycles chapter 19 Viruses 401

29 often is identified as foreign and cut up by cellular enzymes called restriction enzymes, which are so named because they restrict a phage s ability to replicate within the bacterium. (Restriction enzymes are used in molecular biology and DNA cloning techniques; see Concept 20.1.) The bacterium s own DNA is methylated in a way that prevents attack by its own restriction enzymes. A third defense is a system present in both bacteria and archaea called the CRISPR-Cas system. The CRISPR-Cas system was discovered during a study of repetitive DNA sequences present in the genomes of many prokaryotes. These sequences, which puzzled scientists, were named clustered regularly interspaced short palindromic repeats (CRISPRs) because each sequence read the same forward and backward (a palindrome), with different stretches of spacer DNA in between the repeats. At first, scientists assumed the spacer DNA sequences were random and meaningless, but analysis by several research groups showed that each spacer sequence corresponded to DNA from a particular phage that had infected the cell. Further studies revealed that particular nuclease proteins interact with the CRISPR region. These nucleases, called Cas (CRISPR-associated) proteins, can identify and cut phage DNA, thereby defending the bacterium against phage infection. When a phage infects a bacterial cell that has the CRISPR- Cas system, the DNA of the invading phage is integrated into the genome between two repeat sequences. If the cell survives the infection, any further attempt by the same type of phage to infect this cell (or its offspring) triggers transcription of the CRISPR region into RNA molecules (Figure 19.7). These RNAs are cut into pieces and then bound by Cas proteins. The Cas protein uses a portion of the phage-related RNA as a homing device to identify the invading phage DNA and cut it, leading to its destruction. In Concept 20.1, you ll learn how this system is used in the laboratory to alter genes in other cells. Just as natural selection favors bacteria that have receptors altered by mutation or that have enzymes that cut phage DNA, it also favors phage mutants that can bind to altered receptors or that are resistant to enzymes. Thus, the bacterium-phage relationship is in constant evolutionary flux. Replicative Cycles of Animal Viruses Everyone has suffered from viral infections, whether cold sores, influenza, or the common cold. Like all viruses, those that cause illness in humans and other animals can replicate only inside host cells. Many variations on the basic scheme of viral infection and replication are represented among the animal viruses. One key variable is the nature of the viral genome (double- or single-stranded DNA or RNA). Another variable is the presence or absence of a membranous envelope. Whereas few bacteriophages have an envelope or RNA genome, many animal viruses have both. In fact, nearly all Figure 19.7 The CRISPR-Cas system: a type of bacterial immune system. 1 Infection by a phage triggers transcription of the CRISPR region of the bacterial DNA. This region consists of DNA from phages that previously infected the cell, separated by repeats. CRISPR region of DNA RNA transcript 2 The RNA transcript is processed into short RNA strands. Here we focus on RNA that is complementary to the DNA of the invading phage. 3 Each short RNA strand binds to a Cas protein, forming a complex. (Multiple complexes are formed.) 4 Complementary RNA binds to DNA from the invading phage. The Cas protein then cuts the phage DNA. 5 After being cut, the entire phage DNA molecule is degraded and can no longer be replicated. BACTERIAL CELL DNA from previous infection by the same type of phage Transcription Processing Complementary RNA Cas protein RNA Active sites that cut DNA Computer model of CRISPR-Cas9 gene editing complex from Streptococcus pyogenes Phage DNA from invading phage DNA from previous infections by other phages Repeats RNA transcribed from repeat DNA from invading phage Complementary RNA Resulting cut in phage DNA Degraded phage DNA 402 Unit three Genetics

30 animal viruses with RNA genomes have an envelope, as do some with DNA genomes. Rather than consider all the mechanisms of viral infection and replication, we will focus first on the roles of viral envelopes and then on the functioning of RNA as the genetic material of many animal viruses. Viral Envelopes An animal virus equipped with an envelope that is, a membranous outer layer uses it to enter the host cell. Protruding from the outer surface of this envelope are viral glycoproteins that bind to specific receptor molecules on the surface of a host cell. Figure 19.8 outlines the events in the replicative cycle of an enveloped virus with an RNA genome. Ribosomes bound to the endoplasmic reticulum (ER) of the host cell make the protein parts of the envelope glycoproteins; cellular enzymes in the ER and Golgi apparatus then add the sugars. The resulting viral glycoproteins, embedded in membrane derived from the host cell, are transported to the cell surface. In a process much like exocytosis, new viral capsids are wrapped in membrane as they bud from the cell. In other words, the viral envelope is usually derived from the host cell s plasma membrane, although all or most of the molecules of this membrane are specified by viral genes. The enveloped viruses are now free to infect other cells. This replicative cycle does not necessarily kill the host cell, in contrast to the lytic cycles of phages. Some viruses have envelopes that are not derived from plasma membrane. Herpesviruses, for example, are temporarily cloaked in membrane derived from the nuclear envelope of the host; they then shed this membrane in the cytoplasm and acquire a new envelope made from membrane of the Golgi apparatus. These viruses have a double-stranded DNA genome and replicate within the host cell nucleus, using a combination of viral and cellular enzymes to replicate and transcribe their DNA. In the case of herpesviruses, copies of the viral DNA can remain behind as mini-chromosomes in the nuclei of certain nerve cells. There they remain latent until some sort of physical or emotional stress triggers a new round of active virus production. The infection of other cells by these new viruses causes the blisters characteristic of