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Cell_Biology_Alberts. The presence of double-stranded RNA in the cell triggers RNAi by attracting a protein complex containing Dicer, the same nuclease that processes miR-NAs (see Figure 7–75). This protein cleaves the double-stranded RNA into small fragments (approximately 23 nucleotide pairs) called small interfering RNAs (siRNAs). These double-stranded siRNAs are then bound by Argonaute and other components of RISC. As we saw above for miRNAs, one strand of the duplex RNA is then cleaved by Argonaute and discarded. The single-stranded siRNA molecule that remains directs RISC back to complementary RNA molecules produced by the virus or transposable element. Because the match is usually exact, Argonaute cleaves these molecules, leading to their rapid destruction.


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Cell_Biology_Alberts. Each time RISC cleaves a new RNA molecule, the RISC is released; thus, as we saw for miRNAs, a single RNA molecule can act catalytically to destroy many complementary RNAs. Some organisms employ an additional mechanism that amplifies the RNAi response even further. In these organisms, RNA-dependent RNA polymerases use siRNAs as primers to produce additional copies of double-strand RNAs which are then cleaved into siRNAs. This amplification ensures that, once initiated, RNA interference can continue even after all the initiating double-stranded RNA has been degraded or diluted out. For example, it permits progeny cells to continue carrying out the specific RNA interference that was provoked in the parent cells.


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Cell_Biology_Alberts. In some organisms, the RNA interference activity can be spread by the transfer of RNA fragments from cell to cell. This is particularly important in plants (whose cells are linked by fine connecting channels, as discussed in Chapter 19), because it allows an entire plant to become resistant to an RNA virus after only a few of its cells have been infected. In a broad sense, the RNAi response resembles certain aspects of the animal immune system; in both, an invading organism elicits a customized response, and—through amplification of the “attack” molecules—the host becomes systemically protected.


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Cell_Biology_Alberts. Figure 7–76 Human Argonaute protein carrying an miRNA. the protein is folded into four structural domains, each indicated by a different color. the mirnA is held in an extended form that is optimal for forming rnA–rnA base pairs. the active site of Argonaute that “slices” a target rnA, when it is extensively base-paired with the mirnA, is indicated in red. Many Argonaute proteins (three out of the four human proteins, for example) lack the catalytic site and therefore bind target rnAs without slicing them. (Adapted from C.D. Kuhn and l. Joshua-tor, Trends Biochem. Sci. 38:263–271, 2013. with permission from Cell press.)


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Cell_Biology_Alberts. We have seen that although miRNAs and siRNAs are generated in slightly different ways, they rely on the same proteins and seek out their targets in a fundamentally similar manner. Because siRNAs are found in widespread species, they are believed to be the most ancient form of RNA interference, with miRNAs being a later refinement. These siRNA-mediated defense mechanisms are crucial for plants, worms, and insects. In mammals, a protein-based system (described in Chapter 24) has largely taken over the task of fighting off viruses.


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Cell_Biology_Alberts. The siRNA interference pathway just described does not necessarily stop with the destruction of target RNA molecules. In some cases, the RNA interference machinery can also selectively shut off synthesis of the target RNAs. For this to occur, the short siRNAs produced by the Dicer protein are assembled with a group of proteins (including Argonaute) to form the RITS (RNA-induced transcriptional silencing) complex. Using single-stranded siRNA as a guide sequence, this complex binds complementary RNA transcripts as they emerge from a transcribing RNA polymerase II (Figure 7–77). Positioned on the genome in this way, the RITS complex attracts proteins that covalently modify nearby histones and eventually direct the formation of heterochromatin to prevent further transcription initiation. In some cases, an RNA-dependent RNA polymerase and a Dicer enzyme are also recruited by the RITS complex to continually generate additional siRNAs in situ. This positive feedback loop ensures continued


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Cell_Biology_Alberts. some cases, an RNA-dependent RNA polymerase and a Dicer enzyme are also recruited by the RITS complex to continually generate additional siRNAs in situ. This positive feedback loop ensures continued repression of the target gene even after the initiating siRNA molecules have disappeared.


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Cell_Biology_Alberts. RNAi-directed heterochromatin formation is an important cell defense mechanism that limits the spread of transposable elements in genomes by maintaining their DNA sequences in a transcriptionally silent form. However, this same mechanism is also used in some normal processes in the cell. For example, in many organisms, the RNA interference machinery maintains the heterochromatin formed around centromeres. Centromeric DNA sequences are transcribed in both directions, producing complementary RNA transcripts that can base-pair to form double-stranded RNA. This double-stranded RNA triggers the RNA Figure 7–77 RNA interference directed by siRNAs. in many organisms, double-stranded rnA can trigger both the destruction of complementary mrnAs (left) and transcriptional silencing (right). the change in chromatin structure induced by the bound ritS (rnAinduced transcriptional silencing) complex resembles that in figure 7–45.


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Cell_Biology_Alberts. interference pathway and stimulates formation of the heterochromatin that surrounds centromeres, which is necessary for the centromeres to segregate chromosomes accurately during mitosis. pirnAs protect the Germ line from transposable elements


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Cell_Biology_Alberts. pirnAs protect the Germ line from transposable elements A third system of RNA interference relies on piRNAs (piwi-interacting RNAs, named for Piwi, a class of proteins related to Argonaute). piRNAs are made specifically in the germ line, where they block the movement of transposable elements. Found in many organisms, including humans, genes coding for piRNAs consist largely of sequence fragments of transposable elements. These clusters of fragments are transcribed and broken up into short, single-stranded piRNAs. The processing differs from that for miRNAs and siRNAs (for one thing, the Dicer enzyme is not involved), and the resulting piRNAs are slightly longer than miRNAs and siRNAs; moreover, they are complexed with Piwi rather than Argonaute proteins. Once formed, the piRNAs seek out RNA targets by base-pairing and, much like siRNAs, transcriptionally silence intact transposon genes and destroy any RNA (including mRNAs) produced by them.


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Cell_Biology_Alberts. Many mysteries surround piRNAs. Over a million piRNA species are coded in the genomes of many mammals and expressed in the testes, yet only a small fraction seem to be directed against the transposons present in those genomes. Are the piRNAs remnants of past invaders? Do they cover so much “sequence space” that they are broadly protective for any foreign DNA? Another curious feature of piRNAs is that many of them (particularly if base-pairing does not have to be perfect) should, in principle, attack the normal mRNAs made by the organism, yet they do not. It has been proposed that these large numbers of piRNAs may form a system to distinguish “self” RNAs from “foreign” RNAs and attack only the latter. If this is the case, there must be a special way for the cell to spare its own RNAs. One idea is that RNAs produced in the previous generation of an organism are somehow registered and set aside from piRNA attack in subsequent generations. Whether or not this mechanism truly exists, and,


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Cell_Biology_Alberts. idea is that RNAs produced in the previous generation of an organism are somehow registered and set aside from piRNA attack in subsequent generations. Whether or not this mechanism truly exists, and, if so, how it might work, are questions that demonstrate our incomplete understanding of the full implications of RNA interference.


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Cell_Biology_Alberts. Although it likely arose as a defense mechanism against viruses and transposable elements, RNA interference, as we have seen, has become thoroughly integrated into many aspects of normal cell biology, ranging from the control of gene expression to the structure of chromosomes. It has also been developed by scientists into a powerful experimental tool that allows almost any gene to be inactivated by evoking an RNAi response to it. This technique, which can be readily carried out in cultured cells and, in many cases, whole animals and plants, has made possible new genetic approaches in cell and molecular biology. We shall discuss it in detail in the following chapter where we cover modern genetic methods used to study cells (see pp. 499–501). RNAi also has potential in treating human disease. Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise.


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Cell_Biology_Alberts. Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise. Although the mechanism of RNA interference was discovered a few decades ago, we are still being surprised by its mechanistic details and by its broad biological implications.


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Cell_Biology_Alberts. Bacteria Use Small noncoding rnAs to protect themselves from viruses


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Cell_Biology_Alberts. Bacteria make up the vast majority of the Earth’s biomass and, not surprisingly, viruses that infect bacteria greatly outnumber plant and animal viruses. These viruses generally have DNA genomes. A recent discovery revealed that many species of bacteria (and almost all species of archaebacteria) use a repository of small noncoding RNA molecules to seek out and destroy the DNA of the invading viruses. Many features of this defense mechanism, known as the CRISPR system, resemble those we saw above for miRNAs and siRNAs, but there are two important differences. First, when bacteria and archaea are first infected by a virus, they have a mechanism that causes short fragments of that viral DNA to become integrated into their genomes. These serve as “vaccinations,” in the sense that they become the templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus should it reinfect the descendants of the original cell. This aspect of the CRISPR


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Cell_Biology_Alberts. templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus should it reinfect the descendants of the original cell. This aspect of the CRISPR system is similar in principle to adaptive immunity in mammals, in that the cell carries a record of past exposures that is used to protect against future exposures. The second distinguishing feature of the CRISPR system is that these crRNAs then become associated with special proteins that allow them to seek out and destroy double-stranded DNA molecules, rather than single-stranded RNA molecules.


