Introduction to Cells and Cell Research PDF

PART Fundamentals and Foundations Chapter 1 Introduction to Cells and Cell Research Chapter 2 Molecules and Membranes Chapter 3 Bioenergetics and Metabolism Chapter 4 Fundamentals of Molecular Biology Chapter 5 Genomics, Proteomics, and Systems Biology CHAPTER Introduction to Cells and Cell Res...

PART Fundamentals and Foundations Chapter 1 Introduction to Cells and Cell Research Chapter 2 Molecules and Membranes Chapter 3 Bioenergetics and Metabolism Chapter 4 Fundamentals of Molecular Biology Chapter 5 Genomics, Proteomics, and Systems Biology CHAPTER Introduction to Cells and Cell Research U nderstanding the molecular biology of cells is one of the most active and fundamental areas of research in the biological sciences. This is true not only from the standpoint of basic science, but also with respect to the numerous applications of cell and molecular biology to medicine, biotechnology, and agriculture. Especially with the ability to obtain rapid sequences of complete genomes, progress in cell and molecular biology is opening new horizons in the practice of medicine. Striking examples include genome editing; the identification of genes that contribute to susceptibility to a variety of common diseases, such as heart disease, rheumatoid arthritis, and diabetes; the development of new drugs specifically targeted to interfere with the growth of cancer cells; and the potential use of stem cells to replace damaged tissues and treat patients suffering from conditions like diabetes, Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries. Because cell and molecular biology is such a rapidly growing field of research, it is important to understand its experimental basis as well as the current state of our knowledge. This chapter will therefore focus on how cells are studied, as well as review some of their basic properties. Appreciating the similarities and differences between cells is particularly important to understanding cell biology. The first section of this chapter discusses both the unity and the diversity of present-day cells in terms of their evolution from a common ancestor. On the one hand, all cells share common fundamental properties that have been conserved throughout evolution. For example, all cells employ DNA as their genetic material, are surrounded by plasma membranes, and use the same basic mechanisms for energy metabolism. On the other hand, present-day cells have evolved a variety of different lifestyles. Many organisms, such as bacteria, amoebas, and yeasts, consist of single cells that are capable of independent self-replication. More complex organisms are composed of collections of cells that function in a coordinated manner, with different cells specialized to perform particular tasks. The human body, for example, is composed of more than 200 different kinds of cells, each specialized for such distinctive functions as memory, sight, movement, and digestion. The diversity exhibited by the many different kinds of cells is striking; for example, consider the differences between bacteria and the cells of the human brain. The fundamental similarities between different types of cells provide a unifying theme to cell biology, allowing the basic principles learned from experiments with one kind of cell to be extrapolated and generalized to other cell types. Several kinds of cells and organisms are widely used to study different aspects of cell and molecular biology; the second section of this chapter discusses some of the properties of these cells that make them particularly 1.1 The Origin and Evolution of Cells 4 1.2 Experimental Models in Cell Biology 18 1.3 Tools of Cell Biology: Microscopy and Subcellular Fractionation 28 Key Experiment HeLa Cells: The First Immortal Cell Line 25 Molecular Medicine Viruses and Cancer 26 4 Chapter 1 valuable as experimental models. Finally, it is important to recognize that progress in cell biology depends heavily on the availability of experimental tools that allow scientists to make new observations or conduct novel kinds of experiments. This introductory chapter therefore concludes with a discussion of some of the experimental approaches used to study cells, as well as a review of some of the major historical developments that have led to our current understanding of cell structure and function. 1.1 The Origin and Evolution of Cells Learning Objectives You should be able to: • Explain how the first cell originated. • Describe the major steps in evolution of metabolism. • Illustrate the structures of eukaryotic and prokaryotic cells. • Outline the evolution of eukaryotic cells and multicellular organisms. Cells are divided into two main classes, initially defined by whether they contain a nucleus. Prokaryotic cells, such as bacteria, lack a nuclear envelope and are generally smaller and simpler than eukaryotic cells, which include the highly specialized cells of multicellular organisms. In spite of these differences, the same basic molecular mechanisms govern the lives of both prokaryotes and eukaryotes, indicating that all present-day cells are descended from a single primordial ancestor. How did this first cell develop? And how did the complexity and diversity exhibited by present-day cells evolve? How did the first cell arise? Organic molecules formed spontaneously in primitive Earth’s atmosphere. It appears that life first emerged at least 3.8 billion years ago, approximately 750 million years after Earth was formed. How life originated and how the first cell came into being are matters of speculation, since these events cannot be reproduced in the laboratory. Nonetheless, several types of experiments provide important evidence bearing on some steps of the process. It was first suggested in the 1920s that simple organic molecules could form and spontaneously polymerize into macromolecules under the conditions thought to exist in primitive Earth’s atmosphere. At the time life arose, the atmosphere of Earth is thought to have contained little or no free oxygen, instead consisting principally of CO2 and N2 in addition to smaller amounts of gases such as H2, H2S, and CO. Such an atmosphere provides reducing conditions in which organic molecules, given a source of energy such as sunlight or electrical discharge, can form spontaneously. The spontaneous formation of organic molecules was first demonstrated experimentally in the 1950s when Stanley Miller (then a graduate student) showed that the discharge of electric sparks into a mixture of H2, CH4, and NH3, in the presence of water, leads to the formation of a variety of organic molecules, including several amino acids (Figure 1.1). Although Miller’s experiments did not precisely reproduce the conditions of primitive Earth, they clearly demonstrated the plausibility of the spontaneous synthesis of organic molecules, providing the basic materials from which the first living organisms arose. Introduction to Cells and Cell Research The next step in evolution was the formation of macromolecules. The monomeric building blocks of macromolecules have been demonstrated to polymerize spontaneously under plausible prebiotic conditions. Heating dry mixtures of amino acids, for example, results in their polymerization to form polypeptides. But the critical characteristic of the macromolecule from which life evolved must have been the ability to replicate itself. Only a macromolecule capable of directing the