NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

  • By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.
Cover of The Cell

The Cell: A Molecular Approach. 2nd edition.

Show details

Tools of Cell Biology

As in all experimental sciences, research in cell biology depends on the laboratory methods that can be used to study cell structure and function. Many important advances in understanding cells have directly followed the development of new methods that have opened novel avenues of investigation. An appreciation of the experimental tools available to the cell biologist is thus critical to understanding both the current status and future directions of this rapidly moving area of science. Some of the important general methods of cell biology are described in the sections that follow. Other experimental approaches, including the methods of biochemistry and molecular biology, will be discussed in later chapters.

Light Microscopy

Because most cells are too small to be seen by the naked eye, the study of cells has depended heavily on the use of microscopes. Indeed, the very discovery of cells arose from the development of the microscope: Robert Hooke first coined the term “cell” following his observations of a piece of cork with a simple light microscope in 1665 (Figure 1.23). Using a microscope that magnified objects up to about 300 times their actual size, Antony van Leeuwenhoek, in the 1670s, was able to observe a variety of different types of cells, including sperm, red blood cells, and bacteria. The proposal of the cell theory by Matthias Schleiden and Theodor Schwann in 1838 may be seen as the birth of contemporary cell biology. Microscopic studies of plant tissues by Schleiden and of animal tissues by Schwann led to the same conclusion: All organisms are composed of cells. Shortly thereafter, it was recognized that cells are not formed de novo but arise only from division of preexisting cells. Thus, the cell achieved its current recognition as the fundamental unit of all living organisms because of observations made with the light microscope.

Figure 1.23. The cellular structure of cork.

Figure 1.23

The cellular structure of cork. A reproduction of Robert Hooke's drawing of a thin slice of cork examined with a light microscope. The “cells” that Hooke observed were actually only the cell walls remaining from cells that had long since (more...)

The light microscope remains a basic tool of cell biologists, with technical improvements allowing the visualization of ever-increasing details of cell structure. Contemporary light microscopes are able to magnify objects up to about a thousand times. Since most cells are between 1 and 100 μm in diameter, they can be observed by light microscopy, as can some of the larger subcellular organelles, such as nuclei, chloroplasts, and mitochondria. However, the light microscope is not sufficiently powerful to reveal fine details of cell structure, for which resolution—the ability of a microscope to distinguish objects separated by small distances—is even more important than magnification. Images can be magnified as much as desired (for example, by projection onto a large screen), but such magnification does not increase the level of detail that can be observed.

The limit of resolution of the light microscope is approximately 0.2 μm; two objects separated by less than this distance appear as a single image, rather than being distinguished from one another. This theoretical limitation of light microscopy is determined by two factors—the wavelength (λ) of visible light and the light-gathering power of the microscope lens (numerical aperture, NA)—according to the following equation:

Image ch1e1.jpg

The wavelength of visible light is 0.4 to 0.7 μm, so the value of λ is fixed at approximately 0.5 μm for the light microscope. The numerical aperture can be envisioned as the size of the cone of light that enters the microscope lens after passing through the specimen (Figure 1.24). It is given by the equation

Image ch1e2.jpg
where η is the refractive index of the medium through which light travels between the specimen and the lens. The value of η for air is 1.0, but it can be increased to a maximum of approximately 1.4 by using an oil-immersion lens to view the specimen through a drop of oil. The angle α corresponds to half the width of the cone of light collected by the lens. The maximum value of α is 90°, at which sin α = 1, so the highest possible value for the numerical aperture is 1.4.

Figure 1.24. Numerical aperture.

Figure 1.24

Numerical aperture. Light is focused on the specimen by the condenser lens and then collected by the objective lens of the microscope. The numerical aperture is determined by the angle of the cone of light entering the objective lens (α) and by (more...)

The theoretical limit of resolution of the light microscope can therefore be calculated as follows:

Image ch1e3.jpg

Microscopes capable of achieving this level of resolution had been made already by the end of the nineteenth century; further improvements in this aspect of light microscopy cannot be expected.

