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4 Genes and Cells The Fundamental Biological Unit of All Living Organisms Is the Cell To a surprising degree, all cells are similar in design and function, whether in human beings, in plants, or as simple single-celled organisms such as bacteria. One major difference, however, is the presence or absence of a distinct compart- ment, the nucleus, for the genome. Cells with a nucleus, called eukaryotes, are found in advanced single-celled organisms and multicellular organisms. Those without nuclei, called proka~yotes are the simplest single~elled organisms, the bacteria. The importance of the cell as a biological unit is made clear when we consider the life cycle of advanced multicellular organisms such as human beings. The cycle begins with the fusion of egg and sperm, themselves single cells of spe- cialized types, to form the one-celled embryo. At the earliest stage of our life cycle, therefore, we exist as a single cell. This cell divides into two cells, each of those into two more, and so on, to give rise to the adult organism, which may contain as many as 1 million billion cells (see Chapter 5~. Every one of these cells is autonomous in some functions and dependent or interdependent in others. The entire developmental process is regulated by the myriad interactions within and among individual cells. These interactions regulate the capacity of cells to multiply, to differentiate into the hundreds of different cell types that make up our bodies, and to organize themselves into tissues, organs, and finally the human body itself, according to a specific, well-defined architectural plan. This cycle of activity characterizes normal healthy individuals, but the pro- cess can go awry, leading to abno~n~al states characterized by disease in humans, animals, and plants. Since both normal and abnormal progression of the life cycle is governed in a fundamental way by cells, it is logical that to understand and 77

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78 OPPORTUNITIES IN BIOLOGY exert control over these phenomena and, in a real sense, over our own lives and biological environment, it is necessary to learn all we can about the cell and how it conducts its activities. RESEARCH STRATEGIES Since all organisms are related through evolution, we can use information from simple organisms to determine protein and gene functions in higher and more complicated organisms. Furthermore, new technologies allow us to test these inferences by expressing the genes of more complicated species, such as humans, in the cells of simple organisms such as yeast or bacteria. The identification of the genes and the proteins involved in various cellular functions is only part of the story. A deep understanding of the roles played by individual proteins requires detailed characterization of their mechanisms of action. Ultimately this requires knowledge of the three-dimensional structure of each protein and of the rates of its reaction with other molecules. This characteri- zation requires the development of test-tube (in vitro) assays of function. Genet- ics can contribute to this effort by providing components from mutant cells that can be used to identify essential components and to test the molecular roles inferred from the physiology of live cells. Likewise, components identified as part of in vitro mechanisms can be used to isolate the corresponding genes and produce defective mutants to test in the live cell the functions inferred in vitro. Many Cellular Functions Are Carried Out by Macromolecular Assemblies That Can Be Isolated and Reconstitutedirom Their Constituent Molecules Some biological functions such as the fermentation of sugar to alcohol are carried out by individual proteins, alone or in a series of catalytic reactions, but many others including photosynthesis and the beating of sperm tails require complex assemblies consisting of many different types of proteins and other molecules such as lipids and carbohydrates. These subcellular molecular ma- chines include the organelles that can be seen by light microscopy as well as many smaller structures. Perhaps the most successful research strategy in cell biology for the past 30 years has been to purify these molecular machines so that their functions can be studied outside the living cell. Fortunately, the organelles and many smaller macromolecular complexes are so well constructed that they emerge from the trauma of separation still able to perform complex, highly integrated functions such as contraction, transport of molecules across membranes, photosynthesis, the production of adenosine triphosphate (ATP), protein synthesis, and gene tran- scription. This strategy has yielded a biochemical inventory and, in addition, a rather detailed functional characterization of each organelle showing that func

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GENES AND CELLS 79 tional specialization and division of biochemical and biophysical labor prevail at the subcellular level. At the same time, studies of reconstituted cell fractions have demonstrated that most macromolecular machines are formed by the self-assembly of their component molecules. More recently, this approach has been used to demon- strate that cell organelles can interact in vitro to reconstitute a specific intracellu- lar process. Whole organelles such as the nucleus can now be reversibly disas- sembled and reassembled in the test tube. A striking feature of the current state of research in molecular and cell biology is the remarkable degree to which the effectiveness of the reductionist strategy has been confused by experiments. In Each Field of Cell Biology, Particularly Favorable Cells Have Been Identif edfor Experimental Work To a great extent, progress in molecular and cellular biology depends on finding cell types that are suitable for experiments. Fortunately, because of the parsimony of nature, mechanisms for determining the most important cellular processes are shared by cells all along the phylogenetic tree. For example, the force-producing protein myosin from muscle can bind functionally to actin fila- ments from all eukaryotic organisms. Similarly, protein synthesis can be recon- stituted in vitro from different components from plant and animal cells. Fruit flies, sea urchins, and chickens have all been excellent sources of model systems for analyzing cell behavior during embryonic development. Amphibian eggs, because of their large size, are ideal for the production of foreign proteins by microinjection of messenger RNAs (mRNAs). Highly motile amoebas that can be grown in large quantities have yielded much of the basic information about cytoplasmic contractile proteins. Chlamydomonas, an alga with two flagella and well-characterized genetics, has provided much of what we know about the molecular biology of flagella and cilia. The occurrence in yeast of three specific cell types, each of which plays a distinctive role in the cell's life cycle, makes the organism suitable for investiga- tions in several important areas of cell biology, including genetic programming for cell differentiation. Yeasts are also especially valuable for combined bio- chemical and genetic studies of control of the cell cycle, exocytosis (secretion), endocytosis (uptake), and biogenesis of such cell organelles as mitochondria. THE NUCLEUS The Nucleus Is Both the Warehouse and the Factoryfor Most of the Cell's Genetic Material and Activity The nucleus has three main functions: the storage, replication, and expres- sion of the genes. The nucleus in most human cells is a sphere roughly 10

