Assignment
From your course textbook Case Workbook to Accompany Human Genetics: Concepts and Applications, read the assigned case study in the following chapters:
• Chapters 9-12, Questions #21-24
In a 4- to 5-page Microsoft Word document, create a work sheet by answering the Questions for Research and Discussion provided for each case study. (Do not answer the multiple-choice questions).
Cite any sources in APA format.
Learning Outcomes
9.1 Experiments Identify and Describe the Genetic Material
1. Describe the experiments that showed that DNA is the genetic material and protein is not.
2. Explain how Watson and Crick deduced the structure of DNA.
9.2 DNA Structure
3. List the components of a DNA nucleotide building block.
4. Explain how nucleotides are joined into two chains to form the strands of a DNA molecule.
9.3 DNA Replication-Maintaining Genetic Information
5. Explain the semiconservative mechanism of DNA replication.
6. List the steps of DNA replication.
7. Explain how the polymerase chain reaction amplifies DNA outside cells.
9.4 Sequencing DNA
8. Explain the basic strategy used to determine the base sequence of a DNA molecule.
9. Explain how next-generation sequencing improves upon Sanger sequencing.
image The BIG Picture
DNA is the basis of life because of three qualities: It holds information, it copies itself, and it changes.
On the Meaning of Gene
To a biologist, gene has a specific definition-a sequence of DNA that tells a cell how to assemble amino acids into a particular protein. To others, "gene" has different meanings:
To folksinger Arlo Guthrie, gene means aging without signs of the Huntington disease that claimed his father, legendary folksinger Woody Guthrie.
To rare cats in New England, gene means extra toes.
To Adolph Hitler and others who have dehumanized those not like themselves, the concept of gene was abused to justify genocide.
To a smoker, a gene may mean lung cancer develops.
To a redhead in a family of brunettes, gene means an attractive variant.
To a woman whose mother and sisters had breast cancer, gene means escape from their fate-and survivor guilt.
To a lucky few, gene means a mutation that locks HIV out of their cells.
To people with diabetes, gene means safer insulin.
To a forensic entomologist, gene means a clue in the guts of maggots devouring a corpse.
To scientists-turned-entrepreneurs, gene means money.
Collectively, our genes mean that we are very much more alike than different from one another.
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9.1 Experiments Identify and Describe the Genetic Material
"A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way," wrote Francis Crick, codiscoverer with James Watson of the three-dimensional structure of DNA in 1953. Only DNA fulfills these requirements.
DNA was first described in the mid-nineteenth century, when Swiss physician and biochemist Friedrich Miescher isolated nuclei from white blood cells in pus on soiled bandages. He discovered in the nuclei, an unusual acidic substance containing nitrogen and phosphorus. He and others found it in cells from a variety of sources. Because the material resided in cell nuclei, Miescher called it nuclein in an 1871 paper; subsequently, it was called a nucleic acid. Few people appreciated the importance of Miescher's discovery at the time, when inherited disease was widely blamed on protein.
In 1902, English physician Archibald Garrod was the first to provide evidence linking inherited disease and protein. He noted that people who had certain inborn errors of metabolism lacked certain enzymes. Other researchers added evidence of a link between heredity and enzymes from other species, such as fruit flies with unusual eye colors and bread molds with nutritional deficiencies. Both organisms had absent or malfunctioning specific enzymes. As researchers wondered about the connection between enzymes and heredity, they returned to Miescher's discovery of nucleic acids.
DNA Is the Hereditary Molecule
In 1928, English microbiologist Frederick Griffith took the first step in identifying DNA as the genetic material. He was studying pneumonia in the years after the 1918 flu pandemic. Griffith noticed that mice with a certain form of pneumonia harbored one of two types of Streptococcus pneumoniae bacteria. Type R bacteria were rough in texture. Type S bacteria were smooth because they were enclosed in a polysaccharide (a type of carbohydrate) capsule. Mice injected with type R bacteria did not develop pneumonia (figure 9.1a), but mice injected with type S did (figure 9.1b). The polysaccharide coat shielded the bacteria from the mouse immune system, enabling them to cause severe (virulent) infection. Injecting mice with unaltered type R or type S bacteria served as control experiments, which represent the situation without the experimental intervention.
When type S bacteria were heated, which killed them, they no longer could cause pneumonia in mice. However, when Griffith injected mice with a mixture of type R bacteria plus heat-killed type S bacteria-neither of which, alone, was deadly to the mice-the mice died of pneumonia (figure 9.1d). Their bodies contained live type S bacteria, encased in polysaccharide. Griffith termed the apparent conversion of one bacterial type into another "transformation." How did it happen? What component of the dead, smooth bacteria transformed type R to type S?
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Figure 9.1 Discovery of bacterial transformation. Griffith's experiments showed that a molecule in a lethal type of bacteria can transform nonkilling (nonvirulent) bacteria into killers (virulent).
Page 165U.S. physicians Oswald Avery, Colin MacLeod, and Maclyn McCarty hypothesized that a nucleic acid might be Griffith's "transforming principle." They observed that treating broken-open type S bacteria with a protease-an enzyme that dismantles protein-did not prevent the transformation of a nonvirulent to a virulent strain, but treating it with deoxyribonuclease (or DNase), an enzyme that dismantles DNA only, did disrupt transformation. In 1944, they confirmed that DNA transformed the bacteria. They isolated DNA from heat-killed type S bacteria and injected it with type R bacteria into mice (figure 9.2). The mice died, and their bodies contained active type S bacteria. The conclusion: DNA passed from type S bacteria into type R, enabling the type R to manufacture the smooth coat necessary for infection. Once type R bacteria encase themselves in smooth coats, they are no longer type R.