herpes, such as cold sores or genital sores. Once someone acquires a herpesvirus infection, flare-ups may recur throughout the person s life. Figure 19.8 The replicative cycle of an enveloped RNA virus. Shown here is a virus with a single-stranded RNA genome that functions as a template for synthesis of mrna. Some enveloped viruses enter the host cell by fusion of the envelope with the cell s plasma membrane; others enter by endocytosis. For all enveloped RNA viruses, formation of new envelopes for progeny viruses occurs by the mechanism depicted in this figure. Capsid RNA Envelope (with glycoproteins) 1 Glycoproteins on the viral envelope bind to specific receptor molecules (not shown) on the host cell, promoting viral uptake by the cell. 2 The capsid and viral genome enter the cell. Digestion of the capsid by cellular enzymes releases the viral genome. Template HOST CELL Viral genome (RNA) 3 The viral genome (red) functions as a template for synthesis of complementary RNA strands (pink) by a viral RNA polymerase. 5 Complementary RNA strands also function as mrna, which is translated into both capsid proteins (in the cytosol) and glycoproteins for the viral envelope (in the ER and Golgi apparatus). mrna ER Glycoproteins Capsid proteins Copy of genome (RNA) 4 New copies of viral genome RNA are made using the complementary RNA strands as templates. 6 Vesicles transport envelope glycoproteins to the plasma membrane. 7 A capsid assembles around each viral genome molecule. 8 Each new virus buds from the cell, its envelope studded with viral glycoproteins embedded in membrane derived from the host cell. chapter 19 Viruses 403

31 Viral Genetic Material Table 19.1 shows the common classification system for animal viruses, which is based on their genetic material: double- or single-stranded DNA, or double- or single-stranded RNA. Although some phages and most plant viruses are RNA viruses, the broadest variety of RNA genomes is found among the viruses that infect animals. There are three types of singlestranded RNA genomes found in animal viruses (classes IV VI in Table 19.1). The genome of class IV viruses can directly Table 19.1 Classes of Animal Viruses Class/Family Envelope? I. Double-Stranded DNA (dsdna) Adenovirus (see Figure 19.3b) No Examples That Cause Human Diseases Respiratory viruses Papillomavirus No Warts, cervical cancer Polyomavirus No Tumors Herpesvirus Yes Herpes simplex I and II (cold sores, genital sores); varicella zoster (shingles, chicken pox); Epstein-Barr virus (mononucleosis, Burkitt s lymphoma) Poxvirus Yes Smallpox virus; cowpox virus II. Single-Stranded DNA (ssdna) Parvovirus No B19 parvovirus (mild rash) III. Double-Stranded RNA (dsrna) Reovirus No Rotavirus (diarrhea); Colorado tick fever virus IV. Single-Stranded RNA (ssrna); Serves as mrna Picornavirus No Rhinovirus (common cold); poliovirus; hepatitis A virus; other intestinal viruses Coronavirus Yes Severe acute respiratory syndrome (SARS); Middle East respiratory syndrome (MERS) Flavivirus Yes Zika virus (see Figure 19.10c); yellow fever virus; dengue virus; West Nile virus; hepatitis C virus Togavirus Yes Chikungunya virus (see Figure 19.10b); rubella virus; equine encephalitis viruses V. ssrna; Serves as Template for mrna Synthesis Filovirus Yes Ebola virus (hemorrhagic fever; see Figure 19.10a) Orthomyxovirus Yes Influenza virus (see Figure 19.3c) Paramyxovirus Yes Measles virus; mumps virus Rhabdovirus Yes Rabies virus VI. ssrna; Serves as Template for DNA Synthesis Retrovirus Yes Human immunodeficiency virus (HIV/AIDS; see Figure 19.9); RNA tumor viruses (leukemia) serve as mrna and thus can be translated into viral protein immediately after infection. Figure 19.8 shows a virus of class V, in which the RNA genome serves instead as a template for mrna synthesis. The RNA genome is transcribed into complementary RNA strands, which function both as mrna and as templates for the synthesis of additional copies of genomic RNA. All viruses that use an RNA genome as a template for mrna transcription require RNA S RNA synthesis. These viruses use a viral enzyme capable of carrying out this process; there are no such enzymes in most cells. The enzyme used in this process is encoded by the viral genome, and after its synthesis the protein is packaged during viral self-assembly with the genome inside the viral capsid. The RNA animal viruses with the most complicated replicative cycles are the retroviruses (class VI). These viruses have an enzyme called reverse transcriptase that transcribes an RNA template into DNA, an RNA S DNA information flow that is the opposite of the usual direction. This unusual phenomenon is the source of the name retroviruses (retro means backward ). Of particular medical importance is HIV (human immunodeficiency virus), the retrovirus shown in Figure 19.1 that causes AIDS (acquired immunodeficiency syndrome). HIV and other retroviruses are enveloped viruses that contain two identical molecules of single-stranded RNA and two molecules of reverse transcriptase. The HIV replicative cycle (traced in Figure 19.9) is typical of a retrovirus. After HIV enters a host cell, its reverse transcriptase molecules are released into the cytoplasm, where they catalyze synthesis of viral DNA. The newly made viral DNA then enters the cell s nucleus and integrates into the DNA of a chromosome. The integrated viral DNA, called a provirus, never leaves the host s genome, remaining a permanent resident of the cell. (Recall that a prophage, in contrast, leaves the host s genome at the start of a lytic cycle.) The RNA polymerase of the host transcribes the proviral DNA into RNA molecules, which can function both as mrna for the synthesis of viral proteins and as genomes for the new viruses that will be assembled and released from the cell. In Concept 43.4, we describe how HIV causes the deterioration of the immune system that occurs in AIDS. Evolution of Viruses Evolution We began this chapter by asking whether or not viruses are alive. Viruses do not really fit our definition of living organisms. An isolated virus is biologically inert, unable to replicate its genes or regenerate its own ATP. Yet it has a genetic program written in the universal language of life. Do we think of viruses as nature s most complex associations of molecules or as the simplest forms of life? Either way, we must bend our usual definitions. Although viruses cannot replicate or carry out metabolic activities independently, their use of the genetic code makes it hard to deny their evolutionary connection to the living world. 404 Unit three Genetics