synthesis of new copies of itself would have been capable of reproduction and further evolution. Of the two major classes of informational macromolecules in present-day cells (nucleic acids and proteins), only the nucleic acids are capable of directing their own self-replication. Nucleic acids can serve as templates for their own synthesis as a result of specific base pairing between complementary nucleotides (Figure 1.2). A critical step in understanding molecular evolution was thus reached in the early 1980s, when it was discovered in the laboratories of Sid Altman and Tom Cech that RNA is capable of catalyzing a number of chemical reactions, including the polymerization of nucleotides. Further studies have extended the known catalytic activities of RNA, including the description of RNA molecules that direct the synthesis of a new RNA strand from an RNA template. RNA is thus uniquely able to both serve as a template and to catalyze its own replication. Consequently, RNA is generally believed to have been the initial genetic system, and an early stage of chemical evolution is thought to have been based on self-replicating Electrode CH4 NH3 H 2O H2 Electric discharge H2O H2 Water vapor was re uxed through an atmosphere consisting of H2, CH4, and NH3, into which electric sparks were discharged. CH4 NH3 Cooling Water Heat Analysis of the reaction products revealed the formation of a variety of organic molecules, including the amino acids alanine, aspartic acid, glutamic acid, and glycine. Organic molecules Alanine Aspartic acid Glutamic acid Glycine Urea Lactic acid Acetic acid Formic acid Figure 1.1 Spontaneous formation of organic molecules The formation of macromolecules was the next step in evolution, achieved by the polymerization of monomeric building blocks. The critical characteristic of the macromolecule from which life evolved was the ability to replicate itself. Only nucleic acids are capable of directing their own self-replication. RNA is capable of catalyzing a number of chemical reactions, including the polymerization of nucleotides, and can serve as a template and catalyze its own replication. RNA is believed to have been the initial genetic system. C C G A G A U U G A C G A C G C U C A U A C U G C G A G A U U G C U C U A A C G A G A U U G C U C U A A C G A G A U U G A C C U G G A C C U G G A C Figure 1.2 Self-replication of RNA Complementary pairing between nucleotides (adenine [A] with uracil [U] and guanine [G] with cytosine [C]) allows one strand of RNA to serve as a template for the synthesis of a new strand with the complementary sequence. G C U C U G C U C U A A G A C C U G G A U A C U G U C G A G A U U 5 6 Chapter 1 RNA molecules—a period of evolution known as the RNA world. Ordered interactions between RNA and amino acids then evolved into the present-day genetic code, and DNA eventually replaced RNA as the genetic material. As discussed further in Chapter 4, all present-day cells use DNA as the All present-day cells use the same genetic material and employ the same basic mechanisms for DNA replication genetic mechanisms. and expression of the genetic information. Genes are the functional units of inheritance, corresponding to segments of DNA that encode proteins or RNA molecules. The nucleotide sequence of a gene is copied into RNA by a process called transcription. For RNAs that encode proteins, their nucleotide sequence is then used to specify the order of amino acids in a protein by a process called translation. The first cell is presumed to have arisen by the enclosure of self-replicating Phospholipids are the basic RNA in a membrane composed of phospholipids (Figure 1.3). As discussed components of biological in detail in the next chapter, phospholipids are the basic components of all membranes. present-day biological membranes, including the plasma membranes of both prokaryotic and eukaryotic cells. The key characteristic of the phospholipids that form membranes is that they are amphipathic molecules, meaning that one portion of the molecule is soluble in water and another portion is not. 1. **RNA's Early Role:** RNA played a big part in the Phospholipids have long, water-insoluble (hydrophobic) hydrocarbon chains joined to water-soluble (hydrophilic) head groups that contain phosphate. When start of life [RNA World]. It mixed with amino acids, which later turned into the genetic code. placed in water, phospholipids spontaneously aggregate into a bilayer with their phosphate-containing head groups on the outside in contact with water and their 2. **DNA Takes Over:** Later on, DNA replaced hydrocarbon tails in the interior in contact with each other. Such a phospholipid RNA as our main genetic material. bilayer forms a stable barrier between two aqueous compartments—for example, 3. **Genes and Proteins:** Genes are like our body's separating the interior of the cell from its external environment. instruction manuals, written in DNA. They make The enclosure of self-replicating RNA and associated molecules in a proteins through transcription [copying DNA into phospholipid membrane would thus have maintained them as a unit, capable RNA] and translation [turning RNA into proteins]. of self-reproduction and further evolution. RNA-directed protein synthesis RNA can catalyze its own replication. 4. **First Cell Formation:** The very first cell likely formed when self-replicating RNA was enclosed in a protective [phospholipid] membrane. 5. **Role of Phospholipid Membrane:** This special membrane kept everything inside the cell safe, allowing self-reproduction and further evolution. This membrane consists of water-attracting (hydrophilic) and water-repelling (hydrophobic) components, creating a stable barrier between two watery compartments. RNA Phospholipid membrane Water 6. **Protein Making:** RNA helps in making proteins, which is vital for life. Phospholipid molecule: Hydrophilic head group Hydrophobic tail RNA-directed protein synthesis may already have evolved by this time, in which case the first cell wouldhave consisted of self-replicating RNA and its encoded proteins. Water Figure 1.3 Enclosure of self-replicating RNA in a phospholipid membrane The first cell is thought to have arisen by the enclosure of self-replicating RNA and associated molecules in a membrane composed of phospholipids. Each phospholipid molecule has two long hydrophobic tails attached to a hydrophilic head group. The hydrophobic tails are buried in the lipid bilayer; the hydrophilic heads are exposed to water on both sides of the membrane. Introduction to Cells and Cell Research 7 may already have evolved by this time, in which case the first cell would have consisted of self-replicating RNA and its encoded proteins. The evolution of metabolism Because cells originated in a sea of organic molecules, they were able to obtain food and energy directly from their environment. But such a situation is self-limiting, so cells needed to evolve their own mechanisms for generating energy and synthesizing the molecules necessary for their replication. The generation and controlled utilization of metabolic energy is central to all cell activities, and the principal pathways of energy metabolism (discussed in detail in Chapter 3) are highly conserved in present-day cells. All cells use adenosine 5′-triphosphate (ATP) as their source of metabolic energy to drive the synthesis of cell constituents and carry out other energy-requiring activities, such as movement (e.g., muscle contraction). The mechanisms used by cells for the generation of ATP are thought to have evolved in three stages, corresponding to the evolution of glycolysis, photosynthesis, and oxidative metabolism (Figure 1.4). The development of these metabolic pathways changed Earth’s atmosphere, thereby altering the course of further evolution. In the initially anaerobic atmosphere of Earth, the first energy-generating reactions presumably involved the breakdown of organic molecules in the absence of oxygen. These reactions are likely to have been a form of presentday glycolysis—the anaerobic breakdown of glucose to lactic acid, with the net energy gain of two molecules of ATP. In addition to using ATP as their source of intracellular chemical energy, all present-day cells carry out glycolysis, consistent with the notion that these reactions arose very early in evolution. Glycolysis provided a mechanism by which the energy in preformed organic molecules (e.g., glucose) could be converted to ATP, which could then be used as a source of energy to drive other metabolic reactions. The development of photosynthesis is generally thought to have been the next major evolutionary step, which allowed the cell to harness energy from sunlight and provided independence from the utilization of preformed organic molecules. The first photosynthetic bacteria probably utilized H2S to convert CO2 to organic The first cells obtained energy by glycolysis. Photosynthesis made cells independent of organic molecules in the environment. Glycolysis C6H12O6 2 C3H6O3 Glucose Lactic acid Generates 2 ATP Existence of organisms in extreme conditions has led to the hypothesis that life could exist in similar environments elsewhere in the solar system. The field of astrobiology (or exobiology) seeks to find signs of this extraterrestrial life. Photosynthesis 6 CO2 + 6 H2O C6H12O6 + 6 O2 Glucose Oxidative metabolism C6H12O6 + 6 O2 6 CO2 + 6 H2O FYI Generates 36–38 ATP Glucose Figure 1.4 Generation of metabolic energy Glycolysis is the anaerobic breakdown of glucose to lactic acid. Photosynthesis utilizes energy from sunlight to drive the synthesis of glucose from CO2 and H2O, with the release of O2 as a by-product. The O2 released by photosynthesis is used in oxidative metabolism, in which glucose is broken down to CO2 and H2O, releasing much more energy than can be obtained from glycolysis. 8 Chapter 1 The oxidation of glucose to carbon dioxide and water yields much more energy than glycolysis. molecules—a pathway of photosynthesis still used by some bacteria. The use of H2O as a donor of electrons and hydrogen for the conversion of CO2 to organic compounds evolved later and had the important consequence of changing Earth’s atmosphere. The use of H2O in photosynthetic reactions produces the by-product free O2; this mechanism is thought to have been responsible for making O2 abundant in Earth’s atmosphere, which occurred about 2.4 billion years ago. The release of O2 as a consequence of photosynthesis changed the environment in which cells evolved and is commonly thought to have led to the development of oxidative metabolism. Alternatively, oxidative metabolism may have evolved before photosynthesis, with the increase in atmospheric O2 then providing a strong selective advantage for organisms capable of using O2 in energy-producing reactions. In either case, O2 is a highly reactive molecule, and oxidative metabolism, utilizing this reactivity, has provided a mechanism for generating energy from organic molecules that is much more efficient than anaerobic glycolysis. For example, the complete oxidative breakdown of glucose to CO2 and H2O yields energy equivalent to that of 36 to 38 molecules of ATP, in contrast to the 2 ATP molecules formed by anaerobic glycolysis (see Figure 1.4). With few exceptions, present-day cells use oxidative reactions as their principal source of energy. Prokaryotes Plasma membrane Cell wall Prokaryotes are smaller and simpler than eukaryotes. Nucleoid Prokaryotes include cells of two domains, the Archaea and the Bacteria, which diverged early in evolution. The Archaea include cells that live in extreme environments that are unusual today but may have been prevalent in primitive Earth. For example, thermoacidophiles live in hot sulfur springs with temperatures as high as 80°C and pH values as low as 2. The Bacteria include the common forms of present-day prokaryotes—a large group of organisms that live in a wide range of environments, including soil, water, and other organisms (e.g., human pathogens). Prokaryotic cells are smaller and simpler than most eukaryotic cells, their genomes are less complex, and they do not contain nuclei or cytoplasmic organelles (Table 1.1). Most prokaryotic cells are spherical, rod-shaped, or spiral, with diameters of 1 to 10 m. Their DNA contents range from about 0.6 million to 5 million base pairs, an amount sufficient to encode about 5000 different proteins. The largest and most complex prokaryotes are the cyanobacteria—bacteria in which photosynthesis evolved. The structure of a typical bacterial cell is illustrated by Escherichia coli (E. coli), a common inhabitant of the human intestinal tract (Figure 1.5). The cell is rod-shaped, about 1 m in diameter and about 2 m long. Like most other prokaryotes, E. coli is surrounded by a rigid cell wall composed of polysaccharides and peptides. Beneath the cell wall is the plasma membrane, which is a bilayer of phospholipids and associated proteins. Whereas the cell wall is porous and readily penetrated by a variety of molecules, the plasma membrane provides the functional separation between the inside of the cell and its external environment. The DNA of E. coli is a single circular molecule in the nucleoid, which, in contrast to the nucleus of eukaryotes, Figure 1.5 Electron micrograph of E. coli The cell is surrounded by a cell 0.5 m wall, beneath which is the plasma membrane. DNA is located in the nucleoid. Artificial color has been added. (© Biophoto Associates/Science Source.) Introduction to Cells and Cell Research Table 1.1 Prokaryotic and Eukaryotic Cells Characteristic Prokaryote Eukaryote Nucleus Absent Present Diameter of a typical cell ≈1 m 10–100 m Cytoplasmic organelles Absent 6 9 Prokaryotes are smaller and simpler than eukaryotes. Present 6 DNA content (base pairs) 1 × 10 to 5 × 10 1.5 × 107 to 5 × 109 Chromosomes Single circular DNA molecule Multiple linear DNA molecules is not surrounded by a membrane separating it from the cytoplasm. The cytoplasm contains approximately 30,000 ribosomes (the sites of protein synthesis), which account for its granular appearance. Eukaryotic cells Like prokaryotic cells, all eukaryotic cells are surrounded by a plasma membrane and contain ribosomes. However, eukaryotic cells are much more complex and contain a nucleus and a variety of cytoplasmic organelles (Figure 1.6). The largest and most prominent organelle of eukaryotic cells is the nucleus, with a diameter of approximately 5 m. The nucleus contains the genetic information of the cell, which in eukaryotes is organized as linear rather than circular DNA molecules. The nucleus is the site of DNA replication and of RNA synthesis; the translation of RNA into proteins takes place on ribosomes in the cytoplasm. In addition to a nucleus, eukaryotic cells contain a variety of membraneenclosed organelles within their cytoplasm. These organelles provide compartments in which