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80 OPPORTUNITIES IN BIOLOGY micrometers in diameter that contains the almost 2 meters of DNA that makes up the human genome. The DNA contains the code for all cellular proteins and RNAs, as illustrated in the pathway represented by the "central dogma": DNA ~ RNA ~ protein. This information is processed with exquisite precision. In most cells only a tiny fraction of the total number of genes are actually expressed at any given time (or ever). Furthermore, the patterns of gene expression change in precisely pro- grammed ways during development. Thus, cells in kidney and brain express independent repertoires of genes even though they originate from a single fertil- ized ovum and have identical copies of the genetic material. The cells of a developing embryo act like tiny computers that accumulate and remember infor- mation concerning their past and present locations relative to other cells and express genes appropriate to this information. The study of the control of gene expression is an important focus of modern cell biology research. How do the cells of complex multicellular organisms turn on and off their genes? And how is the position of a cell "read out" by the cell to control specific genes? These are difficult but central questions to cell biology that are being actively pursued in many laboratories. DNA is complexed with proteins to form chromosomes. All human body cells (somatic cells) contain 46 chromosomes. The germ cells (sperm in the male and eggs in the female) that contribute to the embryos of the next generation contain only half that number 23. In addition, the nucleus contains an abun- dance of proteins and RNAs, representing various structural elements and the enzymes and products of replication, transcription, and RNA processing. How all of these components are organized within the nucleus is unknown, and research on the three~imensional structure of chromosomes and other nuclear components is needed to better understand how genes function and are controlled. Nuclear Envelope All Molecular Traffic Between the Nucleus and Cytoplasm Is by Way of the Nuclear Envelope Replication of chromosomes and synthesis of most mRNA molecules is restricted to the nucleus, whereas translation of messenger RNA into protein occurs only in the cytoplasm. Separation of chromosomes from the cytoplasmic space is thought to ensure proper regulation of nucleic acid and protein synthesis in the cell. Except at the time of mitosis, when the nuclear envelope breaks down, all molecular traffic between the nucleus and cytoplasm is by way of this enve- lope, which consists of two parallel membranes joined at regions called nuclear pores.

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GENES AI4D CELLS 81 Nuclear pores consist of a precise geometrical arrangement of structural elements. The transport of large molecules across the pores and the direction in which individual molecules are transported (cytoplasm to nucleus or vice versa) seems to be selective. Selective proteins are actively moved from their site of synthesis in the cytoplasm into the nucleus by means of an active transport system in the nuclear pores. Selective RNAs are pumped out of the nucleus through the same pores. For the transported proteins, the specificity is known to reside in a short sequence of their amino acids that seems to be recognized by a component of the nuclear pore. A number of pork complex proteins have been identified. However, the organization of these proteins in the pore complex and their role in regulating traffic in and out of the nucleus are largely unknown. Future analyses of the structure, molecular composition of the pore complex, and the genes encoding its proteins will undoubtedly reveal how this gatekeeper of the nucleus works. Attached to the inner surface of the nuclear envelope with its pores and pore complexes is a fibrous network known as the nuclear lamina. The major proteins of the lamina, called lamins, have been identified and shown to be related to the intermediate filaments found in the cytoplasm. (See the section on cytoskeleton in this chapter.) The nuclear envelope seems to be a major site for anchoring chromosomes and may also facilitate the packing of DNA in the nucleus. The reversible assembly of the lamina may help control the breakdown and reforma- tion of the nuclear envelope during mitosis. Chromosomes Chromosomes Are the Structural Units That Contain the DNA Each chromosome consists of one extraordinarily long DNA molecule com- plexed with a multitude of proteins. An orderly condensation of long DNA molecules into much smaller chromosomes is mediated by nuclear proteins. The first level of folding involves a set of five proteins called histones. DNA interacts with histories to form a regular beadlike structure, the nucleosome. Approxi- mately 160 nucleotide pairs of DNA are wrapped around a core formed from two copies each of four histone molecules (the histone octamer3. This basic structure is repeated over and over to give a beads-on-a-string appearance when chromo- some fibers are viewed in the electron microscope. DNA complexed with histories is generally referred to as chromatin. Chro- matin fibers consisting of nucleosome beads are folded into still more complex structures known as superbeads or supercoils, depending upon one's view of their organization. For technical reasons this higher-order folding of chromatin has been hard to study, and we are only beginning to understand how chromatin folding may be related to important processes such as the turning on and off of genes.

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82 OPPORTUNITIES IN BIOLOGY Beyond superbeads or supercoils, one major level of chromosome folding can be recognized, the so-called loops. Loops have been studied in the lampbrush chromosomes of oocytes for more than 100 years; the giant lampbrush chromo- somes consist of several hundred individually recognizable loops of chromatin extending laterally from the main axis. These lampbrush loops are regions of unusually intense RNA synthesis that provide a unique opportunity to visualize active gene transcription. When RNA synthesis is completed, the loops contract back into the main axis of the chromosomes. The loops are thought to retain their individuality even during mitosis, the time of maximal chromosome condensa- tion. Loops of chromatin similar to those of the lampbrush chromosomes can be seen when normal mitotic chromosomes are chemically treated to loosen their structure. We know very little about the composition (or intranuclear location) of the macromolecular complexes that anchor the DNA into loop-domains inside nuclei. Fortunately, however, the genome does not remain in the dispersed state charac- teristic of physiological activity during the entire cell cycle. At cell division (mitosis), the nuclear envelope disassembles and the genome condenses into discrete chromosomes. These mitotic chromosomes provide an opportunity to study the organization of the DNA loop-domains in the absence of the many soluble components that participate in transcription and replication. Methods are currently being developed to identify proteins that regulate mitotic chromosome architecture. Recently one putative component of the loop-domain anchor com- plex was identified as the enzyme DNA topoisomerase II, which had previously been identified as able to knot and unknot DNA molecules in vitro. Genetic analysis shows that this activity is required at mitosis if the two sets of intertwined DNA molecules are to be successfully partitioned to daughter cells. Future studies should identify other components of the anchor complex and eventually enable us to determine what role they may play in the regulation of a gene's activity. The domain idea has matured sufficiently during the past decade that the genome can now be conceived of as being constrained into loops whose average size is roughly 100,000 base pairs. In theory, the loop-domain model permits the coordinate control of complex arrays of genes, since regions that may be very distant in the DNA sequence may actually be physically adjacent (at the base of a loop) in the nucleus. The nucleolus is an example of the clustering of dispersed DNA regions into a single-functional domain where the genes (often found on different chromosomes) encoding ribosomal RNAs associate physically and are expressed together. It is a striking coincidence that DNA replicates in multiple independent blocks, which again are about 100,000 base pairs long. Thus, loop-domains (or clusters of loop-domains) may constitute the control units for both transcription and replication. This model predicts the existence of a new set of nuclear components that control DNA function the structural components that anchor the loop-domains; at present, however, these ideas have yet to be demonstrated.