32 1 The envelope glycoproteins enable the virus to bind to specific receptors (not shown) on certain white blood cells. Glycoprotein Reverse transcriptase HIV Viral envelope Capsid RNA (two identical strands) Figure 19.9 The replicative cycle of HIV, the retrovirus that causes AIDS. Note in step 5 that DNA synthesized from the viral RNA genome is integrated as a provirus into the host cell chromosomal DNA, a characteristic unique to retroviruses. For simplicity, the cell-surface proteins that act as receptors for HIV are not shown. The photos on the left (artificially colored TEMs) show HIV entering and leaving a human white blood cell. HIV Membrane of white blood cell 2 The virus fuses with the cell s plasma membrane. The capsid proteins are removed, releasing the viral proteins and RNA. HOST CELL 3 Reverse transcriptase catalyzes the synthesis of a DNA strand complementary to the viral RNA. Viral capsid proteins Viral RNA RNA-DNA hybrid Reverse transcriptase 4 Reverse transcriptase catalyzes the synthesis of a second DNA strand complementary to the first. HIV entering a cell 0.25 μm DNA RNA genome for the progeny viruses Chromosomal DNA mrna NUCLEUS Provirus 5 The doublestranded DNA is incorporated as a provirus into the cell s DNA. 6 Proviral genes are transcribed into RNA molecules, which serve as genomes for progeny viruses and as mrnas for translation into viral protein. 7 The viral proteins include capsid proteins and reverse transcriptase (made in the cytosol) and envelope glycoproteins (made in the ER). New HIV leaving a cell 10 New viruses, with viral envelope glycoproteins, bud from the host cell. 9 Capsids are assembled around viral genomes and reverse transcriptase molecules. 8 Vesicles transport the glycoproteins to the cell s plasma membrane. MAKE CONNECTIONS Describe what is known about binding of HIV to immune system cells. (See Figure 7.8.) How was this discovered? Animation: Retrovirus (HIV) Replicative Cycle chapter 19 Viruses 405

33 How did viruses originate? Viruses have been found that infect every form of life not just bacteria, animals, and plants, but also archaea, fungi, and algae and other protists. Because they depend on cells for their own propagation, it seems likely that viruses are not the descendants of precellular forms of life but evolved possibly multiple times after the first cells appeared. Most molecular biologists favor the hypothesis that viruses originated from naked bits of cellular nucleic acids that moved from one cell to another, perhaps via injured cell surfaces. The evolution of genes coding for capsid proteins may have allowed viruses to bind cell membranes, thus facilitating the infection of uninjured cells. Candidates for the original sources of viral genomes include plasmids and transposons. Plasmids are small, circular DNA molecules found in bacteria and in the unicellular eukaryotes called yeasts. Plasmids exist apart from and can replicate independently of the bacterial chromosome and are occasionally transferred between cells. Transposons are DNA segments that can move from one location to another within a cell s genome. Thus, plasmids, transposons, and viruses all share an important feature: They are mobile genetic elements. (We ll discuss plasmids in more detail in Concepts 20.1 and 27.2 and transposons in Concept 21.4.) Consistent with this notion of pieces of DNA shuttling from cell to cell is the observation that a viral genome can have more in common with the genome of its host than with the genomes of viruses that infect other hosts. Indeed, some viral genes are essentially identical to genes of the host. The debate about the origin of viruses was reinvigorated about 15 years ago by reports of one of the largest viruses yet discovered: Mimivirus is a double-stranded DNA (dsdna) virus with an icosahedral capsid that is 400 nm in diameter, the size of a small bacterium. Its genome contains 1.2 million bases (Mb) about 100 times as many as the influenza virus genome and an estimated 1,000 genes. Perhaps the most surprising aspect of mimivirus, however, was that its genome included genes previously found only in cellular genomes. Some of these genes code for proteins involved in translation, DNA repair, protein folding, and polysaccharide synthesis. Whether mimivirus evolved before the first cells and then developed an exploitative relationship with them or evolved more recently and simply scavenged genes from its hosts is not yet settled. Since 2013 several even larger viruses have been discovered that cannot be classified with any existing known virus. One such virus is 1 µm (1,000 nm) in diameter, with a dsdna genome of around Mb, larger than that of some small eukaryotes. What s more, over 90% of its 2,000 or so genes are unrelated to cellular genes, inspiring the name it was given, pandoravirus. A second virus, called Pithovirus sibericum, with a diameter of 1.5 μm and 500 genes, was discovered in permanently frozen soil in Siberia. This virus, once thawed, was able to infect an amoeba after being frozen for 30,000 years! How these and all other viruses fit in the tree of life is an intriguing, unresolved question. The ongoing evolutionary relationship between viruses and the genomes of their host cells is an association that continues to make viruses very useful experimental systems in molecular biology. Knowledge about viruses also allows many practical applications, since viruses have a tremendous impact on all organisms through their ability to cause disease. Concept Check Compare the effect on the host cell of a lytic (virulent) phage and a lysogenic (temperate) phage. 2. MAKE CONNECTIONS Compare the CRISPR-Cas system to the mirna system discussed in Concept 18.3, including their mechanisms and their functions. 3. MAKE CONNECTIONS The RNA virus in Figure 19.8 has a viral RNA polymerase that functions in step 3 of the virus s replicative cycle. Compare this with a cellular RNA polymerase in terms of template and overall function (see Figure 17.10). 4. Why is HIV called a retrovirus? 