Several different types of light microscopy are routinely used to study various aspects of cell structure. The simplest is bright-field microscopy, in which light passes directly through the cell and the ability to distinguish different parts of the cell depends on contrast resulting from the absorption of visible light by cell components. In many cases, cells are stained with dyes that react with proteins or nucleic acids in order to enhance the contrast between different parts of the cell. Prior to staining, specimens are usually treated with fixatives (such as alcohol, acetic acid, or formaldehyde) to stabilize and preserve their structures. The examination of fixed and stained tissues by bright-field microscopy is the standard approach for the analysis of tissue specimens in histology laboratories (Figure 1.25). Such staining procedures kill the cells, however, and therefore are not suitable for many experiments in which the observation of living cells is desired.

Figure 1.25. Bright-field micrograph of stained tissue.

Figure 1.25

Bright-field micrograph of stained tissue. Cross section of a hair follicle in human skin, stained with hematoxylin and eosin. (G. W. Willis/ Biological Photo Service.)

Without staining, the direct passage of light does not provide sufficient contrast to distinguish many parts of the cell, limiting the usefulness of bright-field microscopy. However, optical variations of the light microscope can be used to enhance the contrast between light waves passing through regions of the cell with different densities. The two most common methods for visualizing living cells are phase-contrast microscopy and differential interference-contrast microscopy (Figure 1.26). Both kinds of microscopy use optical systems that convert variations in density or thickness between different parts of the cell to differences in contrast that can be seen in the final image. In bright-field microscopy, transparent structures (such as the nucleus) have little contrast because they absorb light poorly. However, light is slowed down as it passes through these structures so that its phase is altered compared to light that has passed through the surrounding cytoplasm. Phase-contrast and differential interference-contrast microscopy convert these differences in phase to differences in contrast, thereby yielding improved images of live, unstained cells.

Figure 1.26. Microscopic observation of living cells.

Figure 1.26

Microscopic observation of living cells. Photomicrographs of human cheek cells obtained with (A) bright-field, (B) phase-contrast, and (C) differential interference-contrast microscopy. (Courtesy of Mort Abramowitz, Olympus America, Inc.)

The power of the light microscope has been considerably expanded by the use of video cameras and computers for image analysis and processing. Such electronic image-processing systems can substantially enhance the contrast of images obtained with the light microscope, allowing the visualization of small objects that otherwise could not be detected. For example, video-enhanced differential interference-contrast microscopy has allowed visualization of the movement of organelles along microtubules, which are cytoskeletal protein filaments with a diameter of only 0.025 μm (Figure 1.27). However, this enhancement does not overcome the theoretical limit of resolution of the light microscope, approximately 0.2 μm. Thus, although video enhancement allows the visualization of microtubules, the microtubules appear as blurred images at least 0.2 μm in diameter and an individual microtubule cannot be distinguished from a bundle of adjacent structures.

Figure 1.27. Video-enhanced differential interference-contrast microscopy.

Figure 1.27

Video-enhanced differential interference-contrast microscopy. Electronic image processing allows the visualization of single microtubules. (Courtesy of E. D. Salmon, University of North Carolina, Chapel Hill.)

Light microscopy has been brought to the level of molecular analysis by methods for labeling specific molecules so that they can be visualized within cells. Specific genes or RNA transcripts can be detected by hybridization with nucleic acid probes of complementary sequence, and proteins can be detected using appropriate antibodies (see Chapter 3). Both nucleic acid probes and antibodies can be labeled with a variety of tags that allow their visualization in the light microscope, making it possible to determine the location of specific molecules within individual cells.

Fluorescence microscopy is a widely used and very sensitive method for studying the intracellular distribution of molecules (Figure 1.28). A fluorescent dye is used to label the molecule of interest within either fixed or living cells. The fluorescent dye is a molecule that absorbs light at one wavelength and emits light at a second wavelength. This fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye and then using appropriate filters to detect the specific wavelength of light that the dye emits. Fluorescence microscopy can be used to study a variety of molecules within cells. One frequent application is to label antibodies directed against a specific protein with fluorescent dyes, so that the intracellular distribution of the protein can be determined. Proteins in living cells can be visualized by using the green fluorescent protein (GFP) of jellyfish as a fluorescent label. GFP can be fused to a wide range of proteins using standard methods of recombinant DNA, and the GFP-tagged protein can then be introduced into cells and detected by fluorescence microscopy.

