certain interphase nuclei. (A) Fluorescentlight micrograph of interphase
`nuclei from the rapidly growing roottip of a plant. Centromeresare stained
`green and teleomeresred byin situ hybridization of centromere- and
`telomere-specific DNA sequences coupled to the different fluorescent dyes.
`(B) Interpretation of (A) showing chromosomesin the Rab! orientation with
`all the centromeres facing one side of a nucleus andall the telomeres
`pointing toward the opposite side. (A, from R.Abranchesetal.,J. Cell Biol.
`|43:5-12, 1998. © The Rockefeller University Press.)
`
`banding pattern mayberelated to gene expression. Perhapsthe differentiation
`of chromosomesinto G- and R-bandsreflects subtle differences, determined by
`GC content, in the way in which chromatinloops are packagedin these areas. If
`this idea is correct, the rough division of chromosomescan be seen as a form of
`compartmentalization, in which the particular cellular components involved in
`gene expression are more concentrated in the R-bands wheretheir activities are
`required. In anycase,it should be obvious fromthis discussion that we are only
`beginning to glimpsethe principles of large-scale chromosomeorganization.
`
`
`
`
`
`
`
` Figure 4-58 The polarized orientation of chromosomes foundin
`
`Individual Chromosomes OccupyDiscrete Territories
`in an Interphase Nucleus
`Wesaw earlier in this chapter that chromosomesfrom eucaryotes are contained
`in the cell nucleus. However, the nucleusis not simply a bag of chromosomes;
`rather, the chromosomes—aswell as the other componentsinside the nucleus
`which we shall encounter in subsequent chapters—are highly organized. The
`wayin which chromosomesare organized in the nucleus during interphase,
`whenthey are active anddifficult to see, has intrigued biologists since the nine-
`teenth century. Although our understanding todayis far from complete, we do
`knowsomeinteresting features of these chromosome arrangements.
`A certain degree of chromosomal orderresults from the configuration that
`the chromosomesalways haveat the end of mitosis. Just before a cell divides,
`the condensed chromosomesare pulled to each spindle pole by microtubules
`attached to the centromeres; thus, as the chromosomes move, the centromeres
`lead the wayandthedistal arms (terminating in the telomeres) lag behind. The
`chromosomes in some nuclei tend to retain this so-called Rabl orientation
`throughoutinterphase, with their centromeres facing one pole of the nucleus
`and their telomeres pointing toward the opposite pole (Figures 4-58 and 4-59).
`
`(A)
`
`I at
`
`time
`
`
`
`“1\
`anaphase, a stage
`of mitosis
`
`short interphase
`in embryo
`
`long interphase in
`larval tissues
`
`232
`
`Chapter 4: DNA AND CHROMOSOMES
`
`HE GLOBAL STRUCTURE OF CHROMOSOMES
`
`
`Figure 4-60 Selective “painting” of two interphase chromosomes
`in a humanperipheral lymphocyte.The fluorescentlight micrograph
`showsthat the two copies of human chromosome 18 (red) and chromosome
`19 (turquoise) occupy discrete territories of the nucleus. (From J.A. Croft
`et al., J. Cell Biol. 145:1119-1131, 1999. © The Rockefeller University Press.)
`
`The chromosomesin mostinterphase cells are not foundin the Rabl orien-
`tation; instead, the centromeres seemto be dispersed in the nucleus. Mostcells
`have a longer interphase thanthe specialized cells illustrated above, and this
`presumablygives their chromosomestime to assumea different conformation
`(see Figure 4-59). Nevertheless, each interphase chromosome does tend to
`occupya discrete andrelatively small territory in the nucleus: thatis, the differ-
`ent chromosomesare not extensively intertwined (Figure 4-60).
`Onedevice for organizing chromosomesin the nucleus maybethe attach-
`mentof certain portions of each chromosome to the nuclear envelope (Figure
`4-61). For example, in manycells, telomeres seem bound in this way. But the
`exact position of a chromosomein anucleusis not fixed. In the sametissue, for
`example,
`two apparently identical cells can have different chromosomes as
`nearest neighbors.