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Cell_Biology_Alberts. Although many details of CRISPR-mediated immunity remain to be discovered, we can outline the general process in three steps (Figure 7–78). In the first, viral DNA sequences are integrated into special regions of the bacterial genome known as CRISPR (clustered regularly interspersed short palindromic repeat) loci, named for the peculiar structure that first drew the attention of scientists. In its simplest form, a CRISPR locus consists of several hundred repeats of a host DNA sequence interspersed with a large collection of sequences (typically 25–70 nucleotide pairs each) that has been derived from prior exposures to viruses and other foreign DNA. The newest viral sequence is always integrated at the 5ʹend of the CRISPR locus, the end that is transcribed first. Each locus, therefore, carries a temporal record of prior infections. Many bacterial and archaeal species carry several large CRISPR loci in their genomes and are thus immune to a wide range of viruses.


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Cell_Biology_Alberts. In the second step, the CRISPR locus is transcribed to produce a long RNA molecule, which is then processed into the much shorter (approximately 30 nucleotides) crRNAs. In the third step, crRNAs complexed with Cas (CRISPR-associated) proteins seek out complementary viral DNA sequences and direct their destruction by nucleases. Although structurally dissimilar, Cas proteins are analogous to the Argonaute and Piwi proteins discussed above: they hold small single-stranded RNAs in an extended configuration that is optimized, in this case, for seeking and forming complementary base pairs with DNA.


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Cell_Biology_Alberts. We still have much to learn about CRISPR-based immunity in bacteria and archaebacteria. The mechanism through which viral sequences are first identified and integrated into the host genome is poorly understood, as is the way that the crRNAs find their complementary sequences in double-stranded DNA. Moreover, in different species of bacteria and archaebacteria, crRNAs are processed in different ways, and in some cases, the crRNAs can attack viral RNAs as well as DNAs. We shall see in the following chapter that bacterial CRISPR systems have already been artificially “moved” into plants and animals, where they have become very powerful experimental tools for manipulating genomes.


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Cell_Biology_Alberts. Figure 7–78 CRISPR-mediated immunity in bacteria and archaebacteria. After infection by a virus (left panel), a small bit of DnA from the viral genome is inserted into the CriSpr locus. for this to happen, a small fraction of infected cells must survive the initial viral infection. the surviving cells, or more generally their descendants, transcribe the CriSpr locus and process the transcript into crrnAs (middle panel). Upon reinfection with a virus that the population has already been “vaccinated” against, the incoming viral DnA is destroyed by a complementary crrnA (right panel). for a CriSpr system to be effective, the crrnAs must not destroy the CriSpr locus itself, even though the crrnAs are complementary in sequence to it. in many species, in order for crrnAs to attack an invading DnA molecule, there must be additional short nucleotide sequences that are carried by the target molecule. Because these sequences, known as pAMs (protospacer adjacent motifs), lie outside the crrnA


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Cell_Biology_Alberts. DnA molecule, there must be additional short nucleotide sequences that are carried by the target molecule. Because these sequences, known as pAMs (protospacer adjacent motifs), lie outside the crrnA sequences, the host CriSpr locus is spared (see figure 8–55).


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Cell_Biology_Alberts. long noncoding rnAs have Diverse functions in the Cell


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Cell_Biology_Alberts. In this and the preceding chapters, we have seen that noncoding RNA molecules have many functions in the cell. Yet, as is the case with proteins, there remain many noncoding RNAs whose function is still unknown. Many RNAs of unknown function belong to a group known as long noncoding RNA (lncRNA). These are arbitrarily defined as RNAs longer than 200 nucleotides that do not code for protein. As methods have improved for determining the nucleotide sequences of all the RNA molecules produced by a cell line or tissue, the sheer number of lncRNAs (an estimated 8000 for the human genome, for example) came as a surprise to scientists. Most lncRNAs are transcribed by RNA polymerase II and have 5ʹ caps and poly-A tails, and, in many cases, they are spliced. It has been difficult to annotate lncRNAs because low levels of RNA are now known to be made from 75% of the human genome. Most of these RNAs are thought to result from the background “noise” of transcription and RNA processing. According


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Cell_Biology_Alberts. because low levels of RNA are now known to be made from 75% of the human genome. Most of these RNAs are thought to result from the background “noise” of transcription and RNA processing. According to this idea, such nonfunctional RNAs provide no fitness advantage or disadvantage to the organism and are a tolerated by-product of the complex patterns of gene expression that need to be produced in multicellular organisms. For these reasons, it is difficult to estimate the number of lncRNAs that are likely to have a function in the cell and to distinguish them from the background transcription.


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Cell_Biology_Alberts. We have already encountered a few lncRNAs, including the RNA in telomerase (see Figure 5–33), Xist RNA (see Figure 7–52), and an RNA involved in imprinting (see Figure 7–49). Other lncRNAs have been implicated in controlling the enzymatic activity of proteins, inactivating transcription regulators, affecting splicing patterns, and blocking translation of certain mRNAs.


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Cell_Biology_Alberts. In terms of biological function, lncRNA should be considered a catch-all phrase encompassing a great diversity of functions. Nevertheless, there are two unifying features of lncRNAs that can account for their many roles in the cell. The first is that lncRNAs can function as scaffold RNA molecules, holding together groups of proteins to coordinate their functions (Figure 7–79A). We have already seen an example in telomerase, where the RNA molecule holds together and organizes protein components. These RNA-based scaffolds are analogous to protein scaffolds we discussed in Chapter 3 (see Figure 3–78) and Chapter 6 (see Figure 6–47). RNA molecules are well suited to act as scaffolds: small bits of RNA sequence, often those portions that form stem-loop structures, can serve as binding sites for proteins, and these can be strung together with random sequences of RNA in between. This property may be one reason that lncRNAs show relatively little primary-sequence conservation across species.


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Cell_Biology_Alberts. The second key feature of lncRNAs is their ability to serve as guide sequences, binding to specific RNA or DNA target molecules through base-pairing. By doing so, they bring proteins that are bound to them into close proximity with the DNA Figure 7–79 Roles of long noncoding RNA (lncRNA). (A) lncrnAs can serve as scaffolds, bringing together proteins that function in the same process. As described in Chapter 6, rnAs can fold into specific three-dimensional structures that are often recognized by proteins. (B) in addition to serving as scaffolds, lncrnAs can, through formation of complementary base pairs, localize proteins to specific sequences on rnA or DnA molecules. (C) in some cases, lncrnAs act only in cis, for example, when the rnA is held in place by rnA polymerase (top). Other lncrnAs, however, diffuse from their sites of synthesis and therefore act in trans.


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Cell_Biology_Alberts. (A) IncRNA IncRNA IncRNA RNA DNA RNA polymerase controls transcription of genes on same chromosome controls transcription of genes on other chromosomes, chromosome A chromosome A chromosome B ACTS IN CISACTS IN TRANS and RNA sequences (Figure 7–79B). This behavior is similar to that of snoRNAs (see Figure 6–41), crRNAs (see Figure 7–78), and miRNAs (see Figure 7–75), all of which act in this way to guide protein enzymes to specific nucleic acid sequences.


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Cell_Biology_Alberts. In some cases, lncRNAs work simply by base-pairing, without bringing in enzymes or other proteins. For example, a number of lncRNA genes are embedded in protein-coding genes, but they are transcribed in the “wrong direction.” These antisense RNAs can form complementary base pairs with the mRNA (transcribed in the “correct” direction) and block its translation into protein (see Figure 7–66D). Other antisense lncRNAs base-pair with pre-mRNAs as they are synthesized and change the pattern of RNA splicing by masking splice-site sequences. Still others act as “sponges,” base-pairing with miRNAs and thereby reducing their effects.


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Cell_Biology_Alberts. Finally, we note that some lncRNAs can act only in cis; that is, they affect only the chromosome from which they are transcribed. This readily occurs when the transcribed RNA has not yet been released from RNA polymerases (Figure 7–79C). Many lncRNAs, however, diffuse from their site of synthesis and act in trans. Although the best understood lncRNAs work in the nucleus, many are found in the cytosol. The functions—if any—of the great majority of these cytosolic lncRNAs remain undiscovered.


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Cell_Biology_Alberts. RNA molecules have many uses in the cell besides carrying the information needed to specify the order of amino acids during protein synthesis. Although we have encountered noncoding RNAs in other chapters (tRNAs, rRNAs, snoRNAs, for example), the sheer number of noncoding RNAs produced by cells has surprised scientists. One well understood use of noncoding RNAs occurs in RNA interference, where guide RNAs (miRNAs, siRNAs, piRNAs) base-pair with mRNAs. RNA interference can cause mRNAs to be either destroyed or translationally repressed. It can also cause specific genes to be packaged into heterochromatin suppressing their transcription. In bacteria and archaebacteria, RNA interference is used as an adaptive immune response to destroy viruses that infect them. A large family of large noncoding RNAs (lncRNAs) has recently been discovered. Although the function of most of these RNAs is unknown, some serve as RNA scaffolds to bring specific proteins and how is the final rate of


Cell_Biology_Alberts

Cell_Biology_Alberts. noncoding RNAs (lncRNAs) has recently been discovered. Although the function of most of these RNAs is unknown, some serve as RNA scaffolds to bring specific proteins and how is the final rate of transcription of a gene specified by the hundreds of proteins that assemble on its control regions? will we ever be able to predict this rate from inspection of the DnA sequences of control regions?