Protein Is Not the Hereditary Molecule
Science seeks answers by eliminating explanations. It provides evidence in support of a hypothesis, not proof, because conclusions can change when new data become available. To identify the genetic material, researchers also had to show that protein does not transmit genetic information. To do this, in 1953, U.S. microbiologists Alfred Hershey and Martha Chase used Escherichia coli bacteria infected with a virus that consisted of a protein "head" surrounding DNA. Viruses infect bacterial cells by injecting their DNA (or RNA) into them. Infected bacteria may then produce many more viruses. The viral protein coats remain outside the bacterial cells.
Researchers can analyze viruses by growing them on culture medium that contains a radioactive chemical that the viruses take up. The "labeled" viral nucleic acid then emits radiation, which can be detected in several ways. Hershey and Chase knew that protein contains sulfur but not phosphorus, and that nucleic acids contain phosphorus but not sulfur. Both elements also come in radioactive forms. When Hershey and Chase grew viruses in the presence of radioactive sulfur, the viral protein coats took up and emitted radioactivity, but when they ran the experiment using radioactive phosphorus, the viral DNA emitted radioactivity. If protein is the genetic material, then the infected bacteria would have radioactive sulfur. But if DNA is the genetic material, then the bacteria would have radioactive phosphorus.
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Figure 9.2 DNA is the "transforming principle." Avery, MacLeod, and McCarty identified DNA as Griffith's transforming principle. By adding enzymes that either destroy proteins (protease) or DNA (deoxyribonuclease or DNase) to bacteria that were broken apart to release their contents, the researchers demonstrated that DNA transforms bacteria-and that protein does not.
Hershey and Chase grew one batch of virus in a medium containing radioactive sulfur (designated 35S) and another in a medium containing radioactive phosphorus (designated 32P). The viruses grown on sulfur had their protein marked, but not their DNA, because protein incorporates sulfur but DNA does not. Conversely, the viruses grown on labeled phosphorus had their DNA marked, but not their protein, because this element is found in DNA but not protein.
After allowing several minutes for the virus particles to bind to the bacteria and inject their DNA into them, Hershey and Chase agitated each mixture in a blender, shaking free the empty virus protein coats. The contents of each blender were collected in test tubes, then centrifuged (spun at high speed). This settled the bacteria at the bottom of each tube because the lighter virus coats drift down more slowly than bacteria.
At the end of the procedure, Hershey and Chase examined fractions containing the virus coats from the top of each test tube and the infected bacteria that had settled to the bottom (figure 9.3). In the tube containing viruses labeled with sulfur, the virus coats were radioactive, but the virus-infected bacteria, containing viral DNA, were not. In the other tube, where the virus had incorporated radioactive phosphorus, the virus coats carried no radioactive label, but the infected bacteria were radioactive. Therefore, the part of the virus that could enter bacteria and direct them to mass produce more virus was the part that had incorporated phosphorus-the DNA. The genetic material is DNA, and not protein.
Discovering the Structure of DNA
In 1909, Russian-American biochemist Phoebus Levene identified the 5-carbon sugar ribose as part of some nucleic acids, and in 1929, he discovered a similar sugar-deoxyribose-in other nucleic acids. He had revealed a major chemical distinction between RNA and DNA: RNA has ribose, and DNA has deoxyribose. (Recall that RNA serves as a carrier of the information in a DNA molecule that instructs the cell to manufacture a particular protein.)
Levene then discovered that the three parts of a nucleic acid-a sugar, a nitrogen-containing base, and a phosphorus-containing component-are present in equal proportions. He deduced that a nucleic acid building block must contain one of each component. Furthermore, although the sugar and phosphate portions of nucleic acids were always the same, the nitrogen-containing bases were of four types. Scientists at first thought that the bases were present in equal amounts, but if this were so, DNA could not encode as much information as it could if the number of each base type varied. Imagine how much less useful a written language would be if it had to use all the letters with equal frequency.
In the early 1950s, two lines of experimental evidence converged to provide the direct clues that finally revealed DNA's structure. Austrian-American biochemist Erwin Chargaff showed that DNA in several species contains equal amounts of the bases adenine (A) and thymine (T) and equal amounts of the bases guanine (G) and cytosine (C). Next, English physicist Maurice Wilkins and English chemist Rosalind Franklin bombarded DNA with X rays using a technique called X-ray diffraction, then deduced the overall structure of the molecule from the patterns in which the X rays were deflected.
Learning Outcomes
10.1 Transcription Copies the Information in DNA
1. List the major types of RNA molecules and their functions.
2. Explain the importance of transcription factors.
3. List the steps of transcription.
10.2 Translation of a Protein
4. Discuss how researchers deduced the genetic code.
5. List the steps of protein synthesis.
10.3 Processing a Protein
6. Define the four components of a protein's shape.
7. Explain the importance of protein folding.
image The BIG Picture
DNA sequences are the blueprints of life. Cells must maintain this information, yet also access it to manufacture proteins. RNA acts as the go-between, linking DNA sequences to the amino acid sequences of proteins.