`Somecell biologists believe that there is an intranuclear framework,analo-
`gousto the cytoskeleton, on which chromosomesand other componentsof the
`nucleus are organized. The nuclear matrix, or scaffold, has been defined as the
`insoluble material left in the nucleus after a series of biochemical extraction
`steps. Some of the proteins that constitute it can be shownto bind specific DNA
`sequences called SARs or MARs(scaffold-associated or matrix-associated
`regions). These DNA sequenceshave beenpostulated to form the base of chro-
`mosomal loops (see Figure 4-44), or to attach chromosomes to the nuclear
`envelope andotherstructures in the nucleus. By means of such chromosomal
`attachmentsites, the matrix might help to organize chromosomes,localize
`genes, and regulate gene expression and DNAreplication. It still remains
`uncertain, however, whether the matrix isolated bycell biologists represents a
`structure that is present in intact cells.
`
`
`
`
`
`233
`
`
`
`5um
`
`
`
`Figure 4-61 Specific regions of
`interphase chromosomesin close
`proximity to the nuclear envelope.
`This high-resolution microscopic view of
`nuclei from a Drosophila embryo shows
`the localization of two different regions of
`chromosome 2 (yellow and magenta) close
`to the nuclear envelope(stained green
`with antilamina antibodies). Other regions
`of the same chromosome are more
`
`distant from the envelope. (From
`W.F Marshall et al., Mol. Biol. Cell
`7:825-842, 1996.)
`
`EX1010 - pt.2
`
`Figure 4-59 A polymeranalogyfor
`interphase chromosome
`organization. (A) The behavior of a
`Summary
`polymerin solution. Entropy drives a long
`Chromosomes are generally decondensed during interphase, so that their structure
`polymer into a compact conformation 'n
`is difficult to visualize directly. Notable exceptions are the specialized lampbrush
`the absence of an externally applied force.
`If the polymer is subjected to shear or
`chromosomesofvertebrate oocytes and the polytene chromosomesin the giant secre-
`hydrodynamic force, it becomes extended.
`tory cells of insects. Studies of these two types of interphase chromosomes suggest
`But once the force is removed, the
`that each long DNA molecule in a chromosome is divided into a large numberofdis-
`polymer chain returns to a more
`crete domains organized as loops of chromatin, each loop probablyconsisting ofa
`hydrodynamic
`favorable, compact conformation.(B) The
`force
`folded 30-nm chromatin fiber. When genes contained in a loop are expressed, the
`behavior of interphase chromosomes may
`aee)
`loop decondenses andallows the cell’s machinery easy access to the DNA.
`reflect the same simple principles. In
`5 um
`RELAXED
`CATae
`Euchromatin makes up most of interphase chromosomes and probably corre-
`
`Drosophila embryos, for example, mitotic
`sponds to looped domainsof30-nm fibers. However, euchromatinis interrupted by
`divisions occur at intervals of about 10
`
`Stretches of heterochromatin, in which 30-nmfibers are subjected to additionallev-
`minutes; during the short intervening
`
`els ofpacking that usually renderit resistant to gene expression. Heterochromatin is
`interphases, the chromosomeshavelittle
`
`time to relax from the Rabl orientation
`commonly found around centromeres and near telomeres, butit is also present at
`
`induced by their movement during
`other positions on chromosomes. Although considerably less condensed than mitotic
`
`mitosis. However, in later stages of
`chromosomes, interphase chromosomes occupydiscrete territories in the cell nucle-
`
`development, when interphase is much
`Us; thatis, they are not extensively intertwined.
`
`longer, the chromosomeshave time to
`All chromosomes adopt a highly condensed conformation during mitosis. When
`
`fold up. This folding may be strongly
`they are specially stained, these mitotic chromosomes have a bandingstructure that
`
`affected by specific associations between
`allows each individual chromosometo be recognized unambiguously. These bands
`different regions of the same
`
`©ontain millions of DNA nucleotide pairs, and they reflect a poorly-understood
`
`chromosome. (Adapted from A-F.