5. VISUAL SKILLS Looking at Figure 19.9, imagine you are a researcher trying to combat HIV infection. What molecular processes could you attempt to block? For suggested answers, see Appendix A. Concept 19.3 Viruses and prions are formidable pathogens in animals and plants Diseases caused by viral infections afflict humans, agricultural crops, and livestock worldwide. Other smaller, less complex entities known as prions also cause disease in animals. We ll first discuss animal viruses. Viral Diseases in Animals A viral infection can produce symptoms by a number of different routes. Viruses may damage or kill cells by causing the release of hydrolytic enzymes from lysosomes. Some viruses cause infected cells to produce toxins that lead to disease symptoms, and some have molecular components that are toxic, such as envelope proteins. How much damage a virus causes depends partly on the ability of the infected tissue to regenerate by cell division. People usually recover completely from colds because the epithelium of the respiratory tract, which the viruses infect, can efficiently repair itself. In contrast, damage inflicted by poliovirus to mature nerve cells is permanent because these cells do not divide and usually cannot be replaced. Many of the temporary symptoms associated with viral infections, such as fever and body aches, actually result from the body s own efforts to defend itself against infection rather than from cell death caused by the virus. The immune system is a critical part of the body s natural defenses (see Chapter 43). It is also the basis for the major medical tool used to prevent viral infections vaccines. A vaccine is a harmless derivative of a pathogen that stimulates 406 Unit three Genetics

34 the immune system to mount defenses against the harmful pathogen. Smallpox, a viral disease that was once a devastating scourge in many parts of the world, was eradicated by a vaccination program carried out by the World Health Organization (WHO). The very narrow host range of the smallpox virus it infects only humans was a critical factor in the success of this program. Similar worldwide vaccination campaigns are currently under way to eradicate polio and measles. Effective vaccines are also available to protect against rubella, mumps, hepatitis B, and a number of other viral diseases. Although vaccines can prevent some viral illnesses, medical care can do little, at present, to cure most viral infections once they occur. The antibiotics that help us recover from bacterial infections are powerless against viruses. Antibiotics kill bacteria by inhibiting enzymes specific to bacteria but have no effect on eukaryotic or virally encoded enzymes. However, the few enzymes that are encoded only by viruses have provided targets for other drugs. Most antiviral drugs resemble nucleosides and thus interfere with viral nucleic acid synthesis. One such drug is acyclovir, which impedes herpesvirus replication by inhibiting the viral polymerase that synthesizes viral DNA but not the eukaryotic one. Similarly, azidothymidine (AZT) curbs HIV replication by interfering with the synthesis of DNA by reverse transcriptase. In the past 20 years, much effort has gone into developing drugs to treat HIV. Currently, multidrug treatments, sometimes called cocktails, are considered to be most effective. Such treatments commonly include a combination of two nucleoside mimics and a protease inhibitor, which interferes with an enzyme required for assembly of the viruses. Another effective treatment involves a drug called maraviroc, which blocks a protein on the surface of human immune cells that helps bind the HIV virus (see Figure 7.8). This drug has also been used successfully to prevent infection in individuals who either have been exposed to, or are at risk of exposure to, HIV. BBC Video: Know Your Enemy: Bacteria vs. Viruses Emerging Viruses Viruses that suddenly become apparent are often referred to as emerging viruses. HIV, the AIDS virus, is a classic example: This virus appeared in San Francisco in the early 1980s, seemingly out of nowhere, although later studies uncovered a case in the Belgian Congo in A number of other dangerous emerging viruses cause encephalitis, inflammation of the brain. One example is the West Nile virus, which appeared in North America in 1999 and has spread to all 48 contiguous states in the United States, by now resulting in over 40,000 cases and almost 2,000 deaths. Figure Emerging viruses. 500 nm (a) Ebola viruses budding from a monkey cell (colorized SEM). HHMI Video: Interview with Katie Walter The deadly Ebola virus (Figure 19.10a), recognized initially in 1976 in central Africa, is one of several emerging viruses that cause hemorrhagic fever, an often fatal illness characterized by fever, vomiting, massive bleeding, and circulatory system collapse. In 2014, a widespread outbreak of Ebola virus in western Africa caused the World Health Organization to declare an international health emergency. By mid-2015 the outbreak, centered in Guinea, Sierra Leone, and Liberia, had caused over 27,000 illnesses and 11,000 deaths. Another example is the mosquito-borne virus called chikungunya (Figure 19.10b), which causes an acute illness with fever, rashes, and persistent joint pain. Chikungunya has long been considered a tropical virus, but it has now appeared in northern Italy and southeastern France. A more recently emerging virus is the Zika virus (Figure 19.10c), which caused an outbreak of disease in spring 2015 in Brazil. Although symptoms of Zika are often mild, the outbreak was noticed because infection of pregnant women was correlated with a striking increase in the number of babies born with abnormally small brains, a condition called microcephaly. Zika is a mosquito-borne flavivirus (like West Nile virus) that infects neural cells, posing a particular danger to fetal brain development. Because of the neurological defects associated with Zika and its spread to 28 other countries by early 2016, the World Health Organization declared Zika an international health emergency. Types of influenza often emerge as outbreaks of illness. In 2009, a widespread outbreak, or epidemic, of a flu-like illness appeared in Mexico and the United States. The infectious agent was quickly identified as an influenza virus related to viruses that cause the seasonal flu. This particular virus was named H1N1 for reasons that will be explained shortly. The illness spread rapidly, prompting WHO to declare a global epidemic, or pandemic, shortly thereafter. Half a year later, the disease had reached 207 countries, infecting over 600,000 people and killing almost 8, nm (b) Chikungunya viruses emerging from a cell in the upper left and packing together (colorized TEM). 20 nm (c) Computer-generated image of a Zika virus, based on a technique called cryo-electron microscopy. chapter 19 Viruses 407