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Cell_Biology_Alberts. how does the collection of cisregulatory sequences embedded in a genome orchestrate the developmental program of a multicellular organism? how much of the human genome sequence is functional, and why is the remainder retained? which of the thousands of unstudied noncoding rnAs have functions in the cell, and what are these functions? were introns present in early cells (and subsequently lost in some organisms), or did they arise at later times? RNA molecules together to speed up needed reactions. Which statements are true? Explain why or why not. 7–1 In terms of the way it interacts with DNA, the helix–loop–helix motif is more closely related to the leucine zipper motif than it is to the helix–turn–helix motif. 7–2 Once cells have differentiated to their final specialized forms, they never again alter expression of their genes.


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Cell_Biology_Alberts. 7–2 Once cells have differentiated to their final specialized forms, they never again alter expression of their genes. 7–3 CG islands are thought to have arisen during evolution because they were associated with portions of the genome that remained unmethylated in the germ line. 7–4 In most differentiated tissues, daughter cells retain a memory of gene expression patterns that were present in the parent cell through mechanisms that do not involve changes in the sequence of their genomic DNA. Discuss the following problems. 7–5 A small portion of a two-dimensional display of proteins from human brain is shown in Figure Q7–1. These proteins were separated on the basis of size in one dimension and electrical charge (isoelectric point) in the other. Not all protein spots on such displays are products


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Cell_Biology_Alberts. Figure Q7–1 two-dimensional separation of proteins from the human brain (problem 7–5). the proteins were displayed using two-dimensional gel electrophoresis. Only a small portion of the protein spectrum is shown. (Courtesy of tim Myers and leigh Anderson, large Scale Biology Corporation.) of different genes; some represent modified forms of a protein that migrate to different positions. Pick out a couple of sets of spots that could represent proteins that differ by the number of phosphates they carry. Explain the basis for your selection.


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Cell_Biology_Alberts. 7–6 Comparisons of the patterns of mRNA levels across different human cell types show that the level of expression of almost every active gene is different. The patterns of mRNA abundance are so characteristic of cell type that they can be used to determine the tissue of origin of cancer cells, even though the cells may have metastasized to different parts of the body. By definition, however, cancer cells are different from their noncancerous precursor cells. How do you suppose then that patterns of mRNA expression might be used to determine the tissue source of a human cancer? 7–7 What are the two fundamental components of a genetic switch?


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Cell_Biology_Alberts. 7–7 What are the two fundamental components of a genetic switch? 7–8 The nucleus of a eukaryotic cell is much larger than a bacterium, and it contains much more DNA. As a consequence, a transcription regulator in a eukaryotic cell must be able to select its specific binding site from among many more unrelated sequences than does a transcription regulator in a bacterium. Does this present any special problems for eukaryotic gene regulation? Consider the following situation. Assume that the eukaryotic nucleus and the bacterial cell each have a single copy of the same DNA binding site. In addition, assume that the nucleus is 500 times the volume of the bacterium, and has 500 times as much DNA. If the concentration of the transcription regulator that binds the site were the same in the nucleus and in the bacterium, would the regulator occupy its binding site equally as well in the eukaryotic nucleus as it does in the bacterium? Explain your answer.


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Cell_Biology_Alberts. 7–9 Some transcription regulators bind to DNA and cause the double helix to bend at a sharp angle. Such “bending proteins” can affect the initiation of transcription without directly contacting any other protein. Can you devise a plausible explanation for how such proteins might work to modulate transcription? Draw a diagram that illustrates your explanation. 7–10 How is it that protein–protein interactions that are too weak to cause proteins to assemble in solution can nevertheless allow the same proteins to assemble into complexes on DNA?


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Cell_Biology_Alberts. 7–10 How is it that protein–protein interactions that are too weak to cause proteins to assemble in solution can nevertheless allow the same proteins to assemble into complexes on DNA? 7–11 Imagine the two situations shown in Figure Q7–2. In cell 1, a transient signal induces the synthesis of protein A, which is a transcription activator that turns on many genes including its own. In cell 2, a transient signal induces the synthesis of protein R, which is a transcription repressor that turns off many genes including its own. In which, if either, of these situations will the descendants of the original cell “remember” that the progenitor cell had experienced the transient signal? Explain your reasoning. transcription turns on transcription activator protein activator of activator mRNA turns on its own transcription Figure Q7–2 Gene regulatory circuits and cell memory (problem 7–11).


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Cell_Biology_Alberts. transcription turns on transcription activator protein activator of activator mRNA turns on its own transcription Figure Q7–2 Gene regulatory circuits and cell memory (problem 7–11). (A) induction of synthesis of transcription activator A by a transient signal. (B) induction of synthesis of transcription repressor r by a transient signal.


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Cell_Biology_Alberts. (A) induction of synthesis of transcription activator A by a transient signal. (B) induction of synthesis of transcription repressor r by a transient signal. 7–12 Examine the two pedigrees shown in Figure Q7–3. One results from deletion of a maternally imprinted autosomal gene. The other pedigree results from deletion of a paternally imprinted autosomal gene. In both pedigrees, affected individuals (red symbols) are heterozygous for the deletion. These individuals are affected because one copy of the chromosome carries an imprinted, inactive gene, while the other carries a deletion of the gene. Dotted yellow symbols indicate individuals that carry the deleted locus, but do not display the mutant phenotype. Which pedigree is based on paternal imprinting and which on maternal imprinting? Explain your answer.


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Cell_Biology_Alberts. Figure Q7–3 pedigrees reflecting maternal and paternal imprinting (problem 7–12). in one pedigree, the gene is paternally imprinted; in the other, it is maternally imprinted. in generations 3 and 4, only one of the two parents in the indicated matings is shown; the other parent is a normal individual from outside this pedigree. Affected individuals are represented by red circles for females and red squares for males. Dotted yellow symbols indicate individuals that carry the deletion but do not display the phenotype. 7–13 If you insert a β-galactosidase gene lacking its own transcription control region into a cluster of piRNA genes in Drosophila, you find that β-galactosidase expression from a normal copy elsewhere in the genome is strongly inhibited in the fly’s germ cells. If the inactive β-galactosidase gene is inserted outside the piRNA gene cluster, the normal gene is properly expressed. What do you suppose is the basis for this observation? How would you test your hypothesis?


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Cell_Biology_Alberts. Brown tA (2007) Genomes 3. new York: Garland Science. epigenetics (2004) Cold Spring Harb. Symp. Quant. Biol. 69. Gilbert Sf (2013) Developmental Biology, 10th ed. Sunderland, MA: Sinauer Associates, inc. hartwell l, hood l, Goldberg Ml et al. (2010) Genetics: from Genes to Genomes, 4th ed. Boston: McGraw hill. McKnight Sl & Yamamoto Kr (eds) (1993) transcriptional regulation. Cold Spring harbor, nY: Cold Spring harbor laboratory press. Mechanisms of transcription (1998) Cold Spring Harb. Symp. Quant. Biol. 63. ptashne M & Gann A (2002) Genes and Signals. Cold Spring harbor, nY: Cold Spring harbor laboratory press. watson J, Baker t, Bell S et al. (2013) Molecular Biology of the Gene, 7th ed. Menlo park, CA: Benjamin Cummings. An Overview of Gene Control Davidson eh (2006) the regulatory Genome: Gene regulatory networks in Development and evolution. Burlington, MA: elsevier. Gurdon JB (1992) the generation of diversity and pattern in animal development. Cell 68, 185–199.


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Cell_Biology_Alberts. Gurdon JB (1992) the generation of diversity and pattern in animal development. Cell 68, 185–199. Kellis M, wold B, Synder Mp et al. (2014) Defining functional DnA elements in the human genome. Proc. Natl. Acad. Sci. USA 111, 6131–6138. McKnight Sl (1991) Molecular zippers in gene regulation. Sci. Am. 264, 54–64. pabo CO & Sauer rt (1992) transcription factors: structural families and principles of DnA recognition. Annu. Rev. Biochem. 61, 1053–1095. Seeman nC, rosenberg JM & rich A (1976) Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73, 804–808. weirauch Mt & hughes tr (2011) A catalogue of eukaryotic transcription factor types, their evolutionary origin, and species distribution. in A handbook of transcription factors. new York, nY: Springer publishing Company.