An Inborn Error of Arginine Production
Genes instruct cells to build proteins from 20 types of amino acids. An amino acid has four parts: a central carbon atom bonds to a hydrogen atom, an amino group (NH2), an acid group (COOH), and an "R" group that distinguishes the 20 types. The body synthesizes 10 of the 20 amino acids, and must obtain the rest, termed "essential," from food. Some amino acids, such as arginine, are essential only during childhood.
To be healthy, the body must make or obtain all 20 types of amino acids. In argininosuccinic aciduria (ASA; OMIM 207900), the body cannot produce an enzyme required to make arginine. ASA is autosomal recessive and affects 1 in 70,000 newborns.
Lack of arginine causes seizures and coma in children. The brain damage happens because without arginine, nitrogen atoms released from broken-down proteins, instead of being excreted in the urine, bond with hydrogen atoms to form ammonia (NH3). The ammonia harms brain neurons. A different symptom, however, led researchers to a drug already in use for a different disease.
Children with ASA may also have developmental and cognitive delays, very high blood pressure that does not respond to standard medications, and enlarged hearts. Researchers at Baylor College of Medicine working with a teenager with ASA who had all of these symptoms discovered how to help him. They knew that arginine was also necessary to manufacture the tiny but essential molecule nitric oxide (NO). So the researchers tried a drug that heart disease patients use to treat chest pain caused by lack of NO. The drug enables the body to produce the needed NO another way.
Page 181Within days of receiving the heart drug, the young man's blood pressure dropped to normal. Over the following weeks, his heart returned to a normal size, and he began doing much better in school. He started to socialize, and test scores for verbal memory and problem solving increased dramatically.
10.1 Transcription Copies the Information in DNA
Only about 1.5 percent of the DNA of the human genome encodes protein. This part is the exome. Much of the rest of the genome controls how, where, and when proteins are made.
Our genes encode 20,325 types of proteins. A protein consists of one or more long chains of amino acids, called polypeptides. A short sequence of amino acids is a peptide, and the bonds that join amino acids are peptide bonds. Proteins have a great variety of functions; table 10.1 lists some of them.
A cell uses two processes to manufacture proteins using genetic instructions. Transcription first synthesizes an RNA molecule that is complementary to one strand of the DNA double helix for a particular gene. The RNA copy is taken out of the nucleus and into the cytoplasm. There, the process of translation uses the information in the RNA to manufacture a protein by aligning and joining specified amino acids. Finally, the protein folds into a specific three-dimensional form necessary for its function.
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Accessing the genome is a huge, ongoing task. Cells replicate their DNA only during S phase of the cell cycle. In contrast, transcription and translation occur continuously, except during M phase. Transcription and translation supply the proteins essential for life, as well as those that give a cell its specialized characteristics.
Shortly after Watson and Crick published their structure of DNA in 1953, they described the relationship between nucleic acids and proteins as a directional flow of information called the "central dogma" (figure 10.1). As Francis Crick explained in 1957, "The specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and this sequence is a code for the amino acid sequence of a particular protein." This statement inspired more than a decade of intense research to discover exactly how cells make proteins. The process centers around RNA.
RNA Structure and Types
RNA is the bridge between gene and protein. RNA and DNA share an intimate relationship, as figure 10.2 depicts. The bases of an RNA sequence are complementary to those of one strand of the double helix, which is called the template strand. An enzyme, RNA polymerase, builds an RNA molecule. The other, nontemplate strand of the DNA double helix is called the coding strand.
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Figure 10.1 DNA to RNA to protein. Some of the information stored in DNA is copied to RNA (transcription), some of which is used to assemble amino acids into proteins (translation). DNA replication perpetuates genetic information. This figure repeats throughout the chapter, with the part under discussion highlighted.
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Figure 10.2 The relationship among RNA, the DNA template strand, and the DNA coding strand. The RNA sequence is complementary to the DNA template strand and is the same sequence as the DNA coding strand, with uracil (U) in place of thymine (T).
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Figure 10.3 DNA and RNA differences. (a) DNA is double-stranded; RNA is usually single-stranded (b). DNA nucleotides include deoxyribose; RNA nucleotides have ribose (c). Finally, DNA nucleotides include the pyrimidine thymine, whereas RNA has uracil (d).
RNA and DNA have similarities and differences (figure 10.3 and table 10.2). Both are nucleic acids, consisting of sequences of nitrogen-containing bases joined by sugar-phosphate backbones. However, RNA is usually single-stranded, whereas DNA is double-stranded. Also, RNA has the pyrimidine base uracil where DNA has thymine. RNA (ribonucleic acid) nucleotides include the sugar ribose. DNA (deoxyribonucleic acid) nucleotides include the sugar deoxyribose. Functionally, DNA stores genetic information, whereas RNA controls how that information is used. The presence of the-OH at the 5′ position of ribose makes RNA much less stable than DNA, which is critical in its function as a short-lived carrier of genetic information.