`Coarse heterogeneity of chromosomestructure.
`Dernburg etal., Cell 85:745-759, 1996.)
`
`
`00022
`
`EX1010 - pt.2
`
`

`

` References
`
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`Lewin B (2000) GenesVII, Oxford: Oxford University Press.
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`New York: WH Freeman.
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`
`The Structure and Function of DNA
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`Chapter 4: DNA AND CHROMOSOMES
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`and
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`Griffith JD, Comeau L, Rosenfield § et al. (1999) Mammalian telomeres end
`in a large duplex loop. Cell 97, 503-514.
`Grunstein M (1997) Molecular model for telomeric heterochromatin in
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`Hart CM & Laemmli UK (1998) Facilitation of chromatin dynamics by
`SARs. Curr, Opin. Genet. Dev. 8, 519-525.
`Henikoff S (1990) Position-effect variegation after 60 years. Trends Genet
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`Henikoff S (1998) Conspiracy ofsilence among repeated transgenes. Gices-
`says 20, 532-535.
`Hirane T (1999) SMC-mediated chromosome mechanics: a conserved
`scheme from bacteria to vertebrates. Genes Dev.
`|3, 11-19.
`Hirano T (2000) Chromosome cohesion, condensation, and separation.
`Annu. Rev. Biochem. 69,
`| 15-144.
`Lamond Al & Earnshaw WC (1998) Structure and function in the nucleus.
`Science 280, 547-553.
`Lyko F & Paro R (1999) Chromosomal elements conferring epigenctc
`inheritance. Bioessays 21, 824-832.
`Marsden M & Laemmli UK (1979) Metaphase chromosomestructure: €vI-
`dence for a radial loop model. Cell 17, 849-858.
`Pluta AF, Mackay AM, Ainsztein AM etal. (1995) The centromere: hub of
`chromosomal activities. Science 270, 159|-1594.
`Saitoh N, Goldberg | & Earnshaw WC (|995) The SMC proteins anc tne
`coming of age of the chromosomescaffold hypothesis. Bioessays 7
`759-766.
`
`Spector DL (1993) Macromolecular domains within the cell nucleus. 47":
`Rev. Cell Biol. 9, 265-315.
`Thummel CS (1990) Puffs and gene regulation—molecular insights 't°
`the Drosophila ecdysone regulatory hierarchy. Bioessays 12, 56|~568:
`Weiler KS & Wakimoto BT (1995) Heterochromatin and gene expression
`in Drosophila. Annu, Rev. Genet. 29, 577-605.
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`|-566.
`
`
`
`
`
`
`
`
`
`
`
`DNA REPLICATION,
`REPAIR, AND
`RECOMBINATION
`
`
`
`
`
`
`
`
`
`
`
`ability of cells to maintain a high degree of order in a chaotic universe
`ends upon the accurate duplication of vast quantities of genetic information
`
`ed in chemical form as DNA. This process, called DNA replication, must
`
`before a cell can produce two genetically identical daughter cells. Main-
`
`ng order also requires the continued surveillance and repair of this genetic
`
`ation because DNAinsidecells is repeatedly damaged by chemicals and
`
`idiation from the environment, as well as by thermal accidents and reactive
`
`olecules. In this chapter we describe the protein machinesthat replicate and
`
`pe ir the cell’s DNA. These machines catalyze some of the mostrapid and accu-
`
`fe processesthat take place withincells, and their mechanismsclearly demon-
` ate the elegance andefficiency of cellular chemistry.
`
`
`_ While the short-term survival of a cell can depend on preventing changesin
`} DNA,the long-term survival of a species requires that DNA sequences be
`
`langeable over many generations. Despite the great efforts that cells make to
`
`ect their DNA, occasional changes in DNA sequences do occur. Over time,
`
`ese changes provide the genetic variation upon whichselection pressures act
`
`iring the evolution of organisms.