35 How do such viruses burst on the human scene, giving rise to harmful diseases that were previously rare or even unknown? Three processes contribute to the emergence of viral diseases. The first, and perhaps most important, is the mutation of existing viruses. RNA viruses tend to have an unusually high rate of mutation because viral RNA polymerases do not proofread and correct errors in replicating their RNA genomes. Some mutations change existing viruses into new genetic varieties (strains) that can cause disease, even in individuals who are immune to the ancestral virus. For instance, seasonal flu epidemics are caused by new strains of influenza virus genetically different enough from earlier strains that people have little immunity to them. You ll see an example of this process in the Scientific Skills Exercise, where you ll analyze genetic changes in variants of the H1N1 flu virus and correlate them with spread of the disease. A second process that can lead to the emergence of viral diseases is the dissemination of a viral disease from a small, isolated human population. For instance, AIDS went unnamed and virtually unnoticed for decades before it began to spread around the world. In this case, technological and social factors, including affordable international travel, blood transfusions, sexual promiscuity, and the abuse of intravenous drugs, allowed a previously rare human disease to become a global scourge. Interview with David Satcher: The role of the CDC in recognizing AIDS and in public health A third source of new viral diseases in humans is the spread of existing viruses from other animals. Scientists estimate that about three-quarters of new human diseases originate in this way. Animals that harbor and can transmit a particular virus but are generally unaffected by it are said to act as a natural reservoir for that virus. For example, the H1N1 virus that caused the 2009 flu pandemic mentioned earlier was likely passed to humans from pigs; for this reason, the disease it caused was originally called swine flu. In general, flu epidemics provide an instructive example of the effects of viruses moving between species. There are three types of influenza virus: types B and C, which infect only humans and have never caused an epidemic, and type A, which infects a wide range of animals, including birds, pigs, horses, and humans. Influenza A strains have caused four major flu epidemics among humans in the last 100 years. The worst was the first one, the Spanish flu pandemic of , which killed million people, including many World War I soldiers. Different strains of influenza A are given standardized names; for example, both the strain that caused the 1918 flu and the one that caused the 2009 pandemic flu are called H1N1. The name identifies which forms of two viral surface proteins are present: hemagglutinin (HA) and neuraminidase (NA). There are 16 different types of hemagglutinin, a protein that helps the flu virus attach to host cells, and 9 types of neuraminidase, an enzyme that helps release new virus particles from infected cells. Waterbirds have been found that carry viruses with all possible combinations of HA and NA. Variations of the hemagglutinin protein are used each year to generate vaccines against the strains predicted most likely to occur the next year. A likely scenario for the 1918 pandemic and others is that the virus mutated as it passed from one host species to another. When an animal like a pig or a bird is infected with more than one strain of flu virus, the different strains can undergo genetic recombination if the RNA molecules making up their genomes mix and match during viral assembly. Pigs were probably the main hosts for recombination that led to the 2009 flu virus, which turns out to contain sequences from bird, pig, and human flu viruses. Coupled with mutation, these reassortments can lead to the emergence of a viral strain capable of infecting human cells. People who have never been exposed to that particular strain before will lack immunity, and the recombinant virus has the potential to be highly pathogenic. If such a flu virus recombines with viruses that circulate widely among humans, it may acquire the ability to spread easily from person to person, dramatically increasing the potential for a major human outbreak. The many avian flu viruses carried by wild and domestic birds pose a potential long-term threat. A case in point is an H5N1 virus; the first transmission of H5N1 from birds to humans was documented in Hong Kong in Since then, the overall mortality rate due to H5N1 has been greater than 50% of those infected an alarming number. Also, the host range of H5N1 is expanding, which provides increasing chances for reassortment between different strains. If the H5N1 avian flu virus evolves so that it can spread easily from person to person, it could represent a major global health threat akin to that of the 1918 pandemic. How easily could this happen? In 2011, scientists working with ferrets, small mammals that are animal models for human flu, found out that only a few mutations of the avian flu virus would allow infection of cells in the human nasal cavity and windpipe. Furthermore, when the scientists transferred nasal swabs serially from ferret to ferret, the virus became transmissible through the air. Reports of this startling discovery at a scientific conference ignited a firestorm of debate about whether to publish the results and led to an ongoing reevaluation of the federal policies governing this type of experiment in the United States. The risks of doing this type of research (what if the new virus escapes or the procedure falls into the hands of bioterrorists?) must be considered in relation to the risks of not doing it the possibility that we will be unable to combat new, more transmissible viruses because we lack an understanding of how they develop. As we have seen, emerging viruses are generally not new; rather, they are existing viruses that mutate, disseminate more widely in the current host species, or spread to new host species. Changes in host behavior or environmental changes 408 Unit three Genetics