As RNA is synthesized along DNA, it folds into a three-dimensional shape, or conformation, that arises from complementary base pairing within the same RNA molecule. For example, a sequence of AAUUUCC might hydrogen bond to a sequence of UUAAAGG-its complement-elsewhere in the same molecule, a little like touching elbows to knees. Conformation is very important for RNA's functioning. The three major types of RNA are messenger RNA, ribosomal RNA, and transfer RNA (table 10.3). Other classes of RNA control which genes are expressed (transcribed and translated) under specific circumstances. Table 11.1 describes them.
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Learning Outcomes
11.1 Gene Expression Through Time and Tissue
1. Define epigenetics.
2. Explain how globin chain switching, development of organs, and the types of proteins cells make over time illustrate gene expression.
11.2 Control of Gene Expression
3. Explain how small molecules binding to histone proteins control gene expression by remodeling chromatin.
4. Explain how microRNAs control transcription.
11.3 Maximizing Genetic Information
5. Explain how division of genes into exons and introns maximizes the number of encoded proteins.
11.4 Most of the Human Genome Does Not Encode Protein
6. Discuss how viral DNA, noncoding RNAs, and repeated sequences account for large proportions of the human genome.
image The BIG Picture
Discovering the nature of the genetic material, determining the structure of DNA, deciphering the genetic code, and sequencing the human genome led to today's challenge: learning how genes are expressed through tissue and time.
The Dutch Hunger Winter
"Nature versus nurture" implies that genes and the environment work separately, but environmental conditions can greatly affect gene expression, which can affect health. For example, starvation before birth can alter gene expression in a way that may manifest as schizophrenia years later.
From February through April 1945, the "Dutch Hunger Winter," the Nazis blocked all food supplies from entering six large cities in western Holland. As malnutrition weakened and killed people, a cruel experiment took place. Children who had been starved before birth were much more likely to develop schizophrenia years later than their siblings born in better times. The key factor in setting the stage for future poor health was not birth weight, as had been thought, but exposure to dangerous conditions during the first weeks of pregnancy.
In the ongoing Dutch Famine Study, researchers at Columbia University and Leiden University in the Netherlands discovered the link between prenatal malnutrition and schizophrenia because they knew the exact time of the starvation, and the exact calorie intake, from food ration records. They obtained the schizophrenia diagnoses from psychiatric registries and military induction records.
Prenatal nutrition affects an adult phenotype because starvation alters the pattern in which methyl groups (CH3) bind DNA, selectively silencing genes. The DNA of people born in the months after the famine, when studied 60 years later, had different methylation patterns in the gene that encodes insulin-like growth factor 2 (IGF-2), than siblings born following healthier gestations. Because IGF-2 controls expression of genes that affect thinking, researchers hypothesize that schizophrenia may develop in some people born into famine when IGF-2 has too few methyl groups and is overexpressed in the brain.
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11.1 Gene Expression Through Time and Tissue
A genome is like an orchestra. Just as not all of the musical instruments play with the same intensity at every moment, not all genes are expressed continually at the same levels. Some genes are always transcribed and translated, in all cells. Because they keep cells running, these genes are sometimes called "housekeeping genes." Other genes have more specialized roles, and become active as a cell differentiates.
Before the field of genomics began in the 1990s, the study of genetics proceeded one gene at a time, like hearing the separate contributions of a violin, a viola, and a flute. Many genetic investigations today, in contrast, track the crescendos of gene activity that parallel events in an organism's life. This new view introduced the element of time to genetic analysis. Unlike the gene maps of old, which ordered genes linearly on chromosomes, new types of maps are more like networks that depict the timing of gene expression in unfolding programs of development and response to the environment.
The discoveries of the 1950s and 1960s on DNA structure and function answered some questions about the control of gene expression while raising many more. How does a bone cell "know" to transcribe the genes that control the synthesis of collagen and not to transcribe genes that specify muscle proteins? What causes the proportions of blood cell types to shift into leukemia? How do chemical groups "know" to shield DNA from transcription in one circumstance, yet expose it in others?
Changes to the chemical groups that associate with DNA greatly affect which parts of the genome are accessible to transcription factors and under which conditions. Such changes to the molecules that bind to DNA that are transmitted to daughter cells when the cell divides are termed epigenetic, which means "outside the gene." Figure 6.12 shows how methyl groups bind to DNA, causing epigenetic changes.
Epigenetic changes do not alter the DNA base sequence and are reversible. They are passed from one cell generation to the next. These changes may affect the next generation of individuals if the conditions to which a fetus is exposed become dangerous. This is what happened to the survivors of the Dutch Hunger Winter described in the chapter opener. For a few sites in the genome, an epigenetic change may persist through meiosis to a third generation, but this appears to be rare. Specific classes of proteins and RNA molecules carry out epigenetic changes. Much of the genome encodes these modifiers of gene expression. The human genome, then, is a little like a device that comes with a long, detailed instruction manual.
This chapter looks at how cells access the information in DNA. We begin with two examples of gene expression at the molecular and organ levels: (1) hemoglobin switching during development and (2) specialization of the two major parts of the pancreas.
Globin Chain Switching
The globin proteins transport oxygen in the blood. They vividly illustrate control of gene expression because, in a process called globin chain switching, they assemble into different hemoglobin molecules depending upon stage of development (figures 11.1 and 11.2). A hemoglobin molecule in the blood of an adult consists of four polypeptide chains, each wound into a globular conformation. Two of the chains are 146 amino acids long and are called "beta" (β). The other two chains are 141 amino acids long and are termed "alpha" (α). Genes on different chromosomes encode the alpha and beta globin polypeptide chains.