`
`_ We begin this chapter with a brief discussion of the changes that occur in
`
`NA asit is passed down from generation to generation. Next, we discuss the
`
`lular mechanisms—DNAreplication and DNA repair—that are responsible
`
`t keeping these changesto a minimum.Finally, we consider some of the most
`
`triguing ways in which DNA sequencesare altered by cells, with a focus on
`
`recombination and the movementof special DNA sequencesin our chro-
`losomescalled transposable elements.
`
`
`
`HE MAINTENANCE OF DNA SEQUENCES
`
`though the long-term survival of a species is enhanced by occasional genetic
`
`hanges, the survival of the individual demandsgenetic stability. Only rarely do
`
`€ cell's DNA-maintenanceprocessesfail, resulting in permanentchangein the
`NA. Such a changeis called a mutation, and it can destroy an organism if it
`
`
`
`
`
`THE MAINTENANCE OF DNA
`SEQUENCES
`
`DNA REPLICATION MECHANISMS
`
`THE INITIATION AND
`COMPLETION OF DNA
`
`REPLICATION IN CHROMOSOMES
`
`
`DNA REPAIR
`
`GENERAL RECOMBINATION
`
`SITE-SPECIFIC RECOMBINATION
`
`23
`
`
`
`00023
`
`

`

`MANIPULATING PROTEINS,
`DNA, AND RNA
`
`ISOLATING CELLS AND GROWING
`THEM IN CULTURE
`
`FRACTIONATION OF CELLS
`
`ISOLATING. CLONING.AND
`SEQUENCING DNA
`
`ANALY21NG PROTEIN STRUCTURE
`AND FUNCTION
`
`STUDYING GENE EXPRESSION AND
`FUNCTION
`
`Progress in science is often driven by advan ces in technology. Biology, for exam(cid:173)
`ple, entered a new era when Anton van Leeuwenhoek, a Dutch dry-goods dealer,
`ground the first microscope lens. Peering into his marvelous new looking glass,
`van Leeuwenhoek discovered a previously unseen cellular world, where tiny
`creatures tumble and twirl in a small droplet of water (Figure 8-1).
`The 21 ~1 century promises to be a particularly exciting time for biology. ew
`methods for analyzing proteins, DNA, and RNA are fueling an information
`explosion and allowing scientists to study cells a nd their macromolecules in pre(cid:173)
`viously unimagin ed ways. We now have access to the seque nces of many billions
`of nucleotides, providing the complete molecular blueprints for dozens of
`organisms-from microbes and mustard weeds to worms, flies, and humans.
`And powerful new techniques are helping us to decipher that information,
`allowing us not only to compile huge, detailed catalogs of genes and proteins,
`but to begin to unravel how these components work together to form functional
`cells and organisms. The goal is nothing short of obtaining a complete under(cid:173)
`standing of what takes place inside a cell as it responds to its environment and
`interacts with its neighbors. We want to know which genes are switched on,
`which mRNA transcripts are pre ent, and which proteins are active-where they
`are located, with whom they partne r, and to which pathways or networks they
`belong. We also want to understand how the cell successfully manages this stag(cid:173)
`gering number of variables and how it chooses among a n almost unlimited
`number of possibilities in performing its diver e biological tasks. Possession of
`such information will permit us to begin to build a framework for delineating,
`and eventually predicting, how genes and proteins operate to lay the founda(cid:173)
`tions for life.
`In this chapter we brieny review some of the principal methods used to
`study cells and their components, particula rly proteins, D A, and RNA. We con(cid:173)
`sider how cells of different ty pes can be separated from tissues and grown outside
`the body and how cells can be disrupted and thei r organelles a nd constituent
`macromolecules isolated in pure form. We then review the breakthroughs in
`recombinant DNA technology that continue lO revolutionize our understanding
`
`469
`
`EX1014
`
`00024
`
`

`

`~-- A .
`
`'
`'
`
`....
`
`cfe .. C' .