different metabolic activities are localized. Eukaryotic cells are generally much larger than prokaryotic cells, frequently having a cell volume at least a thousandfold greater. The compartmentalization provided by cytoplasmic organelles is what allows eukaryotic cells to function efficiently. Two of these organelles, mitochondria and chloroplasts, play critical roles in energy metabolism. Mitochondria, which are found in almost all eukaryotic cells, are the sites of oxidative metabolism and are thus responsible for generating most of the ATP derived from the breakdown of organic molecules. Chloroplasts are the sites of photosynthesis and are found only in the cells of plants and green algae. Lysosomes and peroxisomes also provide specialized metabolic compartments for the digestion of macromolecules and for various oxidative reactions, respectively. In addition, most plant cells contain large vacuoles that perform a variety of functions, including the digestion of macromolecules and the storage of both waste products and nutrients. Because of the size and complexity of eukaryotic cells, the transport of proteins to their correct destinations within the cell is a formidable task. Two cytoplasmic organelles, the endoplasmic reticulum (ER) and the Golgi apparatus, are specifically devoted to the sorting and transport of proteins destined for secretion, incorporation into the plasma membrane, and incorporation into lysosomes and peroxisomes. The endoplasmic reticulum is an extensive network of intracellular membranes, extending from the nuclear envelope throughout the cytoplasm. It functions not only in the processing and transport of proteins (the rough endoplasmic reticulum, which is covered by ribosomes), but also in the synthesis of lipids (the smooth endoplasmic reticulum). From Eukaryotic cells contain nuclei and cytoplasmic organelles. Animal cell Cytoskeleton Nucleolus Nucleus Rough endoplasmic reticulum Lysosome Ribosomes Mitochondrion Peroxisome Centrioles Golgi apparatus Plant cell Plasma membrane Smooth endoplasmic reticulum Ribosomes Nucleolus Nucleus Cell wall Vacuole Rough endoplasmic reticulum Peroxisome Mitochondrion Plasma membrane Chloroplast Smooth endoplasmic reticulum Plasmodesmata Cytoskeleton Golgi apparatus Introduction to Cells and Cell Research ▼ Figure 1.6 Structures of animal and plant cells Both animal and plant cells are surrounded by a plasma membrane and contain a nucleus, a cytoskeleton, and many cytoplasmic organelles in common. Plant cells are also surrounded by a cell wall and contain chloroplasts and large vacuoles. the endoplasmic reticulum, proteins are transported within small membrane vesicles to the Golgi apparatus, where they are further processed and sorted for transport to their final destinations. In addition to this role in protein transport, the Golgi apparatus serves as a site of lipid synthesis and (in plant cells) as the site of synthesis of some of the polysaccharides that compose the cell wall. The internal organization of eukaryotic cells is maintained by the cytoskeleton, a network of protein filaments extending throughout the cytoplasm. The cytoskeleton provides the structural framework of the cell, determining cell shape and the general organization of the cytoplasm. In addition, the cytoskeleton is responsible for the movements of entire cells (e.g., the contraction of muscle cells) and for the intracellular transport and positioning of organelles and other structures, including the movements of chromosomes during cell division. The origin of eukaryotes Eukaryotic cells are the third domain of life, called the Eukarya, which arose as a branch from the Archaea (Figure 1.7). A critical step in the evolution of eukaryotic cells was the acquisition of membrane-enclosed subcellular organelles, allowing the development of the complexity characteristic of these cells. It is likely that some organelles evolved from invaginations of the plasma membrane. Cyanobacteria Other bacteria Plants Green algae Animals Fungi (yeasts) Protists Archaebacteria Chloroplasts Eukarya Mitochondria Bacteria Archaea First cell Figure 1.7 Evolution of cells Present-day cells evolved from a common ancestor that gave rise to the two prokaryotic domains of life, the Archaea and Bacteria. The evolution of eukaryotic cells (Eukarya) from the Archaea involved the formation of mitochondria by endosymbiosis. Plants and green algae subsequently evolved by the endosymbiotic formation of chloroplasts. 11 12 Chapter 1 Mitochondria and chloroplasts originated by endosymbiosis. FYI Certain present-day marine protists engulf algae to serve as endosymbionts that carry out photosynthesis for their hosts. For example, the nucleus is thought to have been formed by invaginations of the plasma membrane that surrounded the nucleoid of a prokaryotic ancestor. At least two organelles of eukaryotes, mitochondria and chloroplasts, arose by endosymbiosis—one cell living inside another (Figure 1.8). In particular, mitochondria are thought to have evolved from aerobic bacteria living inside the archaeal ancestor of eukaryotes and chloroplasts evolved from photosynthetic bacteria, such as cyanobacteria, living inside the ancestor to plants and green algae. Both mitochondria and chloroplasts are similar to bacteria in size and, like bacteria, they reproduce by dividing in two. Most important, both mitochondria and chloroplasts contain their own DNA, which encodes some of their components. The mitochondrial and chloroplast DNAs are replicated each time the organelle divides, and the genes they encode are transcribed within the organelle and translated on organelle ribosomes. Mitochondria and chloroplasts thus contain their own genetic systems, which are distinct from the nuclear genome of the cell and are more closely related to the genomes of bacteria than to the nuclear genomes of eukaryotes. The acquisition of aerobic bacteria would have provided an anaerobic cell with the ability to carry out oxidative metabolism. The acquisition of photosynthetic bacteria would have provided the ability to perform photosynthesis, thereby affording nutritional independence. Thus, these endosymbiotic associations were highly advantageous to their partners and were selected for in the course of evolution. Through time, most of the genes originally present in these bacteria apparently became incorporated into the nuclear genome of the cell, so only a few components of mitochondria and chloroplasts are still encoded by the organelle genomes. Archaea Bacteria Endosymbiosis Most genes of endocytosed bacterium are transferred to host genome Figure 1.8 Endosymbiosis Mitochondria arose from aerobic bacteria living with the archaeal ancestor to eukaryotes. Most bacterial genes were subsequently transferred to the nuclear genome. Mitochondrion Introduction to Cells and Cell Research It is important to note that the genomes of eukaryotes are mosaics, with some eukaryotic genes more similar to bacterial genes and others more similar to archaeal genes. Curiously, most eukaryotic genes related to informational processes (such as DNA replication, transcription, and protein synthesis) were derived from archaebacteria, whereas most eukaryotic genes related to basic cell operational processes (such as glycolysis and amino acid biosynthesis) were derived from bacteria. One hypothesis to explain the mosaic nature of eukaryotic genomes is that the genome of eukaryotes arose from a