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Figure 11.1 The structure of hemoglobin. A hemoglobin molecule is made up of two globular protein chains from the beta (β) globin group and two from the alpha (α) globin group. Each globin surrounds an iron-containing chemical group called a heme.
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Figure 11.2 Globin chain switching. The subunit composition of human hemoglobin changes as the concentration of oxygen in the environment changes. Each globin quartet has two polypeptide chains encoded by genes in the alpha (α) globin cluster (chromosome 16) and two polypeptide chains from the beta (β) globin cluster (chromosome 11). With the switch from the placenta to the newborn's lungs to obtain oxygen, beta globin begins to replace gamma (γ) globin.
Page 201As a human develops, different globin polypeptide chains are used to make molecules of hemoglobin. The different forms of hemoglobin are necessary because of changes in blood oxygen levels that happen when a newborn begins breathing and no longer receives oxygen through the placenta. The promoters of the globin genes include binding sites for transcription factors, which orchestrate the changing hemoglobin molecules through development. Other DNA sequences in the globin gene clusters turn off expression of genes no longer needed.
The chemical basis for globin chain switching is that different globin polypeptide chains attract oxygen molecules to different degrees. In the embryo, as the placenta forms, hemoglobin consists first of two epsilon (ε) chains, which are in the beta globin group, and two zeta (ζ) chains, which are in the alpha globin group. About 4 percent of the hemoglobin in the embryo includes beta chains. This percentage gradually increases. Globin chains are manufactured first in the yolk sac in the embryo, then in the liver and spleen in the fetus, and finally primarily in the bone marrow after birth.
As the embryo develops into a fetus, the epsilon and zeta globin polypeptide chains decrease in number, as gamma (γ) and alpha chains accumulate. Hemoglobin consisting of two gamma and two alpha chains is called fetal hemoglobin. Because the gamma globin subunits bind very strongly to oxygen released from maternal red blood cells into the placenta, fetal blood carries 20 to 30 percent more oxygen than an adult's blood. As the fetus matures, beta chains gradually replace the gamma chains. At birth, however, the hemoglobin is not fully of the adult type-fetal hemoglobin (two gamma and two alpha chains) comprises from 50 to 85 percent of the blood. By 4 months of age, the proportion drops to 10 to 15 percent, and by 4 years, less than 1 percent of the child's hemoglobin is the fetal form.
Building Tissues and Organs
Blood is a structurally simple tissue that is easy to obtain and study because its components are easily separated. A solid gland or organ, with a distinctive three-dimensional form compared to the fluid blood, and constructed from specialized cells and in many cases more than one type of tissue, is much more complex. Its specific, solid organization must be maintained throughout a lifetime of growth, repair, and changing external conditions. Cells must maintain their specializations.
In all tissues and organs, genes are turned on and off during development, as stem cells self-renew and yield more specialized daughter cells. Researchers isolate individual stem cells and then see which combinations of growth factors, hormones, and other biochemicals must be added to steer development toward a particular cell type.
The pancreas has an interesting organization. It is a dual gland, with two types of cell clusters. The exocrine part releases digestive enzymes into ducts, whereas the endocrine part secretes polypeptide hormones that control nutrient use directly into the bloodstream. The endocrine cell clusters are called pancreatic islets.
The complexity of the pancreas unfolds in the embryo, when ducts form. Within duct walls reside rare stem cells and progenitor cells (see figure 2.20). A transcription factor is activated and controls expression of other genes in a way that stimulates some progenitor cells to divide. Certain daughter cells follow an exocrine pathway and will produce digestive enzymes. Other progenitor cells respond to different signals and divide to yield daughter cells that follow the endocrine pathway.
Figure 11.3 shows the differentiated cell types that form from the two cell lineages in the pancreas. The most familiar pancreatic hormone is insulin, which the beta cells of the pancreas secrete. The absence of insulin, or the inability of cells to recognize it, causes diabetes mellitus. If pancreatic stem cells can be isolated and cultured, it might be possible to coax a person with diabetes to produce new and functional pancreatic beta cells. Many people with diabetes who cannot make insulin take it as a drug.
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Figure 11.3 Building a pancreas. A single type of stem cell theoretically divides to give rise to an exocrine/endocrine progenitor cell that in turn divides to yield more restricted progenitor cells that give rise to both mature exocrine and endocrine cells. The endocrine progenitor cell in turn divides, yielding specialized daughter cells that produce specific hormones.11.4 Most of the Human Genome Does Not Encode Protein
Only about 1.5 percent of human DNA encodes protein. The rest includes viral sequences, sequences that encode RNAs other than mRNA (called noncoding or ncRNAs), introns, promoters and other control sequences, and repeated sequences (table 11.1). In fact, most of the genome is transcribed-a DNA sequence is not "junk" if it does not encode protein.
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Viral DNA
Our genomes include DNA sequences that represent viruses. Viruses are nonliving infectious particles that consist of a nucleic acid (DNA or RNA) encased in a protein coat (see Clinical Connection 17.1). A virus replicates using a cell's transcriptional and translational machinery to mass-produce itself. New viruses may exit the cell, or the viral nucleic acid may remain in a host cell. A DNA virus may take over directly, inserting into a chromosome or remaining outside the nucleus in a circle called an episome. An RNA virus first uses an enzyme (reverse transcriptase) to copy its genetic material into DNA, which then inserts into a host chromosome.