`=
`
`-=-----,
`
`(A)
`
`(B)
`
`Figure 8-1 Microscopic life. A sample
`of "diverse animalcules" seen by van
`Leeuwenhoek using his simple
`microscope. (A) Bacteria seen in material
`he excavated from between his teeth.
`Those in fig. B he described as "swimming
`first forward and then backwards" ( 1692)
`(B) The eucaryotic green alga Volvox
`( 1700). (Courtesy of the John Innes
`Foundation.)
`
`of cellular function. Finally we present the latest techniques used to determine
`the structures and functions of proteins and genes, as well as to dissect their
`complex interactions.
`This chapter serves as a bridge from the basics of cell and molecular biology
`to the detailed discussion of how these macromolecules are organized and
`function together to coordinate the growth, development, and physiology of
`cells and organisms. The techniques and methods described here have made
`possible the discoveries that are presented throughout this book, and they are
`currently being used by tens of thousands of scientists each day.
`
`ISOLATING CELLS AND GROWING THEM
`IN CULTURE
`Although the organelles and large molecules in a cell can be visualized with
`microscopes, understanding how these components function requires a
`detailed biochemical analysis. Most biochemical procedures require obtaining
`large numbers of cells and then physically disrupting them to isolate their com(cid:173)
`ponents. ff the sample is a piece of tissue, composed of different types of cells,
`heterogeneous cell populations will be mixed together. To obtain as much infor(cid:173)
`mation as possible about an individual cell type, biologists have developed ways
`of dissociating cells from tissues and separating the various types. These manip(cid:173)
`ulations result in a relatively homogeneous population of cells that can then be
`analyzed-either directly or after their number has been greatly increased by
`allO'vving the cells to proliferate as a pure culture.
`
`Cells Can Be Isolated from a Tissue Suspension and
`Separated into Different Types
`The first step in isolating cells of a uniform type from a tissue that contains a
`mixture of cell types is to disrupt the extracellular matrix that holds the cells
`together. The best yields of viable dissociated cells are usually obtained from
`fetal or neonatal tissues. The tissue sample is typically treated with proteolytic
`enzymes (such as trypsin and collagenase) to digest proteins in the extracellular
`matrix and with agents (such as ethylenediaminetetraacetic acid, or EDTA) that
`bind, or chelate, the Ca2+ on which cell-cell adhesion depends. The tissue can
`then be teased apart into single living cells by gentle agitation.
`Several approaches are used to separate the different cell types from a mixed
`cell suspension. One exploits differences in physical properties. Large cells can
`be separated from small cells and dense cells from light cells by centrifugation,
`for example. These techniques will be described below in connection with the
`separation of organelles and macromolecules, for which they were originally
`developed. Another approach is based on the tendency of some cell types to
`adhere strongly to glass or plastic, which allows them to be separated from cells
`that adhere less strongly.
`An important refin ement of this last technique depends on the specific
`binding properties of antibodies. Antibodies that bind specifically to the surface
`of only one cell type in a tissue can be coupled to various matrices-such as col(cid:173)
`lagen, polysaccharide beads, or plastic-to form an affinity surface to which
`only cells recognized by the antibodies can adhere. The bound cells are then
`recovered by gentle shaking, by treatment with trypsin to digest the proteins that
`mediate the adhesion, or, in the case of a digestible matrix (such as collagen), by
`degrading the matrix itself with enzymes (such as collagenase).
`One of the most sophisticated cell-separation technique uses an antibody
`coupled to a fluorescent dye to label specific cells. The labeled cells can then be
`separated from the unlabeled ones in an electronic fluorescence-activated cell
`sorter. In this remarkable machine, individual cells traveling single file in a fine
`stream pass through a laser beam and the fluorescence of each cell is rapidly
`measured. A vibrating nozzle generates tiny droplets, most containing either
`one cell or no cells. The droplets containing a single cell are automatically given
`
`•
`
`.....
`
`GM-·B.
`--
`-
`
`Jj:D .