36 Scientific Skills Exercise Analyzing a Sequence-Based Phylogenetic Tree to Understand Viral Evolution How Can Sequence Data Be Used to Track Flu Virus Evolution? In 2009, an influenza A H1N1 virus caused a pandemic, and the virus has continued to resurface in outbreaks across the world. Researchers in Taiwan were curious about why the virus kept appearing despite widespread flu vaccine initiatives. They hypothesized that newly evolved variant strains of the H1N1 virus were able to evade human immune system defenses. To test this hypothesis, they needed to determine if each wave of flu infection was caused by a different H1N1 variant strain. How the Experiment Was Done Scientists obtained the genome sequences for 4,703 virus isolates collected from patients with H1N1 flu in Taiwan. They compared the sequences in different strains for the viral hemagglutinin (HA) gene, and H1N1 flu vaccination. based on mutations that had occurred, arranged the isolates into a phylogenetic tree (see Figure 26.5 for information on how to read phylogenetic trees). Number of viral isolates A/California/07/2009 Group 1 A/Taiwan/T1773/2009 Group 6 A/Taiwan/T1338/2009 A/Taiwan/T0724/2009 A/Taiwan/T1821/2009 A/Taiwan/937/2009 A/Taiwan/T1339/2009 A/Taiwan/940/2009 A/Taiwan/7418/2009 A/Taiwan/8575/2009 A/Taiwan/4909/2009 A/Taiwan/8542/2009 A/Taiwan/2826/2009 A/Taiwan/T0826/2009 A/Taiwan/1017/2009 A/Taiwan/7873/2009 A/Taiwan/11706/2009 Group 8 A/Taiwan/6078/2009 A/Taiwan/6341/2009 A/Taiwan/6200/2009 A/Taiwan/5270/2010 Group 8-1 A/Taiwan/3994/ A/Taiwan/4501/2011 A/Taiwan/67/2011 A/Taiwan/1749/2011 A/Taiwan/4611/2011 A/Taiwan/1164/2010 A/Taiwan/1018/2011 A/Taiwan/552/2011 A/Taiwan/2649/2011 A/Taiwan/1102/2011 Group 9 A/Taiwan/5506/2011 A/Taiwan/1150/2011 A/Taiwan/2883/2011 A/Taiwan/842/2010 A/Taiwan/3697/2011 Wave 1 Wave 2 Interwave Wave 3 Key Groups 1, 3, 6 Group 7 Group 8 Group 8-1 Group 9 Group 10 Group 11 M J J A S O N D J F M A M J J A S O N D J F M A Scientists graphed the number of isolates by the month and year of isolate collection to show the period in which each viral variant was actively causing illness in people. Group 3 Group 10 Group 7 Group 11 Data from the Experiment In the phylogenetic tree, each branch tip is one variant strain of the H1N1 virus with a unique HA gene sequence. The tree is a way to visualize a working hypothesis about the evolutionary relationships between H1N1 variants. Interpret The Data 1. The phylogenetic tree shows the hypothesized evolutionary relationship between the variant strains of H1N1 virus. The more closely connected two variants are, the more alike they are in terms of HA gene sequence. Each fork in a branch, called a node, shows where two lineages separate due to different accumulated mutations. The length of the branches is a measure of how many sequence differences there are between the variants, indicating how distantly related they are. Referring to the phylogenetic tree, which variants are more closely related to each other: A/Taiwan/1018/2011 and A/Taiwan/552/2011 or A/Taiwan/1018/2011 and A/Taiwan/8542/2009? Explain your answer. 2. The scientists arranged the branches into groups made up of one ancestral variant and all of its descendant, mutated variants. They are color-coded in the figure. Using group 11 as an example, trace the lineage of its variants. (a) Do all of the nodes have the same number of branches or branch tips? (b) Are all of the branches in the group the same length? (c) What do these results indicate? 3. The graph at the lower left shows the number of isolates collected (each from an ill patient) on the y-axis and the month and year that the isolates were collected on the x-axis. Each group of variants is plotted separately with a line color that matches the tree diagram. (a) Which group of variants was the earliest to cause the first wave of H1N1 flu in over 100 patients in Taiwan? (b) After a group of variants had a peak number of infections, did members of that same group cause another (later) wave of infection? (c) One variant in group 1 (green, uppermost branch) was used to make a vaccine that was distributed very early in the pandemic. Based on the graphed data, does it look like the vaccine was effective? 4. Groups 9, 10, and 11 all had H1N1 variants that caused a large number of infections at the same time in Taiwan. Does this mean that the scientists hypothesis, that new variants cause new waves of infection, was incorrect? Explain your answer. Instructors: A version of this Scientific Skills Exercise can be assigned in MasteringBiology. Data from J.-R. Yang et al., New variants and age shift to high fatality groups contribute to severe successive waves in the 2009 influenza pandemic in Taiwan, PLoS ONE 6(11): e28288 (2011). chapter 19 Viruses 409

37 can increase the viral traffic responsible for emerging diseases. For instance, new roads built through remote areas can allow viruses to spread between previously isolated human populations. Also, the destruction of forests to expand cropland can bring humans into contact with other animals that may host viruses capable of infecting humans. Finally, genetic mutations and changes in host ranges can allow viruses to jump from one species to another. Many viruses, including chikungunya, mentioned earlier, can be transmitted by mosquitoes. A dramatic expansion of the disease caused by chikungunya occurred in the mid-2000s when a mutation in the virus allowed it to infect not only the mosquito species Aedes aegypti, but also the related Aedes albopictus. Promotion of the use of insecticides and mosquito netting over beds are crucial tools in public health attempts to prevent diseases carried by mosquitoes (Figure 19.11). Figure Mosquitoes as vectors for disease. Mosquitoes transmit viruses when they feed on infected blood from one person and then bite other people. Mosquito netting is an important means of preventing infection in affected areas. Recently, scientists have become concerned about the possible effects of climate change on worldwide viral transmission. Dengue fever, also