About 100,000 sequences in our DNA, of varying lengths and comprising about 8 percent of the genome, were once a type of RNA virus called a retrovirus. The name refers to a retrovirus' direction of genetic information transfer, which is opposite DNA to RNA to protein. Retroviral sequences in our chromosomes are termed "endogenous" because they are carried from generation to generation of the host, rather than acquired as an acute infection. The retroviruses whose genetic material is in our chromosomes are called human endogenous retroviruses, or HERVs.
By comparing HERV sequences to similar viruses in other primates, researchers traced HERVs to a sequence representing a virus that infected our ancestors' genomes about 5 million years ago. Since then, HERV sequences have exchanged parts (recombined) and mutated to the extent that they no longer make us sick. Harmless HERVs silently pass from human generation to generation as parts of our chromosomes. They increase in number with time, as figure 11.13 shows.
Noncoding RNAs
Much more of the human genome is transcribed than would be predicted based on the number and diversity of proteins that a human body can produce. The two general classes of RNAs are coding (the mRNAs) and noncoding (ncRNAs), which include everything else. The best-studied noncoding RNAs are the tRNAs and rRNAs.
The rate of transcription of a cell's tRNA genes is attuned to cell specialization. The proteins of a skeletal muscle cell, for example, require different amounts of certain amino acids than the proteins of a white blood cell, and therefore different amounts of the corresponding tRNAs. Human tRNA genes are dispersed among the chromosomes in clusters. Altogether, our 500 or so types of tRNA genes account for 0.1 percent of the genome.
The 243 types of rRNA genes are grouped on six chromosomes, each cluster harboring 150 to 200 copies of a 44,000-base repeat sequence. Once transcribed from these clustered genes, the rRNAs go to the nucleolus, where another type of ncRNA called small nucleolar RNA (snoRNA) cuts them into their final forms.
Hundreds of thousands of noncoding RNAs are neither tRNA nor rRNA, nor snoRNAs, nor microRNAs, nor the other less abundant types described in table 11.1. Some noncoding RNAs correspond to DNA sequences called pseudogenes. A pseudogene is very similar in sequence to a protein-encoding gene that may be transcribed, but it is not translated into protein. A pseudogene is altered in sequence from an ancestral gene in a way that may impair its translation or folding. Pseudogenes may be remnants of genes past, variants that diverged from the normal sequence too greatly to encode a working protein.
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Figure 11.13 The human genome includes viral DNA sequences. Most, if not all, of them do not harm us.
Since the sequencing of the human genome, a similarly huge project called ENCODE has discovered the functions of non-protein-encoding parts of the genome, including about 12,000 long noncoding RNAs. Many of these sequences likely control gene expression, because they reside in the nucleus, where they are physically associated with chromatin. Long noncoding RNAs represent exons, introns, and regions between genes. A third of them are found only in the genomes of primates, and many are transcribed only in the brain. These findings suggest that the long noncoding RNAs may hold clues to what makes us human-a topic discussed in chapter 16.
Repeats
The human genome is riddled with highly repetitive sequences that may be a different type of information than a protein's amino acid sequence. Perhaps repeat size or number constitute another type of molecular language. Or, perhaps some types of repeats help to hold a chromosome together.
The most abundant type of repeat is a sequence of DNA that can move about the genome. It is called a transposable element, or transposon for short. Geneticist Barbara McClintock originally identified transposons in corn in the 1940s, and they were rediscovered in bacteria in the 1960s. Transposons comprise about 45 percent of the human genome sequence, and typically are present in many copies. Some transposons include parts that encode enzymes that cut them out of one chromosomal site and integrate them into another. Unstable transposons may lie behind inherited diseases that have several symptoms, because they insert into different genes. This is the case for Rett syndrome (see the chapter 2 opener).
An example of a specific type of repeat is an Alu sequence. Each Alu repeat is about 300 bases long, and a human genome may contain 300,000 to 500,000 of them. Alu repeats comprise 2 to 3 percent of the genome, and they have been increasing in number over time because they can copy themselves. We don't know exactly what these common repeats do, if anything. They may serve as attachment points for proteins that bind newly replicated DNA to parental strands before anaphase of mitosis, when replicated chromosomes pull apart.
Rarer classes of repeats comprise telomeres, centromeres, duplications of 10,000 to 300,000 bases, copies of pseudogenes, and simple repeats of one, two, or three bases. In fact, the entire human genome may have duplicated once or even twice.
Repeats may make sense in light of evolution, past and future. Pseudogenes are likely vestiges of genes that functioned in our nonhuman ancestors. Perhaps the repeats that seem to have no obvious function today will serve as raw material from which future genes may arise by mutation.
Discovery of the intricate controls of gene expression has led to a new definition of a gene, greatly expanded from the one-gene, one-protein idea of years past. A gene is a DNA sequence that contributes to a phenotype or function, plus the sequences, both in the gene and outside it, that control its expression. Chapter 12 looks at different types of changes in the DNA sequence-mutations-and their consequences.
Key Concepts Questions 11.4
What can RNA do in addition to encoding protein?