`__,.-
`_,,.,---
`
`ultrasonic nozzle vibrator
`
`-
`
`.:·.·.·~
`
`I //
`
`cell suspension
`
`sh,a<h n,;d
`
`111111
`
`Figure 8-2 A fluorescence-activated
`cell sorter. A cell passing through the
`laser beam is monitored for fluorescence .
`Droplets containing single cells are given a
`negative or positive charge, depending on
`whether the cell is fluorescent or not. The
`droplets are then deflected by an electric
`field into collection tubes according to
`their charge. Note that the cell
`concentration must be adjusted so that
`most droplets contain no cells and flow to
`a waste container together with any cell
`clumps.
`
`laser
`
`small groups of drops
`negatively charged due - - {(cid:173)
`-
`to detection of single
`fluorescent cell
`
`1 - - - --
`
`small groups of drops
`positively charged due
`to detection of single
`nonfluorescent cell
`
`!
`[ } -
`
`+2000V
`
`cell collector
`
`flask for undeflected droplets
`
`a positive or a negative charge at the moment of formation, depending on whether
`the cell they contain is fluorescent; they are then deflected by a strong electric field
`into an appropriate container. Occasional clumps of cells, detected by their
`increased light scattering, are left uncharged and are discarded into a waste con(cid:173)
`tainer. Such machines can accurately select 1 fluorescent cell from a pool of 1000
`unlabeled cells and sort several thousand cells each second (Figure 8-2).
`Selected cells can also be obtained by carefully dissecting them from thin
`tissue slices that have been prepared for microscopic examination (discussed in
`Chapter 9). In one approach, a tissue section is coated with a thin plastic film
`and a region containing the cells of interest is irradiated with a focused pulse
`from an infrared laser. This light pulse melts a small circle of the film, binding
`the cells underneath. These captured cells are then removed for further analysis.
`The technique, called laser capture microdissection, can be used to separate and
`analyze cells from different areas of a tumor, allowing their properties to be
`compared. A related method uses a laser beam to directly cut out a group of cells
`and catapult them into an appropriate container for future analysis (Figure 8-3).
`
`thin section of
`organ containing
`tumor
`
`laser beam cuts around
`reg ion of interest
`
`glass microscope slide
`
`Figure 8-3 Microdissection
`techniques allow selected cells to be
`isolated from tissue slices. This
`method uses a laser beam to excise a
`region of interest and eject it into a
`container. and it permits the isolation of
`even a single cell from a tissue sample.
`
`470
`
`Chapter 8 : MANIPULAT'NG PROTEINS, DNA. AND RNA
`
`ISOLAT ING CELLS AND GROWIN G THEM IN CULTURE
`
`471
`
`00025
`
`

`

`Once a uniform population of cells has been obtained- by microdissection
`or by any of the separation methods just described-
`it can be used directly for
`bioche mical analysis. A homogeneous cell sample also provides a starting
`material for cell culture, thereby allowing the number of cells to be greatly
`increased and their complex behavior to be studied under the strictly defined
`conditions of a culture dish.
`
`C ells C an Be G r own in a Culture Di sh
`
`Given appropriate surround ings, most plant and animal cells can live, multiply,
`and even express differentiated properties in a tissue-culture dish. The cells can
`be watched continuously under the microscope or analyzed biochemically, and
`the effects of adding or removing specific molecules, such as hormones or
`growth factors, can be explored. ln addition, by mixing two cell types, the inte r(cid:173)
`actions between one cell type and another can be studied. Experimen ts pe r(cid:173)
`formed on cultured cells are sometimes said to be carried out in vitro (literally,
`"in glass") to contrast them with experiments using intact organisms, wh ich are
`said to be carried out in vivo (literally, "in the living organism"). These terms can
`be confusing, however, because they a re often used in a very different sense by
`biochemists. In the biochemistry lab, in vitro refers to reactions carried out in a
`test tube in the absence of living cells, wh ereas in vivo refers to any reaction tak(cid:173)
`ing place inside a living cell (even cells that are growing in culture).