mosquito-borne, has appeared in Florida and Portugal, regions where it had not been seen before. The possibility that global climate change has allowed mosquito species carrying these viruses to expand their ranges and interact more is troubling because of the increased chance of a mutation allowing a virus species to jump to a new host. This is an area of active research by scientists applying climate change models to what is known about the habitat requirements of mosquito species. Viral Diseases in Plants More than 2,000 types of viral diseases of plants are known, and together they account for an estimated annual loss of $15 billion worldwide due to their destruction of agricultural and horticultural crops. Common signs of viral infection include bleached or brown spots on leaves and fruits (Figure 19.12), stunted growth, and Figure Immature tomato infected by a virus. damaged flowers or roots, all of which can diminish the yield and quality of crops. Plant viruses have the same basic structure and mode of replication as animal viruses. Most plant viruses discovered thus far, including tobacco mosaic virus (TMV), have an RNA genome. Many have a helical capsid, like TMV, while others have an icosahedral capsid (see Figure 19.3b). Viral diseases of plants spread by two major routes. In the first route, called horizontal transmission, a plant is infected from an external source of the virus. Because the invading virus must get past the plant s outer protective layer of cells (the epidermis), a plant becomes more susceptible to viral infections if it has been damaged by wind, injury, or herbivores. Herbivores, especially insects, pose a double threat because they can also act as carriers of viruses, transmitting disease from plant to plant. Moreover, farmers and gardeners may transmit plant viruses inadvertently on pruning shears and other tools. The other route of viral infection is vertical transmission, in which a plant inherits a viral infection from a parent. Vertical transmission can occur in asexual propagation (for example, through cuttings) or in sexual reproduction via infected seeds. Once a virus enters a plant cell and begins replicating, viral genomes and associated proteins can spread throughout the plant by means of plasmodesmata, the cytoplasmic connections that penetrate the walls between adjacent plant cells (see Figure 36.19). The passage of viral macromolecules from cell to cell is facilitated by virally encoded proteins that cause enlargement of plasmodesmata. Scientists have not yet devised cures for most viral plant diseases. Consequently, research efforts are focused largely on reducing the transmission of such diseases and on breeding resistant varieties of crop plants. Prions: Proteins as Infectious Agents The viruses we have discussed in this chapter are infectious agents that spread diseases, and their genetic material is composed of nucleic acids, whose ability to be replicated is well known. Surprisingly, there are also proteins that are known to be infectious. Proteins called prions appear to cause a number of degenerative brain diseases in various animal species. These diseases include scrapie in sheep; mad cow disease, which has plagued the European beef industry in recent years; and Creutzfeldt-Jakob disease in humans, which has caused the death of some 175 people in the United Kingdom since Prions can be transmitted in food, as may occur when people eat prion-laden beef from cattle with mad cow disease. Kuru, another human disease caused by prions, was identified in the early 1900s among the South Fore natives of New Guinea. A kuru epidemic peaked there in the 1960s, puzzling scientists, who at first thought the disease had a genetic basis. 410 Unit three Genetics

38 Figure Model for how prions propagate. Prions are misfolded versions of normal brain proteins. When a prion contacts a normally folded version of the same protein, it may induce the normal protein to assume the abnormal shape. The resulting chain reaction may continue until high levels of prion aggregation cause cellular malfunction and eventual degeneration of the brain. Animation: Prions: Characteristics Animation: Prions: Diseases Prion Normal protein New prion Original prion Aggregates of prions Eventually, however, anthropological investigations ferreted out how the disease was spread: ritual cannibalism, a widespread practice among South Fore natives at that time. Two characteristics of prions are especially alarming. First, prions act very slowly, with an incubation period of at least ten years before symptoms develop. The lengthy incubation period prevents sources of infection from being identified until long after the first cases appear, allowing many more infections to occur. Second, prions are virtually indestructible; they are not destroyed or deactivated by heating to normal cooking temperatures. To date, there is no known cure for prion diseases, and the only hope for developing effective treatments lies in understanding the process of infection. How can a protein, which cannot replicate itself, be a transmissible pathogen? According to the leading model, a prion is a misfolded form of a protein normally present in brain cells. When the prion gets into a cell containing the normal form of the protein, the prion somehow converts normal protein molecules to the misfolded prion versions. Several prions then aggregate into a complex that can convert other normal proteins to prions, which join the chain (Figure 19.13). Prion aggregation interferes with normal cellular functions and causes disease symptoms. This model was greeted with much skepticism when it was first proposed by Stanley Prusiner in the early 1980s, but it is now widely accepted. Prusiner was awarded the Nobel Prize in 1997 for his work on prions. He has recently proposed that prions are also involved in neurodegenerative diseases such as Alzheimer s and Parkinson s disease. There are many outstanding questions about these small infectious agents. Concept Check Describe two ways in which a preexisting virus can become an emerging virus. 2. Contrast horizontal and vertical transmission of viruses in plants. 