Figure 1.28. Fluorescence microscopy.

Figure 1.28

Fluorescence microscopy. (A) Light passes through an excitation filter to select light of the wavelength (e.g., blue) that excites the fluorescent dye. A dichroic mirror then deflects the excitation light down to the specimen. The fluorescent light emitted (more...)

Confocal microscopy combines fluorescence microscopy with electronic image analysis to obtain three-dimensional images. A small point of light, usually supplied by a laser, is focused on the specimen at a particular depth. The emitted fluorescent light is then collected using a detector, such as a video camera. Before the emitted light reaches the detector, however, it must pass through a pinhole aperture (called a confocal aperture) placed at precisely the point where light emitted from the chosen depth of the specimen comes to a focus (Figure 1.29). Consequently, only light emitted from the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus, a much sharper image than that obtained with standard fluorescence microscopy (Figure 1.30). Moreover, a series of images obtained at different depths can be used to reconstruct a three-dimensional image of the sample.

Figure 1.29. Confocal microscopy.

Figure 1.29

Confocal microscopy. A pinpoint of light is focused on the specimen at a particular depth, and emitted fluorescent light is collected by a detector. Before reaching the detector, the fluorescent light emitted by the specimen must pass through a confocal (more...)

Figure 1.30. Confocal micrograph of mouse embryo cells.

Figure 1.30

Confocal micrograph of mouse embryo cells. Nuclei are stained red and actin filaments underlying the plasma membrane are stained green. (Courtesy of David Albertini, Tufts University School of Medicine.)

Two-photon excitation microscopy is an alternative to confocal microscopy that can be applied to living cells. The specimen is illuminated with a wavelength of light such that excitation of the fluorescent dye requires the simultaneous absorption of two photons (Figure 1.31). The probability of two photons simultaneously exciting the fluorescent dye is only significant at the point in the specimen upon which the input laser beam is focused, so fluorescence is only emitted from the plane of focus of the input light. This highly localized excitation automatically provides three-dimensional resolution, without the need for passing the emitted light through a pinhole aperture, as in confocal microscopy. Moreover, the localization of excitation minimizes damage to the specimen, allowing three-dimensional imaging of living cells.

Figure 1.31. Two-photon excitation microscopy.

Figure 1.31

Two-photon excitation microscopy. Simultaneous absorption of two photons is required to excite the fluorescent dye. This only occurs at the point in the specimen upon which the input light is focused, so fluorescent light is only emitted from the chosen (more...)

Electron Microscopy

Because of the limited resolution of the light microscope, analysis of the details of cell structure has required the use of more powerful microscopic techniques—namely electron microscopy, which was developed in the 1930s and first applied to biological specimens by Albert Claude, Keith Porter, and George Palade in the 1940s and 1950s. The electron microscope can achieve a much greater resolution than that obtained with the light microscope because the wavelength of electrons is shorter than that of light. The wavelength of electrons in an electron microscope can be as short as 0.004 nm—about 100,000 times shorter than the wavelength of visible light. Theoretically, this wavelength could yield a resolution of 0.002 nm, but such a resolution cannot be obtained in practice, because resolution is determined not only by wavelength, but also by the numerical aperture of the microscope lens. Numerical aperture is a limiting factor for electron microscopy because inherent properties of electromagnetic lenses limit their aperture angles to about 0.5 degrees, corresponding to numerical apertures of only about 0.01. Thus, under optimal conditions, the resolving power of the electron microscope is approximately 0.2 nm. Moreover, the resolution that can be obtained with biological specimens is further limited by their lack of inherent contrast. Consequently, for biological samples the practical limit of resolution of the electron microscope is 1 to 2 nm. Although this resolution is much less than that predicted simply from the wavelength of electrons, it represents more than a hundredfold improvement over the resolving power of the light microscope.

Two types of electron microscopy—transmission and scanning—are widely used to study cells. In principle, transmission electron microscopy is similar to the observation of stained cells with the bright-field light microscope. Specimens are fixed and stained with salts of heavy metals, which provide contrast by scattering electrons. A beam of electrons is then passed through the specimen and focused to form an image on a fluorescent screen. Electrons that encounter a heavy metal ion as they pass through the sample are deflected and do not contribute to the final image, so stained areas of the specimen appear dark.