`Tissue culture began in 1907 with an experiment designed to settle a contro(cid:173)
`versy in neurobiology. The hypothesis under examination was known as the neu(cid:173)
`ronal doctrine, which states that each nerve fiber is the outgrowth of a single
`nerve cell and not the product of the fusion of many cells. To test this contention,
`small pieces of spinal cord were placed on clotted tissue fluid in a warm, moist
`chamber and observed at regular intervals under the microscope. After a day or
`so, individual nerve cells could be seen extending long, thin filaments into the
`clot. Thus the neuronal doctrine received strong support, and the foundations
`for the cell-culture revolution were laid.
`The original experiments on nerve fibe rs used cultures of small tissu e frag(cid:173)
`ments called explants. Today, cultures are more commonly made from suspen(cid:173)
`sions of cells dissociated from tissues using the methods described earlier.
`Unlike bacteria, most tissue cells are not adapted to living in suspension and
`require a solid surface on which to grow and divide. For cell cultures, this sup(cid:173)
`port is usually provided by the surface of a plastic tissue-culture dish. Cells vary
`in their requirements, however, and many do not grow or differentiate unless
`the culture dish is coated with specific extracellular matrix co mponents, such
`as collagen or laminin.
`Cultures prepared directly from the tissues of an organ ism, that is, without
`cell proliferation in vitro, are called primary cultures. These can be made with or
`without an initial fractionation step to separate different cell types. In most
`cases, cells in primary cultures can be re moved from the culture dish and made
`to proliferate to form a large number of so-called secondary cultures; in this way,
`they may be repea tedly subcul tured for weeks or months. Such cells often dis(cid:173)
`play many of the differentiated properties a ppropriate to their origin: fibroblasts
`continue to secrete collagen; cells derived from embryonic skeletal muscle fuse
`to form muscle fibers that contract spontaneously in the culture dish; nerve cells
`extend axons that are electrically excitable and make synapses with other nerve
`cells; and epithelial cells form extensive shee ts with many of the properties of an
`intact epithelium (Figure 8-4). Because these phenomena occur in culture, they
`are accessible to study in ways that are often not possible in intact tissues.
`
`Serum-free, Chemically D efined M edia Permit Identification
`of Specific Gro w t h Factor s
`
`Until the early 1970s tissue culture seem ed a blend of scie nce and witchcraft.
`Although fluid clots were replaced by dish es ofliq uid media containing specified
`quantities of small molecules such as salts, glucose, amino acids, and vitamins,
`
`biotin
`choline
`folate
`nicotinamide
`pantothenate
`pyridoxal
`thiamine
`riboflavin
`
`20 µm
`
`100 µm
`
`50 µ m
`
`Figure 8-4 Cells in cult ure. (A) Phase-contrast micrograph of fibroblasts
`in culture. (B) Micrograph of myoblasts in culture shows cells fus ing to form
`multinucleate muscle cells. (C) Oligodendrocyte precursor cells in culture.
`(D) Tobacco cells, from a fast-growing immortal cell line called BY2, in liquid
`culture. Nuclei and vacuoles can be seen in these cells. (A. courtesy of
`Daniel Zicha; B, courtesy of Rosalind Zalin: C, from D.G.Tang et al.,J. Cell Biol.
`148:971 - 984, 2000. © The Rockefeller University Press; D, courtesy of
`Gethin Roberts.)
`
`most media also included either a poorly defined mixture of macromolecules in
`the form of horse or fetal calf serum, or a crude extract made from chick
`embryos. Such media are still used today for most routine cell culture (Table
`8-1), but they make it difficult for the investigator to know which specific macro(cid:173)
`molecules a pa rticular type of cell requires to thrive and to function normally.
`This difficul ty led to the development of various serum-free, chemically
`defined media. In addition to the usual small molecules, such defined media
`contain one or more specific proteins that the cells require lo survive and prolif-
`erate in culture. These added proteins include growth factors, which stimulate
`eel] proliferation, and transferrin, which carries iron into cells. Many of the extra-
`cellular protein si