Cell_Biology_Alberts

Cell_Biology_Alberts. Beckwith J (1987) the operon: an historical account. in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (neidhart fC, ingraham Jl, low KB et al. eds), vol. 2, pp. 1439–1443. washington, DC: ASM press. Gilbert w & Müller-hill B (1967) the lac operator is DnA. Proc. Natl. Acad. Sci. USA 58, 2415. Jacob f & Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356. levine M, Cattoglio C & tjian r (2014) looping back to leap forward: transcription enters a new era. Cell 157, 13–25. narlikar GJ, Sundaramoorthy r & Owen-hughes t (2013) Mechanisms and functions of Atp-dependent chromatin-remodeling enzymes. Cell 154, 490–503. ptashne M (2004) A Genetic Switch: phage and lambda revisited, 3rd ed. Cold Spring harbor, nY: Cold Spring harbor laboratory press. ptashne M (1967) Specific binding of the lambda phage repressor to lambda DnA. Nature 214, 232–234.


Cell_Biology_Alberts

Cell_Biology_Alberts. ptashne M (1967) Specific binding of the lambda phage repressor to lambda DnA. Nature 214, 232–234. St Johnston D & nusslein-volhard C (1992) the origin of pattern and polarity in the Drosophila embryo. Cell 68, 201–219. turner BM (2014) nucleosome signaling: an evolving concept. Biochim. Biophys. Acta 1839, 623–626. Alon U (2007) network motifs: theory and experimental approaches. Nature 8, 450–461. Buganim Y, faddah DA & Jaenisch r (2013) Mechanisms and models of somatic cell reprogramming. Nat. Rev. Genet. 14, 427–439. hobert O (2011) regulation of terminal differentiation programs in the nervous system. Annu. Rev. Cell Dev. Biol. 27, 681–696. lawrence pA (1992) the Making of a fly: the Genetics of Animal Design. new York: Blackwell Scientific publications. Bird A (2011) putting the DnA back into DnA methylation. Nat. Genet. 43, 1050–1051. Gehring M (2013) Genomic imprinting: insights from plants. Annu. Rev. Genet. 47, 187–208.


Cell_Biology_Alberts

Cell_Biology_Alberts. Bird A (2011) putting the DnA back into DnA methylation. Nat. Genet. 43, 1050–1051. Gehring M (2013) Genomic imprinting: insights from plants. Annu. Rev. Genet. 47, 187–208. lawson hA, Cheverud JM & wolf JB (2013) Genomic imprinting and parent-of-origin effects on complex traits. Genetics 14, 609–617. lee Jt & Bartolomei MS (2013) X-inactivation, imprinting, and long noncoding rnAs in health and disease. Cell 152, 1308–1323. li e & Zhang Y (2014) DnA methylation in mammals. Cold Spring Harb. Perspect. Biol. 6, a019133. DiGiammartino DC, nishida K & Manley Jl (2011) Mechanisms and consequences of alternative polyadenylation. Mol Cell 43, 853–866. Gottesman S & Storz G (2011) Bacterial small rnA regulators: versatile roles and rapidly evolving variations. Cold Spring Harb. Perspect. Biol. 3, a003798. hershey JwB, Sonenberg n & Mathews MB (2012) principles of translational control: an overview. Cold Spring Harb. Perspect. Biol. 4, a011528.


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Cell_Biology_Alberts. hershey JwB, Sonenberg n & Mathews MB (2012) principles of translational control: an overview. Cold Spring Harb. Perspect. Biol. 4, a011528. hundley hA & Bass Bl (2010) ADAr editing in double-stranded Utrs and other noncoding rnA sequences. Trends Biochem. Sci. 35, 377–383. Kalsotra A & Cooper tA (2011) functional consequences of developmentally regulated alternative splicing. Nat. Rev. Genet. 12, 715–729. Kortmann J & narberhaus f (2012) Bacterial rnA thermometers: molecular zippers and switches. Nat. Rev. Microbiol. 10, 255–265. parker r (2012) rnA degradation in Saccharomyces cerevisae. Genetics 191, 671–702. popp Mw & Maquat le (2013) Organizing principles of mammalian nonsense-mediated mrnA decay. Annu. Rev. Genet. 47, 139–165. Serganov A & nudler e (2013) A decade of riboswitches. Cell 152, 17–24. thompson Sr (2012) tricks an ireS uses to enslave ribosomes. Trends Microbiol. 20, 558–566. Regulation of Gene Expression By Noncoding RNAs


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Cell_Biology_Alberts. thompson Sr (2012) tricks an ireS uses to enslave ribosomes. Trends Microbiol. 20, 558–566. Regulation of Gene Expression By Noncoding RNAs Bhaya D, Davison M & Barrangou r (2011) CriSpr-Cas systems in bacteria and archaea: versatile small rnAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273–297. Cech tr & Steitz JA (2014) the noncoding rnA revolution–trashing old rules to forge new ones. Cell 157, 77–94. fire A, Xu S, Montgomery MK et al (1998) potent and specific genetic interference by double-stranded rnA in Caenorhabditis elegans. Nature 391, 806–811. Guttman M & rinn Jl (2012) Modular regulatory principles of large non-coding rnAs. Nature 482, 339–346. lee hC, Gu w, Shirayama M et al. (2012) C. elegans pirnAs mediate the genome-wide surveillance of germline transcripts. Cell 150, 78–87. Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459.


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Cell_Biology_Alberts. Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459. rinn Jl & Chang hY (2012) Genome regulation by long noncoding rnAs. Annu. Rev. Biochem. 81, 145–166. tenOever Br (2013) rnA viruses and the host micrornA machinery. Nat. Rev. Microbiol. 11, 169–180. Ulitsky i & Bartel Dp (2013) lincrnAs: genomics, evolution, and mechanisms. Cell 154, 26–46. wiedenheft B, Sternberg Sh & Doudna JA (2012) rnA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338. Analyzing Cells, Molecules, and Systems


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Cell_Biology_Alberts. wiedenheft B, Sternberg Sh & Doudna JA (2012) rnA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338. Analyzing Cells, Molecules, and Systems Progress in science is often driven by advances in technology. The entire field of cell biology, for example, came into being when optical craftsmen learned to grind small lenses of sufficiently high quality to observe cells and their substructures. Innovations in lens grinding, rather than any conceptual or philosophical advance, allowed Hooke and van Leeuwenhoek to discover a previously unseen cellular world, where tiny creatures tumble and twirl in a small droplet of water (Figure 8–1).


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Cell_Biology_Alberts. The twenty-first century is a particularly exciting time for biology. New methods for analyzing cells, proteins, DNA, and RNA are fueling an information explosion and allowing scientists to study cells and their macromolecules in previously unimagined ways. We now have access to the sequences of many billions of nucleotides, providing the complete molecular blueprints for hundreds of organ-isms—from microbes and mustard weeds to worms, flies, mice, dogs, chimpanzees, 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 also to begin to unravel how these components work together to form functional cells and organisms. The long-range goal is nothing short of obtaining a complete understanding of what takes place inside a cell as it responds to its environment and interacts with its neighbors.


Cell_Biology_Alberts

Cell_Biology_Alberts. In this chapter, we present some of the principal methods used to study cells and their molecular components. We consider how to separate cells of different types from tissues, how to grow cells outside the body, and how to disrupt cells and isolate their organelles and constituent macromolecules in pure form. We also present the techniques used to determine protein structure, function, and interactions, and we discuss the breakthroughs in DNA technology that continue to revolutionize our understanding of cell function. We end the chapter with an overview of some of the mathematical approaches that are helping us deal with the enormous complexity of cells. By considering cells as dynamic systems with many moving parts, mathematical approaches can reveal hidden insights into how the many components of cells work together to produce the special qualities of life.


Cell_Biology_Alberts

Cell_Biology_Alberts. 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 that large numbers of cells be physically disrupted to gain access to their components. If the sample is a piece of tissue, composed of different types of cells, heterogeneous cell populations will be mixed together. To obtain as much information as possible about the cells in a tissue, biologists have developed ways of dissociating cells from tissues and separating them according to type. These manipulations result in a relatively homogeneous population of cells that can then be analyzed—either directly or after their number has been greatly increased by allowing the cells to proliferate in culture.


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Cell_Biology_Alberts. Intact tissues provide the most realistic source of material, as they represent the actual cells found within the body. The first step in isolating individual cells is to disrupt the extracellular matrix and cell–cell junctions that hold the cells together. For this purpose, a 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 cells by gentle agitation.


Cell_Biology_Alberts

Cell_Biology_Alberts. For some biochemical preparations, the protein of interest can be obtained in sufficient quantity without having to separate the tissue or organ into cell types. Examples include the preparation of histones from calf thymus, actin from rabbit muscle, or tubulin from cow brain. In other cases, obtaining the desired protein requires enrichment for a specific cell type of interest. Several approaches are used to separate the different cell types from a mixed cell suspension. One of the most sophisticated cell-separation techniques uses an antibody coupled to a fluorescent dye to label specific cells. An antibody is chosen that specifically binds to the surface of only one cell type in the tissue. The labeled cells can then be separated from the unlabeled ones in a 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


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Cell_Biology_Alberts. 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 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 container. 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).