Specimens to be examined by transmission electron microscopy can be prepared by either positive or negative staining. In positive staining, tissue specimens are cut into thin sections and stained with heavy metal salts (such as osmium tetroxide, uranyl acetate, and lead citrate) that react with lipids, proteins, and nucleic acids. These heavy metal ions bind to a variety of cell structures, which consequently appear dark in the final image (Figure 1.32). Alternative positive-staining procedures can also be used to identify specific macromolecules within cells. For example, antibodies labeled with electron-dense heavy metals (such as gold particles) are frequently used to determine the subcellular location of specific proteins in the electron microscope. This method is similar to the use of antibodies labeled with fluorescent dyes in fluorescence microscopy.

Figure 1.32. Positive staining.

Figure 1.32

Positive staining. Transmission electron micrograph of a positively stained white blood cell. (Don W. Fawcett/ Visuals Unlimited.)

Negative staining is useful for the visualization of intact biological structures, such as bacteria, isolated subcellular organelles, and macromolecules (Figure 1.33). In this method, the biological specimen is deposited on a supporting film, and a heavy metal stain is allowed to dry around its surface. The unstained specimen is then surrounded by a film of electron-dense stain, producing an image in which the specimen appears light against a stained dark background.

Figure 1.33. Negative staining.

Figure 1.33

Negative staining. Transmission electron micrograph of negatively stained actin filaments. (Courtesy of Roger Craig, University of Massachusetts Medical Center.)

Metal shadowing is another technique used to visualize the surface of isolated subcellular structures or macromolecules in the transmission electron microscope (Figure 1.34). The specimen is coated with a thin layer of evaporated metal, such as platinum. The metal is sprayed onto the specimen from an angle so that surfaces of the specimen that face the source of evaporated metal molecules are coated more heavily than others. This differential coating creates a shadow effect, giving the specimen a three-dimensional appearance in electron micrographs.

Figure 1.34. Metal shadowing.

Figure 1.34

Metal shadowing. Electron micrograph of actin/myosin filaments of the cytoskeleton prepared by metal shadowing. (Don W. Fawcett, J. Heuser/ Photo Researchers, Inc.)

The preparation of samples by freeze fracture, in combination with metal shadowing, has been particularly important in studies of membrane structure. Specimens are frozen in liquid nitrogen (at -196°C) and then fractured with a knife blade. This process frequently splits the lipid bilayer, revealing the interior faces of a cell membrane (Figure 1.35). The specimen is then shadowed with platinum, and the biological material is dissolved with acid, producing a metal replica of the surface of the sample. Examination of such replicas in the electron microscope reveals many surface bumps, corresponding to proteins that span the lipid bilayer. A variation of freeze fracture called freeze etching allows visualization of the external surfaces of cell membranes in addition to their interior faces.

Figure 1.35. Freeze fracture.

Figure 1.35

Freeze fracture. (A) Freeze fracture splits the lipid bilayer, leaving proteins embedded in the membrane associated with one of the two membrane halves. (B) Micrograph of freeze-fractured plasma membranes of two adjacent cells. Proteins that span the (more...)

The second type of electron microscopy, scanning electron microscopy, is used to provide a three-dimensional image of cells (Figure 1.36). In scanning electron microscopy the electron beam does not pass through the specimen. Instead, the surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen. Electrons that are scattered or emitted from the sample surface are collected to generate a three-dimensional image as the electron beam moves across the cell. Because the resolution of scanning electron microscopy is only about 10 nm, its use is generally restricted to studying whole cells rather than subcellular organelles or macromolecules.

Figure 1.36. Scanning electron microscopy.

Figure 1.36

Scanning electron microscopy. Scanning electron micrograph of a macrophage. (David Phillips/Visuals Unlimited.)

Subcellular Fractionation

Although the electron microscope has allowed detailed visualization of cell structure, microscopy alone is not sufficient to define the functions of the various components of eukaryotic cells. To address many of the questions concerning the function of subcellular organelles, it has proven necessary to isolate the organelles of eukaryotic cells in a form that can be used for biochemical studies. This is usually accomplished by differential centrifugation—a method developed largely by Albert Claude, Christian de Duve, and their colleagues in the 1940s and 1950s to separate the components of cells on the basis of their size and density.