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GENES AND CF~ Centromeres and Kinetochores Play a Key Role in the Migration of Chromosomes During Mitosis 83 During the separation of chromosomes at mitosis, microtubules of the mitotic spindle attach to a specific site on each chromosome known as the centromere or kinetochore. (As now used, centromere refers to the DNA sequences at this site and kinetochore to a rather complex structure of unknown composition visible by electron microscopy.) The centromere is fundamentally important because move- ment of the chromosome at mitosis and meiosis into daughter cells depends on this region; chromosomes without a centromere fail to move normally and are eventually lost from one of the daughter cells. Centromeric DNA has been cloned and sequenced from the yeast Saccharomyces. Remarkably, the DNA region nec- essary for accurate segregation of a chromosome is no more than a few hundred base pairs long in this organism. The centromeres of higher e~caryotes are larger and their characterization will be difficult, but should prove of great interest for comparison with the presumably simpler condition in yeast. The use of antibod- ies produced by patients with an autoimmune disease has made it possible to identify and clone the DNA sequence for a centromeric protein. Characterization of this human protein and others that mediate association of the centromere with the spindle microtubules should give insight into critical questions of chromo- some movement during mitosis and meiosis. Telomeres Maintain the Structural Integrity of Chromosomes The ends of eukaryotic chromosomes, the telomeres, are special in several ways. Telomeres stabilize chromosomes and prevent their fusion with other broken or natural ends. In addition, their structure allows replication without loss of DNA. The ends represent a vanishingly small amount of the total DNA in a typical chromosome. For this reason telomeric sequences were first recognized in certain ciliates, which have thousands of extremely small chromosomes and hence thousands of telomeres. Telomeres have subsequently been identified in yeast and several other organisms, including humans. In all cases they consist of hundreds of nucleotides of simple repeated DNA (such as CCCCAA and CCCCAAAA repeats) associated with unique proteins. Telomere sequences are added to the ends of chromosomes at the time of chromosome replication by a special enzyme or enzymes without the need for a DNA template, and in this way they form a protective cap at each end of a chromosome. Artificial Chromosomes Are Valuable Research Tools The identification of centromeres and telomeres as well as sequences that initiate DNA replication now permits the synthesis of artificial "minichromo- somes" by genetic engineering techniques. Such minichromosomes have already been introduced into yeast cells, where they function normally during both mito

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84 OPPORTUNITIES IN BIOLOGY sis and meiosis. Work on artificial chromosomes in yeast will undoubtedly lead to increased knowledge about chromosome structure and mechanics; eventually similar studies will be possible in higher eukaryotes, simplifying the introduction of specific genes or gene combinations into experimental organisms. One impor- tant use for the artificial yeast chromosomes is in the cloning of very large fragments of DNA (as many as a million nucleotide pairs). The Nucleolus The Nucleolus Is the Site in the Nucleus for the Transcription of Ribosomal RNA The nucleolus is the major structural differentiation seen in nondividing nuclei. It is formed from a specific chromosomal locus, the nucleolar organizer, which contains the genes coding for ribosomal RNA (rRNA). When rRNA is synthesized, it first accumulates in the nucleolus in association with a large number of ribosomal proteins. Eventually the rRNA and proteins are transported to the cytoplasm, where they constitute the mature ribosomes. Although the major features of rRNA synthesis and its relation to the nucleolus were worked out more Can 15 years ago, the nucleolus continues to tee of interest es a model for RNA transcription and processing. In particular, we are just beginning to learn about the nbosomal proteins and the ways they interact with rRNA to form the ribosomes. Ribosomes themselves are crucially unportant to cell function, since they are the machines Hat catalyze all protein synthesis. GENES AND GENE ACTION The Primary Questions That Have Been Askedfor the Past 100 Years by Geneticists Are Still Inspiring Research Innovation Geneticists have always wanted to know how traits are passed from one generation to the next. We now seek to answer these questions at higher levels of resolution. For example, we can ask, What is the structure of genes? How do they replicate? How are they organized on chromosomes? How do they mutate, recombine, and repair themselves? What controls the timing of gene expression and repression? What mechanisms control tissue-specific and cell type-specific gene expression? The answers that we anticipate are at the level of nucleotide sequences, the three-dimensional structure of chromatin, the mechanism of action of enzyme complexes and specific DNA binding proteins. As answers to these questions emerge, we can use the resulting picture of how genes act to ask even more sophisticated questions about their products and functions.