fusion of archaeal and bacterial genomes. According to this proposal, an endosymbiotic association between a bacterium and an archaeum was followed by fusion of the two prokaryotic genomes, giving rise to an ancestral eukaryotic genome with contributions from both bacteria and archaea (see Figure 1.8). The simplest version of this hypothesis is that an initial endosymbiotic relationship of a bacterium living inside an archaeum gave rise not only to mitochondria but also to the genome of eukaryotic cells, containing genes derived from both prokaryotic ancestors. The genomes of eukaryotes are mosaics of archaeal and bacterial genes. The development of multicellular organisms Many eukaryotes are unicellular organisms that, like bacteria, consist of only single cells capable of self-replication. The simplest eukaryotes are the yeasts, which contain only slightly more genes than many bacteria (Table 1.2). Although yeasts are more complex than bacteria, they are much Table 1.2 Cell Genomes Organism Haploid DNA content (millions of base pairs) Protein-coding genes Archaebacteria Methanococcus jannaschii 1.7 1700 Bacteria Mycoplasma 0.6 470 E. coli 4.6 4200 Cyanobacterium 3.6 3200 Unicellular eukaryotes Saccharomyces cerevisiae (yeast) 12 6000 Dictyostelium discoideum 34 12,000 Paramecium 72 39,500 Chlamydomonas 118 14,500 Volvox 138 14,500 Plants Arabidopsis thaliana 125 26,000 Corn 2200 33,000 Apple 740 57,000 97 19,000 Animals Caenorhabditis elegans (nematode) Drosophila melanogaster (fruit fly) 180 14,000 Zebrafish 1700 26,000 Mouse 3000 20,000 Human 3000 20,000 13 14 Chapter 1 Figure 1.9 Scanning electron micrograph of Saccharomyces cerevisiae Yeasts are the simplest eukaryotes. Artificial color has been added to the micrograph. (© Medical-on-Line/Alamy.) 5 m Video 1.1 Paramecium Feeding (A) Paramecium smaller and simpler than most cells of animals or plants. For example, the commonly studied yeast Saccharomyces cerevisiae is about 6 m in diameter and contains 12 million base pairs of DNA (Figure 1.9). Other unicel lular eukaryotes, however, are far more complex cells, with substantially larger and more complex genomes. They include organisms specialized to perform a variety of tasks, including photosynthesis, movement, and the capture and ingestion of other organisms as food. The ciliated protozoan Paramecium, for example, is a large, complex cell that can be up to 350 m in length and is specialized for movement and feeding on bacteria and yeast (Figure 1.10). Surprisingly, the Paramecium genome contains almost twice as many genes as humans (see Table 1.2), illustrating the fact that neither genome size nor gene number is directly related to the complexity of an (B) Chlamydomonas Figure 1.10 Light micrograph of Paramecium and scanning electron micrograph of Chlamydomonas Paramecium and Chlamydomas are examples of unicellular eukaryotes that are more complex than yeast. (A, © M. I. Walker/Science Source; B, © Aaron J. Bell/Science Source.) Introduction to Cells and Cell Research Figure 1.11 Multicellular green algae Volvox consists of approximately 16 germ cells and 2000 somatic cells embedded in a gelatinous matrix. (Courtesy of David Kirk.) organism—an unexpected result of genome sequencing projects that will be discussed further in Chapters 5 and 6. Other unicellular eukaryotes, such as the green alga Chlamydomonas (see Figure 1.10), contain chloroplasts and are able to carry out photosynthesis. The evolution of multicellular organisms from unicellular eukaryotes occurred multiple times, independently for plants and animals. The algae, for example, contain both unicellular and multicellular species. The multicellular green alga Volvox contains cells of two different types: approximately 16 large germ cells and 2000 somatic cells that resemble the unicellular Chlamydomonas (Figure 1.11). Both Chlamydomonas and Volvox have genomes of similar size and complexity, about 10 times larger than that of yeast and containing 14,000–15,000 genes (see Table 1.2). Another example of the transition to multicellularity is provided by the amoeba Dictyostelium discoideum, which is able to alternate between unicellular and multicellular forms depending on the availability of food (Figure 1.12). Increasing cell specialization and division of labor among the cells of simple multicellular organisms then led to the complexity and diversity observed in the many types of cells that make up present-day plants and animals, including human beings. Plants are composed of fewer cell types than are animals, but each different kind of plant cell is specialized to perform specific tasks required by the organism as a whole (Figure 1.13). The cells of plants are organized into three main tissue systems: ground tissue, dermal tissue, and vascular tissue. The ground tissue contains parenchyma cells, which carry out most of the metabolic reactions of the plant, including photosynthesis. Ground tissue also contains two specialized cell types (collenchyma cells and sclerenchyma cells) that are characterized by thick (A) Unicellular amoebae (B) Fruiting body Figure 1.12 Dictyostelium discoideum If food is unavailable, the unicellular amoebae (A) aggregate to form a multicellular fruiting body, specialized for the dispersal of spores (B). (A, courtesy of David Knecht, University of Connecticut; B, © David Scharf/Science Source.) Multicellular organisms evolved from associations between unicellular eukaryotes. Multicellular organisms evolved from associations between unicellular eukaryotes. 15 16 Chapter 1 (A) Collenchyma cells (B) Epidermal cells (C) Xylem vessel elements and tracheids Figure 1.13 Representative plant cells (A) Collenchyma cells (from spinach leaf vein) are specialized for support and have thickened cell walls. (B) Epidermal cells on the surface of a dayflower leaf. Tiny pores (stomata) are flanked by specialized cells called guard cells. (C) Vessel elements and tracheids of a squash stem are elongated cells that are arranged end to end to form vessels of the xylem. (A, © Phil Gates/ Biological Photo Service; B, © Alfred Owczarzak/Biological Photo Service; C, © J. Robert Waaland/Biological Photo Service.) Animal cells evolved to perform specialized functions. (A) Epithelial cells cell walls and provide structural support to the plant. Dermal tissue covers the surface of the plant and is composed of epidermal cells, which form a protective coat and allow the absorption of nutrients. Finally, several types of elongated cells form the vascular system (the xylem and phloem), which is responsible for the transport of water and nutrients throughout the plant. The cells found in animals are considerably more diverse than those of plants. The human body, for example, is composed of more than 200 different kinds of cells, which are generally considered to be components of five main types of tissues: epithelial tissue, connective tissue, blood, nervous tissue, and muscle (Figure 1.14). Epithelial cells form sheets that cover the (B) Fibroblasts (C) Blood cells Erythrocyte Lymphocyte Figure 1.14 Representative animal cells (A) Epithelial cells of the mouth form a thick, multilayered sheet. (B) Fibroblasts are connective tissue cells characterized by their elongated spindle shape. (C) Erythrocytes and lymphocytes in human blood. (A, © G. W. Willis/Visuals Unlimited, Inc.; B, © Biophoto Associates/Science Source; C, © G. W. Willis/Visuals Unlimited, Inc.) Introduction to Cells