The first step in subcellular fractionation is the disruption of the plasma membrane under conditions that do not destroy the internal components of the cell. Several methods are used, including sonication (exposure to high-frequency sound), grinding in a mechanical homogenizer, or treatment with a high-speed blender. All these procedures break the plasma membrane and the endoplasmic reticulum into small fragments while leaving other components of the cell (such as nuclei, lysosomes, peroxisomes, mitochondria, and chloroplasts) intact.

The suspension of broken cells (called a lysate or homogenate) is then fractionated into its components by a series of centrifugations in an ultracentrifuge, which rotates samples at very high speeds (up to 100,000 rpm) to produce forces up to 500,000 times greater than gravity. This force causes cell components to move toward the bottom of the centrifuge tube and form a pellet (a process called sedimentation) at a rate that depends on their size and density, with the largest and heaviest structures sedimenting most rapidly (Figure 1.37). Usually the cell homogenate is first centrifuged at a low speed, which sediments only unbroken cells and the largest subcellular structures—the nuclei. Thus, an enriched fraction of nuclei can be recovered from the pellet of such a low-speed centrifugation while the other cell components remain suspended in the supernatant (the remaining solution). The supernatant is then centrifuged at higher speed to sediment mitochondria, chloroplasts, lysosomes, and peroxisomes. Recentrifugation of the supernatant at still higher speed sediments fragments of the plasma membrane and the endoplasmic reticulum. A fourth centrifugation at still higher speed sediments ribosomes, leaving only the soluble portion of the cytoplasm (the cytosol) in the supernatant.

Figure 1.37. Subcellular fractionation.

Figure 1.37

Subcellular fractionation. Cells are lysed and subcellular components are separated by a series of centrifugations at increasing speeds. Following each centrifugation, the organelles that have sedimented to the bottom of the tube are recovered in the (more...)

The fractions obtained from differential centrifugation correspond to enriched, but still not pure, organelle preparations. A greater degree of purification can be achieved by density-gradient centrifugation, in which organelles are separated by sedimentation through a gradient of a dense substance, such as sucrose. In velocity centrifugation, the starting material is layered on top of the sucrose gradient (Figure 1.38). Particles of different sizes sediment through the gradient at different rates, moving as discrete bands. Following centrifugation, the collection of individual fractions of the gradient provides sufficient resolution to separate organelles of similar size, such as mitochondria, lysosomes, and peroxisomes.

Figure 1.38. Velocity centrifugation in a density gradient.

Figure 1.38

Velocity centrifugation in a density gradient. The sample is layered on top of a gradient of sucrose, and particles of different sizes sediment through the gradient as discrete bands. The separated particles can then be collected in individual fractions (more...)

Equilibrium centrifugation in density gradients can be used to separate subcellular components on the basis of their buoyant density, independent of their size and shape. In this procedure, the sample is centrifuged in a gradient containing a high concentration of sucrose or cesium chloride. Rather than being separated on the basis of their sedimentation velocity, the sample particles are centrifuged until they reach an equilibrium position at which their buoyant density is equal to that of the surrounding sucrose or cesium chloride solution. Such equilibrium centrifugations are useful in separating different types of membranes from one another and are sufficiently sensitive to separate macromolecules that are labeled with different isotopes. A classic example, discussed in Chapter 3, is the analysis of DNA replication by separating DNA molecules containing heavy and light isotopes of nitrogen (15N and 14N) by equilibrium centrifugation in cesium chloride gradients.

Growth of Animal Cells in Culture

The ability to study cells depends largely on how readily they can be grown and manipulated in the laboratory. Although the process is technically far more difficult than the culture of bacteria or yeasts, a wide variety of animal and plant cells can be grown and manipulated in culture. Such in vitro cell culture systems have enabled scientists to study cell growth and differentiation, as well as to perform genetic manipulations required to understand gene structure and function.

Animal cell 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. Most animal cell types, such as fibroblasts and epithelial cells, attach and grow on the plastic surface of dishes used for cell culture (Figure 1.39). 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, some specialized cell types can also be grown in culture, allowing their differentiated properties to be studied in a controlled experimental environment.

Figure 1.39. Animal cells in culture.