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GENES AND CFY15 Genetic Analysis The Combination of Classical Genetics and Biochemustry [las Resulted in an Explosion of Genetic Understanding 85 The field of genetics has made dramatic advances within the past 25 years largely because biologists learned that genes are made out of DNA. In classical genetics, the arrangement of genes on the chromosomes was inferred by analysis of crosses between organisms. In modern genetics the order of genes can in principle be determined directly by sequencing the DNA molecules. The rapidity and certainty with which genes can be sequenced by biochemical procedures, even in organisms having no convenient sexual cycle or, as in humans, having a long cycle, have come to give the illusion that classical genetics has been sub- sumed by biochemistry. The most elegant insights, however, have emerged in the collaboration between classical genetics and biochemistry, which has created the field of molecular genetics. In this collaboration, the goals of genetic research have not changed. Geneticists still seek to learn the set of instructions that specify the architectural plans encoded in the DNA for building a functional organism. Mutations in Essential Metabolic Pathways [rave Contributed to Our Understanding of Both Genetics and Biochemistry Classically, genetic analysis starts with mutations. These are alterations in a single gene that result in changes in an organism's appearance or biochemistry (phenotype). For example, normal yeast cells can grow on a simple medium with no amino acid supplements. Mutations can be found that lead to the requirement that an amino acid, say histidine, which the cells would normally produce itself, be supplied in the growth medium. Mutations of this type are extremely useful because the cells can be maintained and genetic crosses can be made on complete medium, yet the mutation can be detected at any time by plating a sample of cells onto medium that lacks the required nutrient. Such conditional and selectable mutants have been the basis for learning the fundamentals of molecular genetics, the physical nature of the gene, and the structure of chromosomes. These mutations have also been important to our current understanding of biochemistry, The analysis of many mutants, each blocked in the same biochemical pathway, but at different steps, can lead to an understanding of how complex molecules could be formed by sequential chemical reactions. When coupled with genetic crosses, mutant analysis becomes a valuable tool for dissecting some of the most complex biological functions by correlating genotype (the nucleotide sequence) with phenotype (how the organism looks). A decade of intensive research correlated gene structure and function with bio- chemical activities of simple organisms. In some cases a single base-pair change in the DNA can be correlated with the loss of the catalytic function of an enzyme in a biochemical pathway. The metabolite required in the diet of the mutant

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86 OPPORTUNITIES IN BIOLOGY organism reveals the identity of the defective biochemical pathway. The loss of enzyme function easily explains the phenotype of the mutant organism~he failure to grow without a metabolite in the medium since the mutant cannot carry out one of the steps in the biosynthesis of the metabolize. Thus, classical genetic analysis allowed the elucidation of biochemical pathways because the essential steps in the pathways were easy to eliminate: The genes were discov- ered through mutations with a metabolite-requiring phenotype, the functions were inferred by inspection of the phenotype, and the proteins were identified by application of chemical and biochemical analysis of the mutants in comparison with the normal. The Analysis of Mutations in Other Types of Genes Requires a Different Approach Although these approaches permitted an understanding of metabolic path- ways in simple organisms, they are difficult to apply to the direct analysis of cellular structure and to questions involving the complex interactions that deter- mine protein structure and function in higher organisms. The major difficulty lies in our inability to connect genetics and biochemistry at higher levels of complex- ity. Very often the function that we wish to study is part of a subcellular structure and cannot simply be added back from the outside to correct the effect of the mutation. Furthermore, a component of the cell's architecture may be essential for viability, and then a mutation in this function would be lethal. Undaunted, geneticists still made considerable progress through the mutation analysis ap- proach by using conditional lethal mutations (for example, mutations that permit growth at one temperature, but not at another). Despite many imaginative efforts, it became clear that major cellular processes could not be analyzed simply by examining phenotypes in the old ways that had elegantly sufficed for determining basic biochemical pathways. To associate a gene with its function in one of these complex cellular processes required development and exploitation of new experimental strategies that can permit the enumeration of genes controlling the synthesis and function of the cell's architecture. The new methods enable scientists to carry out a new chain of discovery that allows them to associate the gene with a cellular function, with a gene product (usually a protein), and ultimately with a mechanism by which the gene product executes the function. Even with the latest in technologies, one still faces problems in associating genes with products and the products with essential functions related to cellular structure, progress of the cell division cycle, or other functions carried out by macromolecular assemblies. The geneticists can identify candidate genes by observing mutant properties that suggest failure in cell architecture or cell cycle. Biochemists and cell biologists, on the other hand, can find proteins (such as actin and tubulin) that are abundant in structures implicated in basic cell function. The

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GENES AND CFJ1-C 87 challenge to the molecular geneticist is to find ways to bring these lines of endeavor together so that the geneticists' genes can be associated with the bio- chemists' proteins and the cell biologists' structure and function. A Gene Isolation Experiment Usually Begins with the Construction of a Gene Library The gene library is a collection of DNA fragments carrying every gene from the organism, each recombined with a carrier DNA molecule called a vector. The vector contains genes that permit replication and transmission of the vector and any DNA joined to it when the DNA is transformed into the appropriate cloning host (usually a bacterial or yeast cell). The library is made by chopping the chro- mosome into gene-sized pieces of DNA and then joining each piece to its vector. The size of the library depends on the size of the organism's genome. Isolating a Specific Gene from All the Rest of the Recombinant Molecules Can Occur in Two General Ways The more classical route begins with mutations, which define a gene and whose properties (phenotype) indicate failure in a particular cellular function. One can readily isolate the gene as a DNA fragment by using DNA transforma- tion with gene libraries to complement a mutation. Restriction fragment length polymorphisms (RFLPs), to be discussed later, can also be used to localize genes or chromosomes. The isolated gene can be analyzed as a physical entity, after which one can find the gene product and determine, in favorable cases, something about the way in which this protein contributes to cellular function. By this route one eventually obtains all the elements: the gene, the function, and the protein product. This route works well with bacteria, yeast, and cultured cells of higher organisms if the gene desired has a strongly selectable phenotype. The second approach begins with a gene product, perhaps a protein involved in some cellular process. Isolation of the gene for that protein depends on what is already known about that protein and often on the ingenuity of the investigator. If the protein sequence is known, oligonucleotide probes can be chemically synthe- sized on the basis of information contained in the protein's amino acid sequence. These probes can be used to screen the library by hybridization. Other techniques involve isolating mRNA from the cell type of interest, transcribing it into DNA, and cloning it into a vector that is designed to direct transcription of that mRNA when it has been transformed into a bacterial host. Colonies of bacteria producing the desired protein can be identified by antibody binding (if an antibody is available) and then grown to yield quantities of the desired cloned genes. These clones can be used to screen a library of genomic DNA sequences to obtain the gene encoding the mRNA.