What are some types of noncoding RNAs?
What type of noncoding RNA might reflect our past?
Learning Outcomes
12.1 The Nature of Mutations
1. Distinguish between mutation and mutant.
2. Distinguish between mutation and polymorphism.
12.2 A Closer Look at Two Mutations
3. Describe mutations in the genes that encode beta globin and collagen.
12.3 Allelic Disorders
4. Provide examples of how mutations in a single gene can cause more than one illness.
12.4 Causes of Mutation
5. Explain the chemical basis of a spontaneous mutation.
6. Describe ways that researchers induce mutations.
12.5 Types of Mutations
7. Describe the two types of single-base mutations.
8. Explain the consequences of a splice-site mutation.
9. Discuss mutations that add, remove, or move DNA nucleotides.
12.6 The Importance of Position
10. Give examples of how the location of a mutation in a gene affects the phenotype.
11. Describe a conditional mutation.
12.7 DNA Repair
12. What types of damage do DNA repair mechanisms counter?
13. Describe the types of DNA repair.
image The BIG Picture
On a species level, mutations provide the variation necessary for life to continue. On an individual level, mutations cause many illnesses, although a few mutations are helpful. DNA repair mechanisms protect against DNA damage.
One Mutation, Multiple Effects: Osteogenesis Imperfecta
Shirley Banks, 73, became aware of her family's unusual condition as a young child. "My oldest brother had many fractures, and the doctors told him to eat high-calcium foods. We lived on a farm! All the dairy made no difference because of the mutation, but nobody knew." Several cousins easily broke bones too, and Shirley had the family's "brittle bone disease," but didn't yet realize it. Years later, her son Todd would inherit the family legacy: osteogenesis imperfecta (OI) type I (OMIM 166200).
"Todd had his first fracture at 2, when he tripped and broke his leg. I became suspicious because my brother's child broke bones, too. Finally, a doctor who had seen the disease as an intern explained it, and we were diagnosed," Shirley says. She realized her grandmother and a great uncle, who were two of seven in that generation, had the autosomal dominant disease, too.
The Banks family's mutation is in a gene that encodes the connective tissue protein collagen. Their type causes up to 100 fractures in a lifetime, which readily heal, but other forms may break bones before birth, proving lethal in infancy. When genetic testing became available, it was at first for only one type of OI, and some parents who had different types were falsely accused of abusing their children. Eight forms of OI are now recognized.
Only a few cases of OI are known from history. An Egyptian mummy from 1000 B.C. had it, as did ninth-century Viking "Ivan the Boneless," who was reportedly carried into battle aboard a shield and whose remains were exhumed and burnt by King William I, forever obscuring the true diagnosis.
Shirley's only broken bones are in her toes, but the disease causes other problems. She wears digital hearing aids and Page 213the whites of her eyes (sclerae) have a bluish cast. Her tissues are fragile, and she bled profusely during surgery. Shirley and Todd have high pressure in their eyeballs (glaucoma) because their corneas are abnormally thin, which makes the pressure read lower than it actually is. They need higher doses of medication than other people with glaucoma. OI vividly illustrates the fact that one mutation can have multiple effects.
12.1 The Nature of Mutations
A mutation is a change in a DNA sequence that is rare in a population and typically affects the phenotype. "Mutate" refers to the process of altering a DNA sequence. Mutations range from substitution of a single DNA base; to deletion or duplication of tens, hundreds, thousands, or even millions of bases; to missing or extra entire chromosomes. This chapter discusses smaller-scale mutations, and chapter 13 considers mutation at the chromosomal level. However, the extent of mutation is a continuum.
Mutation can affect any part of the genome: sequences that encode proteins or control transcription; introns; repeats; and sites critical to intron removal and exon splicing. Not all DNA sequences are equally likely to mutate.
The effects of mutation vary. Mutations may impair a function, have no effect, or even be beneficial. A deleterious (harmful) mutation can stop or slow production of a protein, overproduce it, or impair the protein's function-such as altering its secretion, location, or interaction with another protein. The effect of a mutation is called a "loss-of-function" when the gene's product is reduced or absent, or a "gain-of-function" when the gene's activity changes. Most mutations are recessive and cause a loss-of-function (see figure 4.8). Gain-of-function mutations tend to be dominant and are also called "toxic."
The terms mutation and polymorphism each denote a genetic change. Recall from chapter 7 that a single nucleotide polymorphism, or SNP, is a single base change. So are many mutations. The distinction between mutation and polymorphism is largely artificial, reflecting frequency in a particular population, in which a mutation is much rarer than a polymorphism. If a genetic change greatly impairs health, individuals with it are unlikely to reproduce, and the mutant allele remains uncommon. A polymorphism that does not harm health, elevates risk of illness only slightly, or is even beneficial, will remain prevalent in a population or even increase in frequency. A genetic change that is a mutation in one population may be a harmless polymorphism in another. This is why considering a patient's ancestry is important in interpreting genetic test results.
Not all mutations are harmful, in contrast to their depiction in science fiction. For example, a mutation protects against HIV infection. About 1 percent of the general population is homozygous for a recessive allele that encodes a cell surface protein called CCR5 (see figure 17.11). To infect an immune system cell, HIV must bind CCR5 and another protein. Because the mutation prevents CCR5 from moving to the cell surface from inside the cell, HIV cannot bind. Heterozygotes for this mutation are partially protected against HIV infection. The opener to chapter 17 describes how mimicking CCR5 mutation treats HIV infection.