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Cell_Biology_Alberts. Although molecules can be extracted from whole tissues, this is often not the most convenient or useful source of material. The complexity of intact tissues and organs is an inherent disadvantage when trying to purify particular molecules. Cells grown in culture provide a more homogeneous population of cells from which to extract material, and they are also much more convenient to work with in the laboratory. Given appropriate surroundings, most plant and animal cells can live, multiply, and even express differentiated properties in a 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 systematically explored.


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Cell_Biology_Alberts. Experiments performed on cultured cells are sometimes said to be carried out in vitro (literally, “in glass”) to contrast them with experiments using intact organisms, which are said to be carried out in vivo (literally, “in the living organism”). 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 eukaryotic green alga Volvox (1700). (Courtesy of the John Innes Foundation.) These terms can be confusing, however, because they are 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, whereas in vivo refers to any reaction taking place inside a living cell, even if that cell is growing in culture.


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Cell_Biology_Alberts. Tissue culture began in 1907 with an experiment designed to settle a controversy in neurobiology. The hypothesis under examination was known as the neuronal 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 (axons) into the clot. Thus, the neuronal doctrine received strong support, and the foundation was laid for the cell-culture revolution.


Cell_Biology_Alberts

Cell_Biology_Alberts. These original experiments on nerve fibers used cultures of small tissue fragments called explants. Today, cultures are more commonly made from suspensions of cells dissociated from tissues. Unlike bacteria, most tissue cells are not adapted to living suspended in fluid and require a solid surface on which to grow and divide. For cell cultures, this support is usually provided by the surface of a plastic culture dish. Cells vary in their requirements, however, and many do not proliferate or differentiate unless the culture dish is coated with materials that cells like to adhere to, such as polylysine or extracellular matrix components.


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Cell_Biology_Alberts. Cultures prepared directly from the tissues of an organism 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 removed from the culture dish and recultured repeatedly in so-called secondary cultures; in this way, they can be repeatedly subcultured (passaged) for weeks or months. Such cells often display many of the differentiated properties appropriate to their 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.


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Cell_Biology_Alberts. Figure 8–3 Light micrographs of cells in culture. (A) Mouse fibroblasts. (B) Chick myoblasts fusing to form multinucleate muscle cells. (C) Purified rat retinal ganglion nerve cells. (D) Tobacco cells in liquid culture. (A, courtesy of Daniel Zicha; B, courtesy of Rosalind Zalin; C, from A. Meyer-Franke et al., Neuron 15:805–819, 1995. With permission from Elsevier; D, courtesy of Gethin Roberts.) origin (Figure 8–3): 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 sheets with many of the properties of an intact epithelium. Because these properties are maintained in culture, they are accessible to study in ways that are often not possible in intact tissues.


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Cell_Biology_Alberts. Cell culture is not limited to animal cells. When a piece of plant tissue is cul-50 µm tured in a sterile medium containing nutrients and appropriate growth regulators, many of the cells are stimulated to proliferate indefinitely in a disorganized manner, producing a mass of relatively undifferentiated cells called a callus. If the nutrients and growth regulators are carefully manipulated, one can induce the formation of a shoot and then root apical meristems within the callus, and, in many species, regenerate a whole new plant. Similar to animal cells, callus cultures can be mechanically dissociated into single cells, which will grow and divide as a suspension culture (see Figure 8–3D). Eukaryotic Cell Lines Are a Widely Used Source of Homogeneous Cells


Cell_Biology_Alberts

Cell_Biology_Alberts. The cell cultures obtained by disrupting tissues tend to suffer from a problem— eventually the cells die. Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called replicative cell senescence (discussed in Chapter 17). Normal human fibroblasts, for example, typically divide only 25–40 times in culture before they stop. In these cells, the limited proliferation capacity reflects a progressive shortening and uncapping of the cell’s telomeres, the repetitive DNA sequences and associated proteins that cap the ends of each chromosome (discussed in Chapter 5). Human somatic cells in the body have turned off production of the enzyme, called telomerase, that normally maintains the telomeres, which is why their telomeres shorten with each cell division. Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase; in this case, they can be propagated as an


Cell_Biology_Alberts

Cell_Biology_Alberts. Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase; in this case, they can be propagated as an “immortalized” cell line.


Cell_Biology_Alberts

Cell_Biology_Alberts. Some human cells, however, cannot be immortalized by this trick. Although their telomeres remain long, they still stop dividing after a limited number of divisions because culture conditions cause excessive mitogenic stimulation, which activates a poorly understood protective mechanism (discussed in Chapter 17) that stops cell division—a process sometimes called “culture shock.” To immortalize these cells, one has to do more than introduce telomerase. One must also inactivate the protective mechanisms, which can be done by introducing certain cancer-promoting oncogenes (discussed in Chapter 20). Unlike human cells, most rodent cells do not turn off production of telomerase and therefore their telomeres do not shorten with each cell division. Therefore, if culture shock can be avoided, some rodent cell types will divide indefinitely in culture. In addition, rodent cells often undergo spontaneous genetic changes in culture that inactivate their protective mechanisms, thereby producing


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Cell_Biology_Alberts. rodent cell types will divide indefinitely in culture. In addition, rodent cells often undergo spontaneous genetic changes in culture that inactivate their protective mechanisms, thereby producing immortalized cell lines.


Cell_Biology_Alberts

Cell_Biology_Alberts. Cell lines can often be most easily generated from cancer cells, but these cultures—referred to as transformed cell lines—differ from those prepared from normal cells in several ways. Transformed cell lines often grow without attaching to a surface, for example, and they can proliferate to a much higher density in a culture dish. Similar properties can be induced experimentally in normal cells by transforming them with a tumor-inducing virus or chemical. The resulting transformed cell lines can usually cause tumors if injected into a susceptible animal.


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Cell_Biology_Alberts. Transformed and nontransformed cell lines are extremely useful in cell research as sources of very large numbers of cells of a uniform type, especially since they can be stored in liquid nitrogen at –196°C for an indefinite period and retain their viability when thawed. It is important to keep in mind, however, that cell lines nearly always differ in important ways from their normal progenitors in the tissues from which they were derived. Some widely used cell lines are listed in Table 8–1. Different lines have different advantages; for example, the PtK epithelial cell lines derived from the rat 444 Chapter 8: Analyzing Cells, Molecules, and Systems kangaroo, unlike many other cell types, remain flat during mitosis, allowing the mitotic apparatus to be readily observed in action.


Cell_Biology_Alberts

Cell_Biology_Alberts. As we see throughout this book, antibodies are particularly useful tools for cell biology. Their great specificity allows precise visualization of selected proteins among the many thousands that each cell typically produces. Antibodies are often produced by inoculating animals with the protein of interest and subsequently isolating the antibodies specific to that protein from the serum of the animal. However, only limited quantities of antibodies can be obtained from a single inoculated animal, and the antibodies produced will be a heterogeneous mixture of antibodies that recognize a variety of different antigenic sites on a macromolecule that differs from animal to animal. Moreover, antibodies specific for the antigen will constitute only a fraction of the antibodies found in the serum. An alternative technology, which allows the production of an unlimited quantity of identical antibodies and greatly increases the specificity and convenience of antibody-based methods, is the


Cell_Biology_Alberts

Cell_Biology_Alberts. serum. An alternative technology, which allows the production of an unlimited quantity of identical antibodies and greatly increases the specificity and convenience of antibody-based methods, is the production of monoclonal antibodies by hybridoma cell lines.


Cell_Biology_Alberts

Cell_Biology_Alberts. This technology, developed in 1975, revolutionized the production of antibodies for use as tools in cell biology, as well as for the diagnosis and treatment of certain diseases, including rheumatoid arthritis and cancer. The procedure requires hybrid cell technology (Figure 8–4), and it involves propagating a clone of cells from a single antibody-secreting B lymphocyte to obtain a homogeneous preparation of antibodies in large quantities. B lymphocytes normally have a limited life-span in culture, but individual antibody-producing B lymphocytes from an immunized mouse, when fused with cells derived from a transformed B lymphocyte cell line, can give rise to hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture. These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody. Each type of monoclonal antibody recognizes a single type of


Cell_Biology_Alberts

Cell_Biology_Alberts. are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody. Each type of monoclonal antibody recognizes a single type of antigenic site—for example, a particular cluster of five or six amino acid side chains on the surface of a protein. Their uniform specificity makes monoclonal antibodies much more useful than conventional antisera for many purposes.