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GENES AND CF-~.r-s 129 Although it seemed unlikely that cyclic AMP would be the only intracellular second messenger of hormones, the next system was not identified until very recently. In this system (Figure 4-5), hormone receptors also act through a G protein to stimulate the activity of phospholipase C (PLC), which cleaves phosphati- dylinositol bisphosphate (PIP2) to form two distinct second-messenger molecules. One of these is diacylglycerol (DAG), a membrane lipid that binds to and stimulates protein kinase C. The other messenger derived from PIP2 is inositol- trisphosphate ALPS), which acts on receptors in the endoplasmic reticulum to release stored calcium ions and raise their cytoplasmic concentration. The two arms of this signaling cascade, calcium ion (Cam) and protein kinase C, act on other cellular components to produce a set of responses that includes contraction of smooth muscle (stimulated by agents such as norepinephnne), modulation of synaptic responses in the central nervous system, a variety of secretory responses, and some, but not all, proliferative responses of cells to growth factors such as PDGF. Hormone _ PtdinsP~ R ~ G?- ~ PLC ~ WAG ~ P K-C aM AT DP ~ Pi E CaM - E* Al Response FIGURE 4-5 Calcium ion as a second messenger. [Adapted from H. R. Boume and A. L. DeFranco, in The Oncogenes, R. Weinberg and M. Wigler, eds. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., in press), chapter 3]

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130 OPPORTUNITIES IN BIOLOGY The discovery of Gs led to the discovery of a whole family of G proteins involved in other kinds of signal transduction. A key feature of the G protein machine is that it allows amplification and regulation of the transduced signal by separating excitation of the receptor from activation of the effecter in both time and space. Thus, the encounter of a neuromodulator such as norepinephrine with its receptor may be short-lived. When the encounter generates a GTP-bound Gs molecule, however, the duration of activation of adenyl cyclase depends on the length of time GTP stays bound to the G protein rather than on the tenacity with which the receptor holds onto norepinephrine. In addition to G. at least five other G proteins have been discovered. They include one that mediates hormonal inhibition of adenyl cyclase, two that mediate retinal phototransduction in rods and cones, and others that mediate hormonal regulation of ion channels and production of second messengers derived from phospholipids. Despite their diversity, each of the G proteins is built on the same general plan, comprising three dissimiliar polypeptide chains, called a, if, and y. The a chains bind and hydrolize GTP and confer on each G protein its specifici- ties for interacting with particular receptors and effecters. The ,3 and ~ polypep- tides anchor the protein to the membrane. In addition, the highly conserved GTP binding sites of the a chains closely resemble similar sites in the elongation and initiation factors of ribosomal protein synthesis and in the protein products (palm) of the ras oncogenes and proto-oncogenes. Thus, the duplication and divergence of genes in evolution have modified a single molecular machine and made it useful to mediate vision, olfaction, control of cell proliferation, ribosomal protein synthesis, and cellular regulation by many hormones and neurotransmitters. The apparent parsimony of evolution encourages investigators to extrapolate information obtained from one class of G proteins to another as well as to other proteins with the conserved GTP binding site, such as p21ra&. Only a few years ago, scientists would have scoffed at the notion that studies of bacterial protein synthesis or retinal phototransduction could reveal anything useful about the cause of cancer. In addition to explaining the cellular actions of a number of important hormones and growth factors, the cascade of events elicited by these substrates has made two other general contributions to the understanding of signal transduc- tion. First, the discovery of a second system of intracellular messengers encour- ages the expectation that more such systems will soon be found. The enormous array of chemically distinct membrane phospholipids provides an almost inex- haustible source of potential precursor molecules for generating second messen- gers. Second, understanding this system has enriched our understanding of the interplay between different second messenger systems in individual cells. For example, epinephrine can stimulate the release of glucose from the liver via two different pathways using different receptors and different G proteins to generate both cyclic AMP and IP3-DAG in the same cell type. The two second-messenger

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GEAlES AND CELl~; 131 ;~ ~ : ~G~PROTEINS~AND HUMAN DISEASE Growing knowledge of G proteins Chase already contributed significantly ~to~mol~ular~lunderstanding of human disease. The pathogenic~tox~ins of vitro choJerae~ (cholera) Andes ~Bor~te//a~pertussis~:: (whooping coughs are now known to Tact as enzymes that attach ~adenosine diphosphat~ribose (AD~P-ribose) to~t~he~a polype~ptide chain~s~of~two specific G proteins, LOG and G. ~respectively. The modification of G by cholera toxin stabilizes the G l' Q protein in its~GTP-bound form and thereby increases activation of adeny cyclas~ei (AC);I~the resulting cyclic AMP rise in mucosal cellsl;of the intestine causes~the profuse secretion of salt and water responsible for the often fatal Diarrhea of cholera. In contrast,~attach~ment~by pertussis toxin of ADP-ribose~ to the a chain of G. produces a G protein that~i~is uncoupled from inte~ract~on w~h~hormone~receptors asa~rQsuit~pertuss~isl~ioxinblocks~hormonalinhibi , ~ ~ bisons Of AC. ~ In ~ othe~rl~cell types, pQ~4U"iS ~ toxin s ~ anions on GO or ~ a Gj-like HI molecule block ~ligand-stimulatdd pho~spholipid~ metabolism anal elevation of cytoplas~mk calciums (thereby inhibiting,~;for; example, release of allergic ;~ rnediators~from mast cells). ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ In the other direction the ability of these toxins toll introduce radioac J tively labeled ADP-ribose into the G protein cz chains has made them valuable tools for studying G ~proteins. ~ Indeed, the G molecule was discov- ~e~red by investigators primarily interested in the mechanisms of action of ~pertuss~istoxin~ratherthanin~hormone~action. The pivotal biological roles Of the G proteins suggest that mutations in the corresponding genes~s~houl~d produce inherited disorders of signal transduction. One of these~possible diseases has Ben identified: inheri- tance of a mutant allele of the gene that encodes the a chain of Produces An inherited human disorder called pseudohypoparathyroid~ism, type ~1 (PHP-~ 1~. The hypocalcomia, mental retardation, convulsions, and other clinical manifestations of PHP-I result from partial resistance to hormones that utilize cyclic AMP as a second messenger. Many endocrine responses mediated by cyclic AMP are only mildly affected in PUPA resistance to parathyroid hormone, the master hormone of calcium ion homeostasis, is a prominent feature of PHP-I, suggesting that the amount or activity of Gs is normally more rate-limiting for actions of this hormone than of others.