The term mutation refers to genotype-that is, a change at the DNA or chromosome level. The familiar term mutant refers to phenotype. The nature of a mutant phenotype depends upon how the mutation affects the gene's product or activity, and usually connotes an abnormal or unusual characteristic. However, a mutant phenotype may also be an uncommon variant that is nevertheless "normal," such as red hair.
In an evolutionary sense, mutation has been essential to life, because it produces individuals with variant phenotypes who are better able to survive specific environmental challenges, including illnesses. Our evolutionary relatedness to other species enables us to learn from mutations in nonhuman species (figure 12.1).
A mutation may be present in all the cells of an individual or just in some cells. In a germline mutation, the change occurs during the DNA replication that precedes meiosis. The resulting gamete and all the cells that descend from it after fertilization have the mutation-that is, every cell in the body. Germline mutations are transmitted to the next generation of individuals. In contrast, a somatic mutation happens during DNA replication before a mitotic cell division, and is passed to the next generation of cells, not individuals. All the cells that descend from the original changed cell are altered, but they might only comprise a small part of the body. Somatic mutations are more likely to occur in cells that divide often, such as skin and blood cells, because there are more opportunities for replication errors. Such errors occur spontaneously or in response to exposure to toxins.
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Figure 12.1 Animal models of human disease. This dog has amyotrophic lateral sclerosis (Lou Gehrig's disease), which also affects humans. Mutation in the same gene-superoxide dismutase 1-causes about 2 percent of human cases, as well as the canine cases.
Page 214New tools that enable researchers to detect mutations and polymorphisms throughout the genome, and in single cells from different organs of the same individual, reveal that the body has more genomically different cell populations than had been thought. A person may have cells from a twin that died before birth, or that descend from a cell that underwent a somatic mutation. Most women who have been pregnant retain cells from their fetuses. Most of us are, in some way, genomic mosaics.
Key Concepts Questions 12.1
Where do mutations occur in the genome?
How are mutations and polymorphisms alike and how do they differ?
Why is the ability of DNA to mutate important in evolution?
Distinguish between the consequences of a germline versus a somatic mutation.
12.2 A Closer Look at Two Mutations
Identifying how a mutation causes symptoms has clinical applications, and also reveals the workings of biology. Following are two examples of well-studied mutations that cause disease.
The Beta Globin Gene Revisited
The first genetic illness understood at the molecular level was sickle cell disease. The tiny mutation responsible for sickle cell disease is a substitution of the amino acid valine for the glutamic acid that is normally the sixth amino acid in the beta globin polypeptide chain (figure
12.2). At the DNA level, the change was even smaller-a CTC to a CAC, corresponding to RNA codons GAG and GUG. Valine at this position changes the surfaces of hemoglobin molecules so that in low-oxygen conditions they attach at many more points than they would if the wild type glutamic acid were at the site. The aggregated hemoglobin molecules form ropelike cables that at first make red blood cells sticky and able to deform. Then the red blood cells bend into rigid, fragile, sickle-shaped structures. The misshapen cells lodge in narrow blood vessels, cutting off local blood supplies. Once a blockage occurs, sickling speeds up and spreads, as the oxygen level falls. The result is great pain in the blocked body parts, particularly the hands, feet, and intestines. The bones ache, and depletion of normal red blood cells causes the great fatigue of anemia.
Sickle cell disease was the first inherited illness linked to a molecular abnormality, but it wasn't the first known condition that results from a mutation in the beta globin gene. In 1925, Thomas Cooley and Pearl Lee described severe anemia in Italian children, and in the decade following, others described a milder version of "Cooley's anemia," also in Italian children. The disease was named thalassemia, from the Greek for "sea," in light of its high prevalence in the Mediterranean area. The two disorders turned out to be the same. The severe form, sometimes called thalassemia major, results from a homozygous mutation in the beta globin gene at a site other than the one that causes sickle cell disease. The milder form, called thalassemia minor, affects some individuals who are heterozygous for the mutation.
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Figure 12.2 Sickle cell disease results from a single DNA base change that substitutes one amino acid in the protein (valine replaces glutamic acid). This changes the surfaces of the molecules, and they aggregate into long, curved rods that deform the red blood cell. The illustration shows the appearance of sickled cells.
Once researchers had worked out the structure of hemoglobin, and learned that different globins function in the embryo and fetus (see figure 11.2), the molecular basis of thalassemia became clear. The disorder that is common in the Mediterranean is more accurately called beta thalassemia (OMIM 141900), because the symptoms result from too few beta globin chains. Without them, not enough hemoglobin molecules are assembled to effectively deliver oxygen to tissues. Fatigue and bone pain arise during the first year of life as the child depletes fetal hemoglobin, and the "adult" beta globin genes are not transcribed and translated on schedule.
As severe beta thalassemia progresses, red blood cells die because the excess of alpha globin chains prevents formation of hemoglobin molecules. Liberated iron slowly destroys the heart, liver, and endocrine glands. Periodic blood transfusions can control the anemia, but they hasten iron buildup and organ damage. Drugs called chelators that entrap the iron can extend life past early adulthood, but they are very costly and not available in some nations.