Figure 1.39

Animal cells in culture. Scanning electron micrograph of human fibroblasts attached to the surface of a culture dish. (David M. Phillips/Visuals Unlimited.)

The culture media required for the propagation of animal cells are much more complex than the minimal media sufficient to support the growth of bacteria and yeasts. Early studies of cell culture utilized media consisting of undefined components, such as plasma, serum, and embryo extracts. A major advance was thus made in 1955, when Harry Eagle described the first defined media that supported the growth of animal cells. In addition to salts and glucose, the media used for animal cell cultures contain various amino acids and vitamins, which the cells cannot make for themselves. The growth media for most animal cells in culture also include serum, which serves as a source of polypeptide growth factors that are required to stimulate cell division. Several such growth factors have been identified. They serve as critical regulators of cell growth and differentiation in multicellular organisms, providing signals by which different cells communicate with each other. For example, an important function of skin fibroblasts in the intact animal is to proliferate when needed to repair damage resulting from a cut or wound. Their division is triggered by a growth factor released from platelets during blood clotting, thereby stimulating proliferation of fibroblasts in the neighborhood of the damaged tissue. The identification of individual growth factors has made possible the culture of a variety of cells in serum-free media (media in which serum has been replaced by the specific growth factors required for proliferation of the cells in question).

The initial cell cultures established from a tissue are called primary cultures (Figure 1.40). 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, normal human fibroblasts can usually be cultured for 50 to 100 population doublings, after which they stop growing and die. In contrast, cells derived from tumors frequently proliferate indefinitely in culture and are referred to as immortal cell lines. In addition, a number of immortalized rodent cell lines have been isolated from cultures of normal fibroblasts. Instead of dying as most of their counterparts do, a few cells in these cultures continue proliferating indefinitely, forming cell lines like those derived from tumors. Such permanent cell lines have been particularly useful for many types of experiments because they provide a continuous and uniform source of cells that can be manipulated, cloned, and indefinitely propagated in the laboratory.

Figure 1.40. Culture of animal cells.

Figure 1.40

Culture of animal cells.

Even under optimal conditions, the division time of most actively growing animal cells is on the order of 20 hours—ten times longer than the division time of yeasts. Consequently, experiments with cultured animal cells are more difficult and take much longer than those with bacteria or yeasts. For example, the growth of a visible colony of animal cells from a single cell takes a week or more, whereas colonies of E. coli or yeast develop from single cells overnight. Nonetheless, genetic manipulations of animal cells in culture have been indispensable to our understanding of cell structure and function.

Culture of Plant Cells

Plant cells can also be cultured in nutrient media containing appropriate growth regulatory molecules. In contrast to the polypeptide growth factors that regulate the proliferation of most animal cells, the growth regulators of plant cells are small molecules that can pass through the plant cell wall. When provided with appropriate mixtures of these growth regulatory molecules, many types of plant cells proliferate in culture, producing a mass of undifferentiated cells called a callus (Figure 1.41).

Figure 1.41. Plant cells in culture.

Figure 1.41

Plant cells in culture. An undifferentiated mass of plant cells (a callus) growing on a solid medium. (John N. A. Lott/Biological Photo Service.)

A striking feature of plant cells that contrasts sharply to the behavior of animal cells is the phenomenon called totipotency. Differentiated animal cells, such as fibroblasts, cannot develop into other cell types, such as nerve cells. Many plant cells, however, are capable of forming any of the different cell types and tissues ultimately needed to regenerate an entire plant. Consequently, by appropriate manipulation of nutrients and growth regulatory molecules, undifferentiated plant cells in culture can be induced to form a variety of plant tissues, including roots, stems, and leaves. In many cases, even an entire plant can be regenerated from a single cultured cell. In addition to its theoretical interest, the ability to produce a new plant from a single cell that has been manipulated in culture makes it easy to introduce genetic alterations into plants, opening important possibilities for agricultural genetic engineering.