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132 OPPORTUNITIES IN BIOLOGY systems act in parallel to stimulate the breakdown of glycogen in the liver cell. In contrast, the IP3-DAG and cyclic AMP work in opposite directions in platelets. By imagining only one or Pro more second-messenger systems in addition to cyclic AMP and IP3-DAG, we could account for almost any degree of complexity in cellular responses to extracellular signals. Regulation of cell proliferation involves just such a complex interplay of transduction pathways; these pathways are just beginning to be understood. Cytoplasrruc Free Calcium Acts as a Second Messenger Three mechanisms for raising the free calcium ion concentration in cyto- plasm have been found: the voltage-gated calcium channel of the plasma mem- brane, release of calcium ions from the endoplasmic reticulum of muscle when the surface membrane is depolarized in response to a nerve impulse, and release of calcium ions from the endoplasmic reticulum in response to hormonal signals acting on plasma membrane receptors. We understand how {P3 iS produced, but not what it does to the endoplasmic reticulum to release calcium. Because of the central role of calcium ions in signaling, the mechanisms of excitation-contrac- tion coupling in muscle and that of calcium ion mobilization by ~3 in many cells needs vigorous investigation. Two processes regulated by intracellular calcium also need to be studied. One is the release of vesicles of packaged neurotransmitters, hormones, or en- zymes. Although we know that calcium entry is required, we do not understand any of the subsequent steps that, for example, cause the transmitter ACh to be released within 0.1 millisecond of the excitation of a nerve terminal. Both botulism and tetanus are bacterial diseases whose lethal consequences are due to toxins that interfere with this calcium-dependent secretion by nerve cells. An- other calcium-dependent process is cell motility, including muscle contraction. The calcium-sensitive component molecules of the contractile machinery have been characterized in some detail, but except for muscle the physiological mecha- nisms coordinating motility are poorly understood. Progress is now possible in the analysis of calcium-stimulated events with the advent of new optical methods to measure intracellular calcium ion concentra- tions. Newly synthesized calcium-indicator dyes can be injected or allowed to permeate into cells. The time course of changes within a single cell can now be monitored reliably. Computer-aided spectroscopic techniques permit making accurate maps of the changing free calcium in each pixel of a video image of the cell observed under a high-power light microscope. The technique has already been used to observe the fertilization of eggs and the stimulation of liver cells by peptide hormones.

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GENES AND CF-~-' ~ 133 Transduction Mechanisms Use a Variery of Strategies to Alter Cellular Chemistry in Response to Messenger Molecules More intracellular second messengers remain to be discovered, and the means by which they and the "established" second messenger systems integrate control of cell function are still to be elucidated. Thus, a large number of hormones and other chemical signals act on receptors whose transduction mecha- nisms are unknown. These include growth hormone, many of the lymphokines, nerve growth factor, and tumor necrosis factor. Understanding the actions of these ligands may uncover new molecular mechanisms of signal transduction. Moreover, each transduction system can alter its sensitivity to extracellular shm- uli. Determining the molecular mechanisms that regulate sensitivity represents important new opportunities in research. Oncogenes A New Class of Genes Can Transform Host Cells An oncogene (the src gene) was first identified in the transforming Rous sarcoma retrovirus. When suitably expressed, this gene alone was capable of transforming avian cells. Normal cellular homologues of oncogenes, or cancer genes, were found subsequently in virtually every organism analyzed, which suggested that transposed DNA from normal hosts had been incorporated into the retrovirus genome, under control of viral promoters, and was capable of initiating and maintaining transformation of infected cells. Subsequently, more than 25 oncogenes have been identified along with the normal cellular counterparts, the cellular proto-oncogenes. Oncogenes may be resolved into families that include elements of many of the cell-regulatory systems reviewed above: (1) the protein products of the src gene family appear to be tyrosine-specific protein kineses (and thus similar to growth-factor receptors) that are located at the plasma membrane or in cytoplasm; (2) other oncogenes are related to growth factors or to their transmembrane receptors; (3) the ras gene proteins bind and hydrolyze GTP and seem to act as signal transducers at the plasma membrane and cytoplasm; and (4) nuclear oncogenes probably act as transcription factors or DNA-binding proteins. Research opportunities include the identification and functional characteriza- tion of additional proteins encoded by oncogenes. Once defined, the pathways by which these proteins influence cell growth and those that regulate proto-onco- genes should provide insight into the cell cycle, cell proliferation, and control by pharmacological means. Research into oncogenes should help us better under- stand normal cellular proliferation, the abnormal proliferation characteristic of the cancer cell, and pathways of significance to cellular differentiation. Other re