and Cell Research surface of the body and line the internal organs. There are many different types of epithelial cells, each specialized for a specific function, including protection (the skin), absorption (e.g., cells lining the small intestine), and secretion (e.g., cells of the salivary gland). Connective tissues include bone, cartilage, and adipose tissue, each of which is formed by different types of cells (osteoblasts, chondrocytes, and adipocytes, respectively). The loose connective tissue that underlies epithelial layers and fills the spaces between organs and tissues in the body is formed by another cell type, the fibroblast. Blood contains several different types of cells: red blood cells (erythrocytes) function in oxygen transport, and white blood cells (granulocytes, monocytes, macrophages, and lymphocytes) function in inflammatory reactions and the immune response. Nervous tissue is composed of supporting cells and nerve cells, or neurons, which are highly specialized to transmit signals throughout the body. Various types of sensory cells, such as cells of the eye and ear, are further specialized to receive external signals from the environment. Finally, several different types of muscle cells are responsible for the production of force and movement. The evolution of animals clearly involved the development of considerable diversity and specialization at the cellular level. Understanding the mechanisms that control the growth and differentiation of such a complex array of specialized cells, starting from a single fertilized egg, is one of the major challenges facing contemporary cell and molecular biology. 1.1 Review The first cell is thought to have arisen at least 3.8 billion years ago by the enclosure of self-replicating RNA in a phospholipid membrane. The earliest reactions for generation of metabolic energy were a form of anaerobic glycolysis, followed by the evolution of photosynthesis and oxidative metabolism. Two domains of prokaryotic cells, Bacteria and Archaea, diverged early in evolution. Eukaryotic cells, which are larger and more complex than prokaryotic cells, contain a nucleus and cytoplasmic organelles. They evolved as a branch from the Archaea, with mitochondria and chloroplasts originating by endosymbiosis. Multicellular organisms then evolved from associations between unicellular eukaryotes, and division of labor led to the development of the many kinds of specialized cells that make up present-day plants and animals. Questions 1. What properties of RNA support the hypothesis that it was the first self-replicating molecule in early evolution? 2. How did the evolution of photosynthesis affect the development of oxidative metabolism? 3. Assume that the diameter of a eukaryotic cell is 50 m. How much larger in volume is that cell compared to a bacterium with a diameter of 2 m? What is the function of subcellular organelles in helping eukaryotic cells function despite their large size? 4. What is the evidence that mitochondria originated from bacteria that were engulfed by the precursor of eukaryotic cells? 17 18 Chapter 1 5. What present-day organisms do you expect the DNA sequence of chloroplasts to most closely resemble? 6. How would you compare the evolutionary origins of mitochondria and the endoplasmic reticulum? 7. How does genome complexity relate to the development of multicellular organisms? 1.2 Experimental Models in Cell Biology Learning Objectives You should be able to: • Explain the advantages of E. coli for studying basic concepts of molecular biology. • Contrast yeast with E. coli as a model system. • Summarize the simple models for studying plant and animal development. • Describe the advantages and disadvantages of studying vertebrates. • Summarize the principles of animal cell culture. • Explain how viruses can be used to study cell biology. The evolution of present-day cells from a common ancestor has important implications for cell and molecular biology as an experimental science. Because the fundamental properties of all cells have been conserved during evolution, the basic principles learned from experiments performed with one type of cell are generally applicable to other cells. On the other hand, because of the diversity of present-day cells, many kinds of experiments can be more readily undertaken with one type of cell than with another. Several different kinds of cells and organisms are commonly used as experimental models to study various aspects of cell and molecular biology. The features of some of these cells that make them particularly advantageous as experimental models are discussed in the sections that follow. The availability of complete genome sequences further enhances the value of these organisms as model systems in understanding the molecular biology of cells. E. coli Because of their comparative simplicity, bacteria are ideal models for studying many fundamental aspects of biochemistry and molecular biology. The most thoroughly studied species of bacteria is Escherichia coli (E. coli), which has long been the favored organism for investigation of the basic mechanisms of molecular genetics. Most of our present concepts of molecular biology—including our understanding of DNA replication, the genetic code, gene expression, and protein synthesis—derive from studies of this humble bacterium. E. coli has been especially useful to molecular biologists because of both its relative simplicity and the ease with which it can be propagated and studied in the laboratory. The genome of E. coli, for example, consists of approximately 4.6 million base pairs and contains about 4000 genes. The human genome is Introduction to Cells and Cell Research nearly a thousand times larger (approximately 3 billion base pairs) and is thought to contain about 20,000 protein-coding genes (see Table 1.2). The small size of the E. coli genome provides obvious advantages for genetic analysis. Molecular genetic experiments are further facilitated by the rapid growth of E. coli under well-defined laboratory conditions. Under optimal culture conditions, E. coli divide every 20 minutes. Moreover, a clonal population of E. coli, in which all cells are derived by division of a single cell of origin, can be readily isolated as a colony grown on semisolid agar-containing medium (Figure 1.15). Because bacterial colonies containing as many as 108 cells can develop overnight, selecting genetic variants of an E. coli strain—for example, mutants that are resistant to an antibiotic such as penicillin—is easy and rapid. The ease with which such mutants can be selected and analyzed was critical to the success of experiments that defined the basic principles of molecular genetics, discussed in Chapter 4. The nutrient mixtures in which E. coli divide most rapidly include glucose, salts, and various organic compounds, such as amino acids, vitamins, and nucleic acid precursors. However, E. coli can also grow in much simpler media consisting only of salts, a source of nitrogen (such as ammonia), and a source of carbon and energy (such as glucose). In such a medium, the bacteria grow a little more slowly (with a division time of about 40 minutes) because they must synthesize all their own amino acids, nucleotides, and other organic compounds. The ability of E. coli to carry out these biosynthetic reactions in simple defined media has made them extremely useful in elucidating the biochemical pathways involved. Thus, the rapid growth and simple nutritional requirements of E. coli have greatly facilitated fundamental experiments in both molecular biology and biochemistry. 