3. WHAT IF? TMV has been isolated from virtually all commercial tobacco products. Why, then, is TMV infection not an additional hazard for smokers? For suggested answers, see Appendix A. 19 Chapter Review Go to MasteringBiology for Videos, Animations, Vocab Self-Quiz, Practice Tests, and more in the Study Area. Summary of Key Concepts Concept 19.1 A virus consists of a nucleic acid surrounded by a protein coat (pp )? Researchers discovered viruses in the late 1800s by studying a plant disease, tobacco mosaic disease. A virus is a small nucleic acid genome enclosed in a VOCAB SELF-QUIZ goo.gl/6u55ks protein capsid and sometimes a membranous viral envelope. The genome may be single- or double-stranded DNA or RNA. Are viruses generally considered living or nonliving? Explain. Concept 19.2 Viruses replicate only in host cells (pp ) Viruses use enzymes, ribosomes, and small molecules of host cells to synthesize progeny viruses during replication. Each type of virus has a characteristic host range, affected by whether cell-surface proteins are present that viral surface proteins can bind to. Phages (viruses that infect bacteria) can replicate by two alternative mechanisms: the lytic cycle and the lysogenic cycle. Phage DNA Lytic cycle Virulent or temperate phage Destruction of host DNA Production of new phages Lysis of host cell causes release of progeny phages Bacterial chromosome The phage attaches to a host cell and injects its DNA. Prophage Lysogenic cycle Temperate phage only Genome integrates into bacterial chromosome as prophage, which (1) is replicated and passed on to daughter cells and (2) can be induced to leave the chromosome and initiate a lytic cycle chapter 19 Viruses 411

39 ? Bacteria have various ways of defending themselves against phage infections, including the CRISPR-Cas system. Many animal viruses have an envelope. Retroviruses (such as HIV) use the enzyme reverse transcriptase to copy their RNA genome into DNA, which can be integrated into the host genome as a provirus. Since viruses can replicate only within cells, they probably evolved after the first cells appeared, perhaps as packaged fragments of cellular nucleic acid. Describe enzymes that are not found in most cells but are necessary for the replication of certain types of viruses. Concept 19.3 Viruses and prions are formidable pathogens in animals and plants (pp )? Symptoms of viral diseases may be caused by direct viral harm to cells or by the body s immune response. Vaccines stimulate the immune system to defend the host against specific viruses. An epidemic, a widespread outbreak of a disease, can become a pandemic, a global epidemic. Outbreaks of emerging viral diseases in humans are usually not new, but rather are caused by existing viruses that expand their host territory. The H1N flu virus was a new combination of pig, human, and avian viral genes that caused a pandemic. The H5N1 avian flu virus has the potential to cause a highmortality flu pandemic. Viruses enter plant cells through damaged cell walls (horizontal transmission) or are inherited from a parent (vertical transmission). Prions are slow-acting, virtually indestructible infectious proteins that cause brain diseases in mammals. What aspect of an RNA virus makes it more likely than a DNA virus to become an emerging virus? Test Your Understanding Level 1: Knowledge/Comprehension 1. Which of the following characteristics, structures, or processes is common to both bacteria and viruses? (A) metabolism (B) ribosomes (C) genetic material composed of nucleic acid (D) cell division PRACTICE TEST goo.gl/cuygkd 2. Emerging viruses arise by (A) mutation of existing viruses. (B) the spread of existing viruses to new host species. (C) the spread of existing viruses more widely within their host species. (D) all of the above. 3. To cause a human pandemic, the H5N1 avian flu virus would have to (A) spread to primates such as chimpanzees. (B) develop into a virus with a different host range. (C) become capable of human-to-human transmission. (D) become much more pathogenic. Level 2: Application/Analysis 4. A bacterium is infected with an experimentally constructed bacteriophage composed of the T2 phage protein coat and T4 phage DNA. The new phages produced would have (A) T2 protein and T4 DNA. (B) T4 protein and T2 DNA. (C) T2 protein and T2 DNA. (D) T4 protein and T4 DNA. 5. RNA viruses require their own supply of certain enzymes because (A) host cells rapidly destroy the viruses. (B) host cells lack enzymes that can replicate the viral genome. (C) these enzymes translate viral mrna into proteins. (D) these enzymes penetrate host cell membranes. 6. DRAW IT Redraw Figure 19.8 to show the replicative cycle of a virus with a single-stranded genome that can function as mrna (a class IV virus). Level 3: Synthesis/Evaluation 7. EVOLUTION CONNECTION The success of some viruses lies in their ability to evolve rapidly within the host. Such viruses evade the host s defenses by mutating and producing many altered progeny viruses before the body can mount an attack. Thus, the viruses present late in infection differ from those that initially infected the body. Discuss this as an example of evolution in microcosm. Which viral lineages tend to predominate? 8. SCIENTIFIC INQUIRY When bacteria infect an animal, the number of bacteria in the body increases in an exponential fashion (graph A). After infection by a virulent animal virus with a lytic replicative cycle, there is no evidence of infection for a while. Then the number of viruses rises suddenly and subsequently increases in a series of steps (graph B). Explain the difference in the curves. Number of bacteria A Time Number of viruses B Time 9. WRITE ABOUT A THEME: ORGANIZATION While viruses are considered by most scientists to be nonliving, they do show some characteristics of life, including the correlation of structure and function. In a short essay ( words), discuss how the structure of a virus correlates with its function. 10. SYNTHESIZE YOUR KNOWLEDGE Oseltamivir (Tamiflu), an antiviral drug prescribed for influenza, inhibits the enzyme neuraminidase. Explain how this drug could prevent infection in someone exposed to the flu or could shorten the course of flu in an infected patient (the reasons for which it is prescribed). For selected answers, see Appendix A. For additional practice questions, check out the Dynamic Study Modules in MasteringBiology. You can use them to study on your smartphone, tablet, or computer anytime, anywhere! 412 Unit three Genetics