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134 OPPORTUNITIES IN BIOLOGY search using genetically engineered molecular hybrids should provide under- standing at the molecular level of the function of each of the protein domains of these gene products responsible for transformation. GENERAL PLANT CELL BIOLOGY Plant Cells Differ from Animal Cells in Many Ways Cells of plants and animals have many features in common, such as nuclei, endoplasmic reticula, Golgi complexes, mitochondria, plasmalemmas, coated vesicles, microfilaments, and microtubules; but in some features and processes, cells of these two kingdoms are distinct from one another. For example, plant, but not animal, cells contain plastics (the generic term that includes chloroplasts and modified chloroplasts such as amyloplasts that produce and store starch), large vacuoles that serve as the lyric compartment (lysosome-lilce) and contain most of the plant cell's water; they also have relatively rigid cell walls composed of strands of cellulose embedded in amorphous carbohydrate polymers together with a small amount of protein. At higher levels of organization, plant cells are connected to one another by strands of cytoplasm (plasmodesmata), rather than by junctional elements of the type found in animal cells. The tissues and organs of plants and animals are not organized along similar functional lines. Leaves, stems, roots, and buds have no conspicuous counterparts in hearts, livers, skins, or lungs. At many fundamental levels-biosynthetic pathways and structures of pro- teins and nucleic acids-plant and animal cells are often, but not always, the same. Thus, differences, when they occur in similar processes, often reveal much about the basic nature of a process. It is often profitable to determine whether newly discovered features of plants occur in animals and vice versa. The features of plant cells and tissues that differ distinctively from those of animals each require direct investigation. For example, in plants, mitochondria perform the same respiratory functions (and perhaps some other, as yet undiscov- ered functions) as they do in animals, but they generally contain much more DNA. They also seem to use the standard genetic code, whereas the mitochondria of animals and yeast use a nonstandard one. In addition to photosynthesis, a number of phenomena are unique to plants, and some are targets of active research. For example, each plant cell contains three rather than two gene-containing compartments (the nucleus, mitochondria, and plastics), thus greatly complicating protein targeting and the integration of genome expression. Other research is focused on the plant's rigid cell walls, which are constituted in different ways at different stages of development and for various specific functions of different cell types. The composition of the cell walls is reasonably well understood, but many questions remain unanswered. Self-incompatibility, the inability of some plants to fertilize themselves, is an mportant mechanism for regulating outcrossing. The expression of plant genes 1]

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GENES AND CF! I-S 135 can be affected by symbiotic or pathogenic microorganisms through complex interactions. In this area, important advances have been made, especially in studies of symbiotic nitrogen fixation, but the total effort is still very small. Some transposable genetic elements have been characterized molecularly, but how they are integrated into and excised from the genome remains to be understood. Chloroplasts Photosynthesis Is the Biological Process That Connects Life on Earth to the Sun Through photosynthesis, light energy is converted to chemical-bond energy stored in sugar molecules. In higher plants and algae, photosynthesis is carried out in chloroplasts-organelles containing vesicles bounded by energy-transduc- ing membranes in which the chlorophyll is localized. In primitive, noncompa~t- mentalized cells-prokaryotes-these vesicles lie free in the cytoplasm. Progress in understanding photosynthesis has been intertwined with ad- vances in chemistry, photophysics, and biology. The path followed by carbon during photosynthesis from carbon dioxide to sugar is now known in detail, and most of the enzymes have been identified. The enzymes themselves are being studied, and, surprisingly, a number of them are found to be regulated (in ways yet to be understood) by light and by certain of the small molecules that are interme- diates in the biosynthetic chains of photosynthetic carbohydrate production. The regulatory mechanisms and how each enzyme interacts with other proteins and with its substrates are questions currently being addressed. Chloroplast Genes Are Being Mapped and Sequenced at a Rapid Rate The chloroplast genomes of tobacco and liverwort have been fully sequenced, and restriction maps of the chloroplast genome have been completed for at least 40 to 50 species of plants. Most gene mapping is limited to genes that were located and initially mapped and sequenced in maize, spinach, tobacco, and the green algae Chlamydomonas and Euglena. As more and more chloroplast DNA is sequenced, interest will grow in identifying the gene products of unrecognized proteins; their identification should move us toward a better understanding of photosynthesis and plastic metabolism. This DNA sequencing will also reveal features of plastic genes. At least one type of promoter sequence-resembling the proka~yotic Me-has been recognized. The existence of other kinds of control sequences remains to be established. The identification of these and other controlling elements of plastic or nuclear-cyto- plasmic origin that constitute the parts of the machinery for control of differential gene expression may well be the most interesting and outstanding problem in this research area. Its resolution is likely to illuminate the mechanisms underlying the transcriptional control of chloroplast gene expression and to reveal research approaches leading toward an understanding of intergenomic integration.

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136 Gene Expression in C~oroplasts Is Both Developmentally and Functionally Regulated OPPORTUNITIES IN BIOLOGY Like mitochondria, plastics contain genetic material, but not all of their components are products of these genes. Many proteins of the chloroplast are imported from the cytoplasm, and these are encoded in nuclear genes. For example, the larger of the two subunits of ribulose bisphosphate carboxylase/ oxygenase (rubisco) is the product of a chloroplast gene, whereas the smaller subunit is the product of a nuclear gene. How the photosynthetic apparatus of chloroplasts is produced, that is, its biogenesis, is a question fundamental to understanding the life of a plant or alga cell. A number of related questions are under active investigation: What are the special characteristics of chloroplast genes? What are the enzymes and mecha- nisms for their replication? What are the mechanisms for controlling the expres- sion of sets of developmentally regulated chloroplast genes? How is the expres- sion of these genes integrated with the expression of nuclear genes for chloroplast components? How are nuclear gene-coded, cytoplasmically synthesized proteins targeted to plastics, and what are the mechanisms for their uptake and integration into the life of the plastic? How does the machinery for chloroplast gene expression interact with the machinery of the nuclear-cytoplasmic compartment? Among the most interesting aspects of the plant eukaryotic cell is the integration of the activities of its multiple compartmentalized genomes-of nuclei, plastics, and mitochondria. The nature of the integrating mechanisms can now be investi- gated. The Origin of Plastids Is Still a Mystery One of the great puzzles of modern biology is how euk~uyotic cells origi- nated. A unique characteristic of eukaryotes is the presence of multiple compart- mentalized genomes. In plants, these include the nucleus, mitochondria, and plastics. The question of how the expression of these genomes is integrated has already been raised. An older unanswered question is, How did the multiple genomes come into existence? There are two obvious possibilities. One is that membranous compartments formed in the structural equivalent of a modern proka~yotic cell, and some genes then became sequestered in each compartment. A second possibility, favored by most available evidence, is that the major organelles characteristic of eukaryotic cells-mitochondria (which are found in the cells of all but a very few eukaryotes) and chloroplast~are the descendants of symbiotic bacteria that entered early eukaryotic cells. To account for the genetic organization of contemporary eukalyotic cells it is necessary to assume the movement of genes or gene functions from the symbiont to the host genome. Many researchers have interpreted the available evidence as suggesting multiple origins, involving different groups of bacteria, for chloroplasts and perhaps for mitochondria also. These hypotheses require further analysis.