19 Figure 1.15 Bacterial colonies Photograph of colonies of E. coli growing on the surface of an agar-containing medium. (© A. M. Siegelman/Visuals Unlimited, Inc.) The ease of working with E. coli made it the fundamental model for molecular biology. Yeasts Although bacteria have been an invaluable model for studies of many conserved properties of cells, they obviously cannot be used to study aspects of cell structure and function that are unique to eukaryotes. Yeasts, the simplest eukaryotes, have a number of experimental advantages similar to those of E. coli. Consequently, yeasts have provided a crucial model for studies of many fundamental aspects of eukaryotic cell biology. The genome of the most frequently studied yeast, Saccharomyces cerevisiae, consists of 12 million base pairs of DNA and contains about 6000 genes. Although the yeast genome is approximately three times larger than that of E. coli, it is far more manageable than the genomes of more complex eukaryotes, such as humans. Yet, even in its simplicity, the yeast cell exhibits the typical features of eukaryotic cells (Figure 1.16): It contains a distinct nucleus surrounded by a nuclear membrane, its genomic DNA is organized as 16 linear chromosomes, and its cytoplasm contains subcellular organelles. Yeasts can be readily grown in the laboratory and can be studied by many of the same molecular genetic approaches that have proved so successful with E. coli. Although yeasts do not replicate as rapidly as bacteria, they still divide as frequently as every 2 hours and they can easily be grown as colonies from a single cell. Consequently, yeasts can be used for a variety of genetic manipulations similar Figure 1.16 Transmission electron microto those that can be performed using bacteria. graph of Saccharomyces cerevisiae Yeasts These features have made yeast cells the most approachable are the simplest model for studying eukaryotic cells. (© Biophoto Associates/Science Source.) eukaryotic cells from the standpoint of molecular biology. Yeast 20 Chapter 1 Figure 1.17 Caenorhabditis elegans The nematode is widely used for studies of animal development. (From J. E. Sulston and H. R. Horvitz, 1977. Dev. Biol. 56: 110.) Ovary Pharynx Eggs Intestine Vulva Rectum Anus 1 mm Yeasts are the simplest model for eukaryotic cells. mutants have been important in understanding many fundamental processes in eukaryotes, including DNA replication, transcription, RNA processing, protein sorting, and the regulation of cell division, as will be discussed in subsequent chapters. The unity of molecular cell biology is made abundantly clear by the fact that the general principles of cell structure and function revealed by studies of yeasts apply to all eukaryotic cells. Caenorhabditis elegans and Drosophila melanogaster C. elegans is a simple model for studies of animal development and differentiation. The genetics of Drosophila have made it a key model in developmental biology. The unicellular yeasts are important models for studies of eukaryotic cells, but understanding the development of multicellular organisms requires the experimental analysis of plants and animals—organisms that are more complex. The nematode Caenorhabditis elegans (Figure 1.17) possesses several notable features that make it one of the most widely used models for studies of animal development and cell differentiation. Although the genome of C. elegans (close to 100 million base pairs) is larger than those of unicellular eukaryotes, it is smaller and more manageable than the genomes of most animals. Despite its relatively small size, however, the genome of C. elegans contains approximately 19,000 genes—more than three times the number of genes in yeast, and nearly the same number of protein-coding genes in humans. Biologically, C. elegans is a relatively simple multicellular organism: Adult worms consist of only 959 somatic cells, plus 1000–2000 germ cells. In addition, C. elegans can be easily grown and subjected to genetic manipulations in the laboratory. The simplicity of C. elegans has enabled the course of its development to be studied in detail by microscopic observation. Such analyses have successfully traced the embryonic origin and lineage of all the cells in the adult worm. Genetic studies have also identified many of the mutations responsible for developmental abnormalities, leading to the isolation and characterization of critical genes that control nematode development and differentiation. Importantly, similar genes have also been found to function in complex animals (including humans), making C. elegans an important model for studies of animal development. Like C. elegans, the fruit fly Drosophila melanogaster (Figure 1.18) has been a crucial model organism in developmental biology. The genome of Drosophila is 180 million base pairs, larger than that of C. elegans, but the Drosophila genome only contains about 14,000 genes. Furthermore, Drosophila can be easily maintained and bred in the laboratory, and its short reproductive cycle (about 2 weeks) makes it a very useful organism for genetic experiments. Introduction to Cells and Cell Research 21 Many fundamental concepts of genetics—such as the relationship between genes and chromosomes—were derived from studies of Drosophila early in the twentieth century (see Chapter 4). Extensive genetic analysis of Drosophila has uncovered many genes that control development and differentiation, and current methods of molecular biology have allowed the functions of these genes to be analyzed in detail. Consequently, studies of Drosophila have led to striking advances in understanding the molecular mechanisms that govern animal development, particularly with respect to formation of the body plan of complex multicellular organisms. As with C. elegans, similar genes and mechanisms exist in vertebrates, validating the use of Drosophila as a major experimental model in contemporary developmental biology. Arabidopsis thaliana The study of plant molecular biology and development is an active and expanding field of considerable economic importance as well as intellectual interest. Since the genomes of plants cover a range of complexity comparable to that of animal genomes (see Table 1.2), an optimal model for studies of plant development would be a relatively simple organism with some of the advantageous properties of C. elegans and Drosophila. The small flowering plant Arabidopsis thaliana (mouse-ear cress) (Figure 1.19) meets these criteria and is therefore widely used as a model to study the molecular biology of plants. Arabidopsis is notable for its genome of only about 125 million base pairs. Although Arabidopsis contains a total of about 26,000 genes, many of these are repeated, so its number of unique genes is approximately 15,000—a complexity similar to that of C. elegans and Drosophila. In addition, Arabidopsis is relatively easy to grow in the laboratory, and methods for molecular genetic manipulations of this plant have been developed. These studies have led to the identification of genes involved in various aspects of plant development, such as the development of flowers. Analysis of these genes points to many similarities, but also to striking differences, between the mechanisms that control the development of plants and animals. Vertebrates