Cell_Biology_Alberts

Cell_Biology_Alberts. An important advantage of the hybridoma technique is that monoclonal antibodies can be made against molecules that constitute only a minor component of a complex mixture. In an ordinary antiserum made against such a mixture, the proportion of antibody molecules that recognize the minor component would be too small to be useful. But if the B lymphocytes that produce the various components of this antiserum are made into hybridomas, it becomes possible to screen individual hybridoma clones from the large mixture to select one that produces the desired type of monoclonal antibody and to propagate the selected hybridoma SUSPENSION OF TWO CELL three clones of hybrid cellsTYPES CENTRIFUGED WITH A FUSING AGENT ADDED SELECTIVE MEDIUM FORMATION OF PROLIFERATE. THESE HETEROKARYONS, BECOME HYBRID WHICH ARE CELLS, WHICH ARE THEN CULTURED THEN CLONED


Cell_Biology_Alberts

Cell_Biology_Alberts. FORMATION OF PROLIFERATE. THESE HETEROKARYONS, BECOME HYBRID WHICH ARE CELLS, WHICH ARE THEN CULTURED THEN CLONED Figure 8–4 The production of hybrid cells. It is possible to fuse one cell with another to form a heterokaryon, a combined cell with two separate nuclei. Typically, a suspension of cells is treated with certain inactivated viruses or with polyethylene glycol, each of which alters the plasma membranes of cells in a way that induces them to fuse. Eventually, a heterokaryon proceeds to mitosis and produces a hybrid cell in which the two separate nuclear envelopes have been disassembled, allowing all the chromosomes to be brought together in a single large nucleus. Such hybrid cells can give rise to immortal hybrid cell lines. If one of the parent cells was from a tumor cell line, the hybrid cell is called a hybridoma.


Cell_Biology_Alberts

Cell_Biology_Alberts. indefinitely so as to produce that antibody in unlimited quantities. In principle, therefore, a monoclonal antibody can be made against any protein in a biological sample. Once an antibody has been made, it can be used to localize the protein in cells and tissues, to follow its movement, and to purify the protein to study its structure and function.


Cell_Biology_Alberts

Cell_Biology_Alberts. Tissues can be dissociated into their component cells, from which individual cell types can be purified and used for biochemical analysis or for the establishment of cell cultures. Many animal and plant cells survive and proliferate in a culture dish if they are provided with a suitable culture medium containing nutrients and appropriate signal molecules. Although many animal cells stop dividing after a finite number of cell divisions, cells that have been immortalized through spontaneous mutations or genetic manipulation can be maintained indefinitely as cell lines. Hybridoma cells are widely employed to produce unlimited quantities of uniform monoclonal antibodies, which are used to detect and purify cell proteins, as well as to diagnose and treat diseases.


Cell_Biology_Alberts

Cell_Biology_Alberts. The challenge of isolating a single type of protein from the thousands of other proteins present in a cell is a formidable one, but must be overcome in order to study protein function in vitro. As we shall see later in this chapter, recombinant DNA technology can enormously simplify this task by “tricking” cells into producing large quantities of a given protein, thereby making its purification much easier. Whether the source of the protein is an engineered cell or a natural tissue, a purification procedure usually starts with subcellular fractionation to reduce the complexity of the material, and is then followed by purification steps of increasing specificity.


Cell_Biology_Alberts

Cell_Biology_Alberts. To purify a protein, it must first be extracted from inside the cell. Cells can be broken up in various ways: they can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or ground up in a blender. These procedures break many of the membranes of the cell (including the plasma membrane and endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. If carefully carried out, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension of cells is thereby reduced to a thick slurry (called a homogenate or extract) that contains a variety of membrane-enclosed organelles, each with a distinctive size, charge, and density. Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called


Cell_Biology_Alberts

Cell_Biology_Alberts. Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called microsomes—retain most of their original biochemical properties.


Cell_Biology_Alberts

Cell_Biology_Alberts. The different components of the homogenate must then be separated. Such cell fractionations became possible only after the commercial development in the early 1940s of an instrument known as the preparative ultracentrifuge, which rotates extracts of broken cells at high speeds (Figure 8–5). This treatment separates cell components by size and density: in general, the largest objects experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first the small closed vesicles and then the ribosomes can be collected (Figure 8–6). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 8–5 The preparative ultracentrifuge. (A) The sample is contained in tubes that are inserted into a ring of angled cylindrical holes in a metal rotor. Rapid rotation of the rotor generates enormous centrifugal forces, which cause particles in the sample to sediment against the bottom sides of the sample tubes, as shown here. The vacuum reduces friction, preventing heating of the rotor and allowing the refrigeration system to maintain the sample at 4°C. (B) Some fractionation methods require a different type of rotor called a swinging-bucket rotor. In this case, the sample tubes are placed in metal tubes on hinges that allow the tubes to swing outward when the rotor spins. Sample tubes are therefore horizontal during spinning, and samples are sedimented toward the bottom, not the sides, of the tube, providing better separation of differently sized components (see Figures 8–6 and 8–7).


Cell_Biology_Alberts

Cell_Biology_Alberts. Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate, in a process called velocity sedimentation (Figure 8–7A). For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the solution denser than any solution above it, and it thereby prevents


Cell_Biology_Alberts

Cell_Biology_Alberts. by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation.


Cell_Biology_Alberts

Cell_Biology_Alberts. When sedimented through sucrose gradients, different cell components separate into distinct bands that can be collected individually. The relative rate at which each component sediments depends primarily on its size and shape—normally being described in terms of its sedimentation coefficient, or S value. Present-day ultracentrifuges rotate at speeds of up to 80,000 rpm and produce forces as high as 500,000 times gravity. These enormous forces drive even small macromolecules, such as tRNA molecules and simple enzymes, to sediment at an appreciable rate and allow them to be separated from one another by size. The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case, the


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 8–6 Cell fractionation by centrifugation. Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it. Typical values for the various centrifugation steps referred to in the figure are: low speed: 1000 times gravity for 10 minutes medium speed: 20,000 times gravity for 20 minutes high speed: 80,000 times gravity for 1 hour very high speed: 150,000 times gravity for 3 hours steep(e.g., 5–20%) sucrose gradient (e.g., 20–70%) sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8–7A, but it eventually reaches a position where the density of the solution is equal to its own density. At this point, the component floats and can move no farther. A series of distinct bands is thereby


Cell_Biology_Alberts

Cell_Biology_Alberts. but it eventually reaches a position where the density of the solution is equal to its own density. At this point, the component floats and can move no farther. A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the components of highest buoyant density (Figure 8–7B). This method, called equilibrium sedimentation, is so sensitive that it can separate macromolecules that have incorporated heavy isotopes, such as 13C or 15N, from the same macromolecules that contain the lighter, common isotopes (12C or 14N). In fact, the cesium-chloride method was developed in 1957 to separate the labeled from the unlabeled DNA produced after exposure of a growing population of bacteria to nucleotide precursors containing 15N; this classic experiment provided direct evidence for the semiconservative replication of DNA (see Figure 5–5).


Cell_Biology_Alberts

Cell_Biology_Alberts. Cell Extracts Provide Accessible Systems to Study Cell Functions Studies of organelles and other large subcellular components isolated in the ultracentrifuge have contributed enormously to our understanding of the functions of different cell components. Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy into forms that the cell can use. Similarly, resealed vesicles formed from fragments of rough and smooth endoplasmic reticulum


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 8–7 Comparison of velocity sedimentation and equilibrium sedimentation. (A) In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose, which increases in concentration toward the bottom of the tube (typically from 5% to 20% sucrose). After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom, as illustrated here.


Cell_Biology_Alberts

Cell_Biology_Alberts. (B) In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings. Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride. The final bands, at equilibrium, can be collected as in (A). (microsomes) have been separated from each other and analyzed as functional models of these compartments of the intact cell. Similarly, highly concentrated cell extracts, especially extracts of Xenopus laevis (African clawed frog) oocytes, have played a critical role in the study of such complex and highly organized processes as the cell-division cycle, the separation of chromosomes on the mitotic spindle, and the vesicular-transport steps involved in the movement of proteins from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane.


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Cell_Biology_Alberts. Cell extracts also provide, in principle, the starting material for the complete separation of all of the individual macromolecular components of the cell. We now consider how this separation is achieved, focusing on proteins. Proteins are most often fractionated by column chromatography, in which a mixture of proteins in solution is passed through a column containing a porous solid matrix. Different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separately as they flow out of the bottom of the column (Figure 8–8). Depending on the choice of matrix, proteins can be separated according to their charge (ion-exchange chromatography), their hydrophobicity (hydrophobic chromatography), their size (gel-filtration chromatography), or their ability to bind to particular small molecules or to other macromolecules (affinity chromatography).


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Cell_Biology_Alberts. Many types of matrices are available. Ion-exchange columns (Figure 8–9A) are packed with small beads that carry either a positive or a negative charge, so that proteins are fractionated according to the arrangement of charges on their surface. Hydrophobic columns are packed with beads from which hydrophobic side chains protrude, selectively retarding proteins with exposed hydrophobic regions. Gel-filtration columns (Figure 8–9B), which separate proteins according to their size, are packed with tiny porous beads: molecules that are small enough to enter the pores linger inside successive beads as they pass down the column, while larger molecules remain in the solution flowing between the beads and therefore move more rapidly, emerging from the column first. Besides providing applied to the top of applied reservoir of solvent


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Cell_Biology_Alberts. Figure 8–8 The separation of molecules by column chromatography. The sample, a solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose. A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. Because various components of the sample travel at different rates through the column, they are fractionated into different tubes. a means of separating molecules, gel-filtration chromatography is a convenient way to estimate their size.