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GENES AND CELLS 137 What evolutionary pressures led to the existence of the different information- processing systems now found in the nuclear-cytoplasmic and organelle compart- ments? Are the different systems relics of the independent evolution of two progenitor cell types that subsequently joined to become the ancestral form of the modem eukaryotic cell? Alternatively, did two or three information storage and processing systems evolve within a single cell? What we learn about the molecu- lar biology of the organelle and the nuclear-cytoplasmic systems may lead to a better understanding of the origin and evolution of eukaryotic cells as well as of the forces and mechanisms underlying the shifts of genes among genomes. Evolutionary relations among genomes are much better understood now than they were half a dozen years ago because of the accumulation of nucleic acid sequence data. However, the forces that have led to the segregation of genes in nuclei, mitochondria, and plastics are unknown. Gene-transfer methods are beginning to be used to explore these questions from the focus of the nuclear genome. Efficient organelle gene transfer (transformation) methods that would greatly aid such investigations remain to be developed. The Plant Cell Wall Research on the Extracellular Matrix (Cell Wall) of Plants Is Crucial to Understanding [low Plants Grow Lacking a skeletal structure and subjected to a fluctuating osmotic environ- ment, plant cells rely on rigid cell walls to serve as cementing substances between cells, to provide mechanical strength, and to support high internal osmotic pres- sures. For many years, it has been known that the ultimate shape and strength of such walls are largely determined by the pattern and extent of deposition of extended fibrils composed of cellulose (glucose molecules joined in p-1,4 link- age). In recent years, the concept has evolved that a second framework, com- posed of cross-linked extended rods of a hydroxyproline-rich glycoprotein called extensin, also contributes to strength and shape in some plant cell ~es. Inter- spersed within these frameworks exist a variety of ma~x polysaccharides, some having complex structures. One example is the recently discovered, highly branched rhamnogalacturonan II polymer that contains a number of variously linked sugars including a novel monosaccharide called aceric acid, which was previously unknown in nature. Progress in determining the structure of these polysaccharides has advanced considerably in recent years, in part as a result of vastly improved techniques for the analysis of complex carbohydrates. These techniques require the use of expensive instrumentation such as mass and nuclear magnetic resonance spectrometers. Increased access to such instrumentation would hasten progress in the study of extracellular matrices of both plants and animals. Understanding plant cell-wall structure is crucial for the ultimate understand- ing of how plants grow. Plants increase in size by expanding their rigid cell walls,

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138 OPPORTUNITIES IN BIOLOGY but our understanding of the mechanism by which this occurs is still limited. The process is known to be under hormonal control; wall loosening is believed to occur by processes of breakage and reformation of crucial linkages and by turnover of some wall polymers, but the specific processes remain elusive. Re- cent structural analyses have provided evidence for intra- and interchain linkages between extensin molecules through isodityrosine residues, presumably formed in the wall by a peroxidase-mediated reaction. Similar enzymes may also be responsible for the formation of cross-links between phenolic components at- tached in ester-linkage to matrix polysaccharides. Other recent studies have implicated hormonally regulated degradative enzymes involved in the turnover of some wall polysaccharides, such as xyloglucan, in the process of wall extension. On the basis of current knowledge of wall structure, one can estimate that there must be more than a hundred different enzymes required for wall assembly. Not one of these enzymes has yet been purified and characterized in detail, although a number have been detected and assayed in crude membrane prepara- tions isolated from plants. Similarly, no gene responsible for coding for these enzymes has been identified, mapped, or cloned. Exciting progress has been made recently for the cell-wall protein extensin, since a gene for this protein has recently been cloned and characterized. Coupled with previous structural infor- mation on the protein, data from the gene sequence now give the entire amino acid sequence of the protein so that we can identify sites of glycosylation, cross- linking, and possible areas of interaction of this polymer with other wall compo- nents. We now recognize that the cell wall is a dynamic structure. Not only do changes occur during normal growth and development, but the wall also responds quickly to external perturbations such as mechanical damage, water, or tempera- ture stress and upon interaction with symbionts, pathogens, or parasites. Recent work has defined some of these "wound" responses in some detail. Specific changes identified are a cessation of cellulose synthesis coupled with induction of synthesis of a related beta-glucan called callose, which seals off areas of assault; induction of lignin or suberin synthesis; and elevation of synthesis of the soluble precursor to cell-wall extensin. This latter compound in some cases seems to serve as an agglutinin for invading pathogens. Most exciting is the recent work implicating fragments of wall polysaccharides as regulatory molecules. One example is an oligogalacturonide released from the wall by invading pathogens; this oligosaccharide serves as a specific inducer of the synthesis of phytoalex- ins-plant-made antibiotics that are toxic to invading microorganisms. Other examples include small phenolic compounds that may be released from the wall and that signal interactions crucial for the establishment of plant parasites or symbiotic associations with bacteria (see Chapter 11~. Recent advances in our ability to analyze the structure of wall components indicate that the time is rapidly approaching when data from structural studies can be integrated with growth physiology studies to achieve an overall understanding

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GENES AND CFI.! C 139 of plant growth. Recent development of specific dyes and monoclonal antibodies that interact with specific linkages in the wall should lead to a much better under- standing of the localization and interaction of the various polymers in the wall. Cloning of the extensin genes will now allow a study of the regulation of various members of this family of genes; the patterns of expression of these genes should help clarify the various functions of this unusual polymer. The exciting discovery that fragments of wall components serve as regulatory signals will undoubtedly open a whole new area of study on ways plants communicate and interact with other organisms. Finally, recent advances in the biochemistry of membrane proteins may lead to breakthroughs in the identification and isolation of enzymes and their corresponding genes involved in wall synthesis. Since plant cell walls are a major sink for biomass, much of which is only poorly digestible, it is hoped that our ultimate ability to modify wall structure by modifying the expression of genes controlling wall assembly will lead to a greater understanding of just how flexible such wall structure can be, and perhaps also to the development of plants with improved agronomic or nutritional value.