The most complex animals are the vertebrates, including humans and other mammals. The human genome is approximately 3 billion base pairs—about 20 to 30 times larger than the genomes of C. elegans, Drosophila, or Arabidopsis—and contains about 20,000 protein-coding genes. Moreover, the human body is composed of more than 200 different kinds of specialized cell types. This complexity makes the vertebrates difficult to study from the standpoint of cell and molecular biology, but much of the interest in biological sciences nonetheless stems from the desire to understand the human organism. Moreover, an understanding of many questions of immediate practical importance (e.g., in medicine) must be based directly on studies of human (or closely related) cell types. The specialized properties of some highly differentiated cell types have made them important models for studies of particular aspects of cell biology. Figure 1.19 Arabidopsis thaliana Arabidopsis is a model for studying the molecular biology of plants. (Photo by David McIntyre.) Figure 1.18 Drosophila melanogaster The fruit fly is a key model for genetics and developmental biology. (Photo by David McIntyre.) Arabidopsis thaliana is the basic plant model system. 22 Chapter 1 (A) (B) Figure 1.20 Zebrafish (A) A 24-hour-old embryo. (B) An adult fish. (A, courtesy of Charles Kimmel, University of Oregon; B, photo by David McIntyre.) The zebrafish is an important model for vertebrate development. The mouse is the closest model for human biology. Muscle cells, for example, are highly specialized to undergo contraction, producing force and movement. Because of this specialization, muscle cells are crucial models for studying cell movement at the molecular level. Another example is provided by nerve cells (neurons), which are specialized to conduct electrochemical signals over long distances. In humans, nerve cell axons may be more than a meter long, and some invertebrates, such as the squid, have giant neurons with axons as large as 1 mm in diameter. Because of their highly specialized structure and function, these giant neurons have provided important models for studies of ion transport across the plasma membrane, and of the role of the cytoskeleton in the transport of cytoplasmic organelles. The zebrafish (Figure 1.20) possesses a number of advantages for genetic studies of vertebrate development. These small fish are easy to maintain in the laboratory and they reproduce rapidly, with a generation time of 3–4 months. In addition, the embryos develop outside of the mother and are transparent, so that early stages of development can be easily observed. Powerful methods have been developed to facilitate the isolation of mutations affecting zebrafish development, and several thousand such mutations have now been identified. Because the zebrafish is an easily studied vertebrate, it promises to bridge the gap between humans and the simpler invertebrate systems, such as C. elegans and Drosophila. Among mammals, the mouse is the most suitable for genetic analysis. Although the technical difficulties in studying mouse genetics (compared, for example, with the genetics of yeasts or Drosophila) are formidable, many mutations affecting mouse development have been identified. Most important, recent advances in molecular biology have enabled the production of genetically engineered mice in which specific mutant genes have been introduced into the mouse germ line, allowing the functions of these genes to be studied in the context of the whole animal. The suitability of the mouse as a model for human development is indicated not only by the similarity of the mouse and human genomes but also by the fact that mutations in homologous genes result in similar developmental defects in both species—piebaldism (a defect in pigmentation) offering a striking example (Figure 1.21). Introduction to Cells and Cell Research Figure 1.21 The mouse as a model for human development A child and a mouse show similar defects in pigmentation (piebaldism) as a result of mutations in a gene required for normal migration of melanocytes (the cells responsible for skin pigmentation) during embryonic development. (From R. A. Fleischman et al., 1991. Proc. Natl. Acad. Sci. USA 88: 10885; courtesy of R. A. Fleischmann, Markey Cancer Center, University of Kentucky.) Animal cell culture One important approach to studying the cells of multicellular organisms is to grow isolated cells in culture, where they can be manipulated under controlled laboratory conditions. The use of cultured cells has allowed studies of many aspects of mammalian cell biology, including experiments that have elucidated the mechanisms of DNA replication, gene expression, protein synthesis and processing, and cell division. Moreover, the ability to grow animal cells in culture has allowed studies of the signaling mechanisms that control cell growth and differentiation within the intact organism. Although the process is technically far more difficult than the culture of bacteria or yeasts, a wide variety of animal cells can be grown and manipulated in culture. Cultures are initiated by the dispersion of a piece of tissue into a suspension of its component cells, which is then added to a culture dish containing nutrient media (Figure 1.22). Most animal cell types, such as fibroblasts and epithelial cells, attach to and grow on the plastic surface of dishes used for cell culture. Because they contain rapidly growing cells, embryos or tumors are frequently used as starting material. Embryo fibroblasts grow particularly well in culture and consequently are one of the most widely studied types of animal cells. Under appropriate conditions, however, many specialized cell types can also be grown in culture, allowing their differentiated properties to be studied in a controlled experimental environment. Embryonic stem (ES) cells are a particularly notable example. These cells are established in culture from early embryos and maintain their ability to differentiate into all of the cell types present in adult organisms. Consequently, embryonic stem cells have played an important role in studying development and differentiation, as well as The growth of cells in culture allows them to be manipulated outside of intact organisms. 23 24 Chapter 1 Figure 1.22 Culture of animal cells on culture dishes in nutrient medium. Tissue A piece of tissue is dispersed into a suspension The growthcells. of cells in culture of individual allows them to be manipulated outside of intact animals. Cell suspension The cells are plated in a culture dish in nutrient medium. Liquid medium Primary culture The cells in this primary culture attach to the dish and grow until they cover the culture dish surface. The cells can then be removed from the culture dish and replated at a lower density to form a secondary culture. Secondary culture Cells obtained from a tissue are grown offering the possibility of contributing to the treatment of human diseases by providing a source of tissue for transplantation therapies. The initial cell cultures established from a tissue are called primary cultures (see Figure 1.22). The cells in a primary culture usually grow until they cover the culture dish surface. They can then be removed from the dish and replated at a lower density to form secondary cultures. This process can be repeated many times, but most normal cells cannot be grown in culture indefinitely. For example,