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Cell_Biology_Alberts. a means of separating molecules, gel-filtration chromatography is a convenient way to estimate their size. Affinity chromatography (Figure 8–9C) takes advantage of the biologically important binding interactions that occur on protein surfaces. If a substrate molecule is covalently coupled to an inert matrix such as a polysaccharide bead, the enzyme that operates on that substrate will often be specifically retained by the matrix and can then be eluted (washed out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically designed sequence can be immobilized in this way and used to purify DNA-binding proteins that normally recognize this sequence of nucleotides in chromosomes. Alternatively, specific antibodies can be coupled to a matrix to purify protein molecules recognized by the antibodies. Because of the great specificity of all such affinity columns, 1000to 10,000-fold purifications can sometimes be achieved in a single pass.


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Cell_Biology_Alberts. If one starts with a complex mixture of proteins, a single passage through an ion-exchange or a gel-filtration column does not produce very highly purified fractions, since these methods individually increase the proportion of a given protein in the mixture no more than twentyfold. Because most individual proteins represent less than 1/1000 of the total cell protein, it is usually necessary to use several different types of columns in succession to attain sufficient purity, with affinity chromatography being the most efficient (Figure 8–10).


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Cell_Biology_Alberts. Inhomogeneities in the matrices (such as cellulose), which cause an uneven flow of solvent through the column, limit the resolution of conventional column chromatography. Special chromatography resins (usually silica-based) composed of tiny spheres (3–10 μm in diameter) can be packed with a special apparatus to form a uniform column bed. Such high-performance liquid chromatography (HPLC) columns attain a high degree of resolution. In HPLC, the solutes equilibrate very rapidly with the interior of the tiny spheres, and so solutes with different affinities for the matrix are efficiently separated from one another even at very fast flow rates. HPLC is therefore the method of choice for separating many proteins and small molecules.


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Cell_Biology_Alberts. Immunoprecipitation is a useful variation on the theme of affinity chromatography. Specific antibodies that recognize the protein to be purified are attached to small agarose beads. Rather than being packed into a column, as in affinity chromatography, a small quantity of the antibody-coated beads is simply added to a protein extract in a test tube and mixed in suspension for a short period of time— thereby allowing the antibodies to bind the desired protein. The beads are then collected by low-speed centrifugation, and the unbound proteins in the supernatant are discarded. This method is commonly used to purify small amounts of enzymes from cell extracts for analysis of enzymatic activity or for studies of associated proteins. bead with covalently attached substrate


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Cell_Biology_Alberts. Figure 8–9 Three types of matrices used for chromatography. (A) In ion-exchange chromatography, the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged. Analogous matrices based on agarose or other polymers are also frequently used. The strength of the association between the dissolved molecules and the ion-exchange matrix depends on both the ionic strength and the pH of the solution that is passing down the column, which may therefore be varied systematically (as in Figure 8–10) to achieve an effective separation. (B) In gel-filtration chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the


Cell_Biology_Alberts

Cell_Biology_Alberts. chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the column than larger molecules that cannot penetrate. Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various molecular mass, from less than 500 daltons to more than 5 × 106 daltons.


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Cell_Biology_Alberts. (C) Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody–antigen complex with concentrated salt solutions or solutions of high or low pH. High degrees of purification can be achieved in a single pass through an affinity column. pool these fractions and apply them to the next column below fraction number pool these fractions and apply them to the next column below pool these fractions, which now contain the highly purifed protein Genetically Engineered Tags Provide an Easy Way to Purify Proteins


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Cell_Biology_Alberts. Using the recombinant DNA methods discussed in subsequent sections, any gene can be modified to produce its protein with a special recognition tag attached to it, so as to make subsequent purification of the protein simple and rapid. Often the recognition tag is itself an antigenic determinant, or epitope, which can be recognized by a highly specific antibody. The antibody can then be used to purify the protein by affinity chromatography or immunoprecipitation (Figure 8–11). Other types of tags are specifically designed for protein purification. For example, a repeated sequence of the amino acid histidine binds to certain metal ions, including nickel and copper. If genetic engineering techniques are used to attach a short string of histidines to one end of a protein, the slightly modified protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used to purify the modified protein from a complex molecular


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Cell_Biology_Alberts. protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used to purify the modified protein from a complex molecular mixture.


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Cell_Biology_Alberts. Figure 8–10 Protein purification by chromatography. Typical results obtained when three different chromatographic steps are used in succession to purify a protein. In this example, a homogenate of cells was first fractionated by allowing it to percolate through an ion-exchange resin packed into a column (A). The column was washed to remove all unbound contaminants, and the bound proteins were then eluted by pouring a solution containing a gradually increasing concentration of salt onto the top of the column. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to their affinity for the resin—those proteins binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The


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Cell_Biology_Alberts. binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The fractions with activity were pooled and then applied to a gel-filtration column (B). The elution position of the still-impure protein was again determined by its enzymatic activity, and the active fractions were pooled and purified to homogeneity on an affinity column (C) that contained an immobilized substrate of the enzyme.


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Cell_Biology_Alberts. gene for protein of interest rapid purifcation of tagged protein and any associated proteins Figure 8–11 epitope tagging for the purification of proteins. Using standard genetic engineering techniques, a short peptide tag can be added to a protein of interest. If the tag is itself an antigenic determinant, or epitope, it can be targeted by an appropriate antibody, which can be used to purify the protein by immunoprecipitation or affinity chromatography. In other cases, an entire protein is used as the recognition tag. When cells are engineered to synthesize the small enzyme glutathione S-transferase (GST) attached to a protein of interest, the resulting fusion protein can be purified from the other contents of the cell with an affinity column containing glutathione, a substrate molecule that binds specifically and tightly to GST.


Cell_Biology_Alberts

Cell_Biology_Alberts. As a further refinement of purification methods using recognition tags, an amino acid sequence that forms a cleavage site for a highly specific proteolytic enzyme can be engineered between the protein of choice and the recognition tag. Because the amino acid sequences at the cleavage site are very rarely found by chance in proteins, the tag can later be cleaved off without destroying the purified protein.


Cell_Biology_Alberts

Cell_Biology_Alberts. This type of specific cleavage is used in an especially powerful purification methodology known as tandem affinity purification tagging (TAP-tagging). Here, one end of a protein is engineered to contain two recognition tags that are separated by a protease cleavage site. The tag on the very end of the construct is chosen to bind irreversibly to an affinity column, allowing the column to be washed extensively to remove all contaminating proteins. Protease cleavage then releases the protein, which is then further purified using the second tag. Because this two-step strategy provides an especially high degree of protein purification with relatively little effort, it is used extensively in cell biology. Thus, for example, a set of approximately 6000 yeast strains, each with a different gene fused to DNA that encodes a TAP-tag, has been constructed to allow any yeast protein to be rapidly purified. Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions


Cell_Biology_Alberts

Cell_Biology_Alberts. Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions Purified cell-free systems provide a means of studying biological processes free from all of the complex side reactions that occur in a living cell. To make this possible, cell homogenates are fractionated with the aim of purifying each of the individual macromolecules that are needed to catalyze a biological process of interest. For example, the experiments to decipher the mechanisms of protein synthesis began with a cell homogenate that could translate RNA molecules to produce proteins. Fractionation of this homogenate, step by step, produced in turn the ribosomes, tRNAs, and various enzymes that together constitute the protein-synthetic machinery. Once individual pure components were available, each could be added or withheld separately to define its exact role in the overall process.


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Cell_Biology_Alberts. A major goal for cell biologists is the reconstitution of every biological process in a purified cell-free system. Only in this way can we define all of the components needed for the process and control their concentrations, which is required to work out their precise mechanism of action. Although much remains to be done, a great deal of what we know today about the molecular biology of the cell has been discovered by studies in such cell-free systems. They have been used, for example, to decipher the molecular details of DNA replication and DNA transcription, RNA splicing, protein translation, muscle contraction, and particle transport along microtubules, and many other processes that occur in cells.


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Cell_Biology_Alberts. Populations of cells can be analyzed biochemically by disrupting them and fractionating their contents, allowing functional cell-free systems to be developed. Highly purified cell-free systems are needed for determining the molecular details of complex cell processes, and the development of such systems requires extensive purification of all the proteins and other components involved. The proteins in soluble cell extracts can be purified by column chromatography; depending on the type of column matrix, biologically active proteins can be separated on the basis of their molecular weight, hydrophobicity, charge characteristics, or affinity for other molecules. In a typical purification, the sample is passed through several different columns in turn, with the enriched fractions obtained from one column being applied to the next. Recombinant DNA techniques (described later) allow special recognition tags to be attached to proteins, thereby greatly simplifying their purification.