Viruses are intracellular parasites that cannot replicate on their own. They reproduce by infecting host cells and usurping the cellular machinery to produce more virus particles. In their simplest forms, viruses consist only of genomic nucleic acid (either DNA or RNA) surrounded by a protein coat (Figure 1.42). Viruses are important in molecular and cellular biology because they provide simple systems that can be used to investigate the functions of cells. Because virus replication depends on the metabolism of the infected cells, studies of viruses have revealed many fundamental aspects of cell biology. Studies of bacterial viruses contributed substantially to our understanding of the basic mechanisms of molecular genetics, and experiments with a plant virus (tobacco mosaic virus) first demonstrated the genetic potential of RNA. Animal viruses have provided particularly sensitive probes for investigations of various activities of eukaryotic cells.

Figure 1.42. Structure of an animal virus.

Figure 1.42

Structure of an animal virus. (A) Papillomavirus particles contain a small circular DNA molecule enclosed in a protein coat (the capsid). (B) Electron micrograph of human papillomavirus particles. Artificial color has been added. (B, Alfred Pasieka/Science (more...)

The rapid growth and small genome size of bacteria make them excellent subjects for experiments in molecular biology, and bacterial viruses (bacteriophages) have simplified the study of bacterial genetics even further. One of the most important bacteriophages is T4, which infects and replicates in E. coli. Infection with a single particle of T4 leads to the formation of approximately 200 progeny virus particles in 20 to 30 minutes. The initially infected cell then bursts (lyses), releasing progeny virus particles into the medium, where they can infect new cells. In a culture of bacteria growing on agar medium, the replication of T4 leads to the formation of a clear area of lysed cells (a plaque) in the lawn of bacteria (Figure 1.43). Just as infectious virus particles are easy to grow and assay, viral mutants—for example, viruses that will grow in one strain of E. coli but not another—are easy to isolate. Thus, T4 is manipulated even more readily than E. coli for studies of molecular genetics. Moreover, the genome of T4 is 20 times smaller than that of E. coli—approximately 0.2 million base pairs—further facilitating genetic analysis. Some other bacteriophages have even smaller genomes—the simplest consisting of RNA molecules of only about 3600 nucleotides. Bacterial viruses have thus provided extremely facile experimental systems for molecular genetics. Studies of these viruses are largely what have led to the elucidation of many fundamental principles of molecular biology.

Figure 1.43. Bacteriophage plaques.

Figure 1.43

Bacteriophage plaques. T4 plaques are visible on a lawn of E. coli. Each plaque arises by the replication of a single virus particle. (E. C. S. Chen/ Visuals Unlimited.)

Because of the increased complexity of the animal cell genome, viruses have been even more important in studies of animal cells than in studies of bacteria. Many animal viruses replicate and can be assayed by plaque formation in cell cultures, much as bacteriophages can. Moreover, the genomes of animal viruses are similar in complexity to those of bacterial viruses (ranging from approximately 3000 to 300,000 base pairs), so animal viruses are far more manageable than are their host cells.

There are many diverse animal viruses, each containing either DNA or RNA as their genetic material (Table 1.3). One family of animal viruses—the retroviruses—contain RNA genomes in their virus particles but synthesize a DNA copy of their genome in infected cells. These viruses provide a good example of the importance of viruses as models, because studies of the retroviruses are what first demonstrated the synthesis of DNA from RNA templates—a fundamental mode of genetic information transfer now known to occur in both prokaryotic and eukaryotic cells. Other examples in which animal viruses have provided important models for investigations of their host cells include studies of DNA replication, transcription, RNA processing, and protein transport and secretion.

Table 1.3. Examples of Animal Viruses.

Table 1.3

Examples of Animal Viruses.

It is particularly noteworthy that infection by some animal viruses, rather than killing the host cell, converts a normal cell into a cancer cell. Studies of such cancer-causing viruses, first described by Peyton Rous in 1911, not only have provided the basis for our current understanding of cancer at the level of cell and molecular biology, but also have led to the elucidation of many of the molecular mechanisms that control animal cell growth and differentiation.

Box Icon


Key Experiment : Animal Cell Culture. Nutrition Needs of Mammalian Cells in Tissue Culture Harry Eagle

Box Icon


Molecular Medicine : Viruses and Cancer. Cancer is a family of diseases characterized by uncontrolled cell proliferation. The growth of normal animal cells is carefully regulated to meet the needs of the complete organism. In contrast, cancer cells grow (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, Geoffrey M Cooper.
Bookshelf ID: NBK9941


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...