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Cell_Biology_Alberts. Proteins perform most cellular processes: they catalyze metabolic reactions, use nucleotide hydrolysis to do mechanical work, and serve as the major structural elements of the cell. The great variety of protein structures and functions has stimulated the development of a multitude of techniques to study them.


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Cell_Biology_Alberts. Proteins usually possess a net positive or negative charge, depending on the mixture of charged amino acids they contain. An electric field applied to a solution containing a protein molecule causes the protein to migrate at a rate that depends on its net charge and on its size and shape. The most popular application of this property is SDS polyacrylamide-gel electrophoresis (SDS-PAGE). It uses a highly cross-linked gel of polyacrylamide as the inert matrix through which the proteins migrate. The gel is prepared by polymerization of monomers; the pore size of the gel can be adjusted so that it is small enough to retard the migration of the protein molecules of interest. The proteins are dissolved in a solution that includes a powerful negatively charged detergent, sodium dodecyl sulfate, or SDS (Figure 8–12). Because this detergent binds to hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains, the individual protein molecules are


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Cell_Biology_Alberts. or SDS (Figure 8–12). Because this detergent binds to hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains, the individual protein molecules are released from their associations with other proteins or lipid molecules and rendered freely soluble in the detergent solution. In addition, a reducing agent such as β-mercaptoethanol (see Figure 8–12) is usually added to break any S–S linkages in the proteins, so that all of the constituent polypeptides in multisubunit proteins can be analyzed separately.


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Cell_Biology_Alberts. What happens when a mixture of SDS-solubilized proteins is run through a slab of polyacrylamide gel? Each protein molecule binds large numbers of the negatively charged detergent molecules, which mask the protein’s intrinsic charge and cause it to migrate toward the positive electrode when a voltage is applied. Proteins of the same size tend to move through the gel with similar speeds because (1) their native structure is completely unfolded by the SDS, so that their shapes are the same, and (2) they bind the same amount of SDS and therefore have the same amount of negative charge. Larger proteins, with more charge, are subjected to larger electrical forces but also to a larger drag. In free solution, the two effects would cancel out, but, in the mesh of the polyacrylamide gel, which acts as a molecular sieve, large proteins are retarded much more than small ones. As a result, a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of


Cell_Biology_Alberts

Cell_Biology_Alberts. as a molecular sieve, large proteins are retarded much more than small ones. As a result, a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of molecular weight (Figure 8–13). The major proteins are readily detected by staining the proteins in the gel with a dye such as Coomassie blue. Even minor proteins are seen in gels treated with a silver stain, so that as little as 10 ng of protein can be detected in a band. For some purposes, specific proteins can also be labeled with a radioactive isotope tag; exposure of the gel to film results in an autoradiograph on which the labeled proteins are visible (see Figure 8–16).


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Cell_Biology_Alberts. SDS-PAGE is widely used because it can separate all types of proteins, including those that are normally insoluble in water—such as the many proteins in membranes. And because the method separates polypeptides by size, it provides information about the molecular weight and the subunit composition of proteins. Figure 8–14 presents a photograph of a gel that has been used to analyze each of the successive stages in the purification of a protein. Because different proteins can have similar sizes, shapes, masses, and overall charges, most separation techniques such as SDS polyacrylamide-gel electrophoresis or ion-exchange chromatography cannot typically separate all the proteins Figure 8–12 The detergent sodium dodecyl sulfate (SDS) and the reducing agent β-mercaptoethanol. These two chemicals are used to solubilize proteins for SDS polyacrylamide-gel electrophoresis. The SDS is shown here in its ionized form.


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Cell_Biology_Alberts. sample loaded onto gel protein with two by pipette subunits, A and B, joined by a disulfde single-subunit bridge protein


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Cell_Biology_Alberts. Figure 8–13 SDS polyacrylamide-gel electrophoresis (SDS-PAGe). (A) An electrophoresis apparatus. (B) Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS–protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as A well as the subunit composition of a protein. If the protein contains a large amount of carbohydrate, however, it will move anomalously on the gel and its apparent molecular weight estimated by SDS-PAGE will be misleading. Other modifications, such as phosphorylation, can also cause small changes in a protein’s migration in the gel. slab of polyacrylamide gel in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation


Cell_Biology_Alberts

Cell_Biology_Alberts. changes in a protein’s migration in the gel. slab of polyacrylamide gel in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation procedures, can resolve up to 2000 proteins in the form of a two-dimensional protein map.


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Cell_Biology_Alberts. In the first step, the proteins are separated by their intrinsic charges. The sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, together with β-mercaptoethanol and the denaturing reagent urea. This solution solubilizes, denatures, and dissociates all the polypeptide chains but leaves their intrinsic charge unchanged. The polypeptide chains are then separated in a pH gradient by a procedure called isoelectric focusing, which takes advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution. Every protein has a characteristic isoelectric point, the pH at which the protein has no net charge and therefore does not migrate in an electric field. In isoelectric focusing, proteins are separated electrophoretically in a narrow tube of polyacrylamide gel in which a gradient of pH is established by a mixture of special buffers. Each protein moves to a position in the gradient that


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Cell_Biology_Alberts. Figure 8–14 Analysis of protein samples by SDS polyacrylamide-gel electrophoresis. The photograph shows a Coomassie-stained gel that has been used to detect the proteins present at successive stages in the purification of an enzyme. The leftmost lane (lane 1) contains the complex mixture of proteins in the starting cell extract, and each succeeding lane analyzes the proteins obtained after a chromatographic fractionation of the protein sample analyzed in the previous lane (see Figure 8–10). The same total amount of protein (10 μg) was loaded onto the gel at the top of each lane. Individual proteins normally appear as sharp, dye-stained bands; a band broadens, however, when it contains a large amount of protein. (From T. Formosa and B.M. Alberts, J. Biol. Chem. 261:6107–6118, 1986.) mass(daltons)100,00040,00015,000 Figure 8–15 Separation of protein molecules by isoelectric focusing. at low pH,


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Cell_Biology_Alberts. T. Formosa and B.M. Alberts, J. Biol. Chem. 261:6107–6118, 1986.) mass(daltons)100,00040,00015,000 Figure 8–15 Separation of protein molecules by isoelectric focusing. at low pH, At low pH (high H+ concentration), the charged the protein carboxylic acid groups of proteins tend to at the isoelectric example, –NH3+), giving most proteins point, the protein a net positive charge. At high pH, the and therefore no carboxylic acid groups are negatively at high pH, charged (–COO–) and the basic groups the electric feld; tend to be uncharged (for example, –NH2), for the protein giving most proteins a net negative charge. shown, thethe protein


Cell_Biology_Alberts

Cell_Biology_Alberts. shown, thethe protein At its isoelectric pH, a protein has no net is 6.5 charge since the positive and negative charges balance. Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isoelectric corresponds to its isoelectric point and remains there (Figure 8–15). This is the pH, as shown. first dimension of two-dimensional polyacrylamide-gel electrophoresis.


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Cell_Biology_Alberts. In the second step, the narrow tube gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size, as in one-dimensional SDS-PAGE: the original tube gel is soaked in SDS and then placed along the top edge of an SDS polyacrylamide-gel slab, through which each polypeptide chain migrates to form a discrete spot. This is the second dimension of two-dimensional polyacrylamide-gel electrophoresis. The only proteins left unresolved are those that have both identical sizes and identical isoelectric points, a relatively rare situation. Even trace amounts of each polypeptide chain can be detected on the gel by various staining procedures—or by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8–16). The technique has such great resolving power that it can distinguish between two proteins


Cell_Biology_Alberts

Cell_Biology_Alberts. by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8–16). The technique has such great resolving power that it can distinguish between two proteins that differ in only a single charged amino acid, or a single negatively charged phosphorylation site.


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Cell_Biology_Alberts. Specific Proteins Can Be Detected by Blotting with Antibodies A specific protein can be identified after its fractionation on a polyacrylamide gel by exposing all the proteins present on the gel to a specific antibody that has been labeled with a radioactive isotope or a fluorescent dye. This procedure is normally carried out after transferring all of the separated proteins present in the 100 50 25SDS migration (mol. mass in kilodaltons) Figure 8–16 Two-dimensional polyacrylamide-gel electrophoresis.


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Cell_Biology_Alberts. All the proteins in an E. coli bacterial cell are separated in this gel, in which each spot corresponds to a different polypeptide chain. The proteins were first separated on the basis of their isoelectric points by isoelectric focusing in the horizontal dimension. They were then further fractionated according to their molecular mass by electrophoresis from top to bottom in the presence of SDS. Note that different proteins are present in very different amounts. The bacteria were fed with a mixture of radioisotope-labeled amino acids so that all of their proteins were radioactive and could be detected by autoradiography. (Courtesy of Patrick O’Farrell.) gel onto a sheet of nitrocellulose paper or nylon membrane. Placing the membrane over the gel and driving the proteins out of the gel with a strong electric current transfers the protein onto the membrane. The membrane is then soaked in a solution of labeled antibody to reveal the protein of interest. This method of detecting proteins