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Opportunities in Biology (1989)

Chapter: 4. Genes and Cells

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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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Suggested Citation:"4. Genes and Cells." National Research Council. 1989. Opportunities in Biology. Washington, DC: The National Academies Press. doi: 10.17226/742.
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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

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

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

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.

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.

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.

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

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.

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

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

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.

88 OPPORTUNITIES IN BIOLOGY Once the gene is cloned, much can be learned from its sequence. Much more can be learned if the gene can be put back into the species of origin. In many organisms it is now possible to introduce DNA, usually by injection, that will become stably integrated into chromosomes and frequently be normally ex- pressed. It is often possible to use such transformation to show that a cloned gene will repair a mutation. Specific features of transformation vary significantly with the organism. In yeast and in the slime mold Dictyosteli~n, the introduced gene can displace the resident gene by homologous recombination (recombination that requires se- quence similarity). This feature makes it possible to analyze the new gene without interference by the preexisting one and also ensures that only one copy of the transformed gene will be added. Homologous gene replacements are not yet routinely possible in other organisms, although techniques are being actively sought. In spite of some limitations, gene transformation has proved to be a powerful research tool that has opened analysis of complex function by direct genetic approaches. The Genome The Genome Refers to the Assemblage of All of the Genes of an Organism The size of the genome (that is, the total amount of DNA) varies widely among organisms (Figure 4-1~. Even though the number of genes in different multicellular animals is probably reasonably constant (totaling perhaps tens of thousands), the total amount of DNA in their genomes varies by a factor of 100 or more. Paradoxically, the size of the genome is not directly correlated with the complexity of the organism. Thus, much of the DNA in organisms with high DNA values (such as mammals, including humans) is noncoding DNA. What is the function of this DNA? Some of it may have no function at all, although we are beginning to learn about functions of DNA segments that do not contain conven- tional genes. One intriguing class of"extra" DNA includes transposons (transposable elements). These are short pieces of DNA that have the ability to move from one place in the genome to another or from one genome to another. The transposon usually contains sequences that specify the machinery required for its own mobil- ity and perhaps one or two other genes that can affect the host. Although the transposons may appear to be innocuous passengers in the genome, they play a significant role in generating evolutionary diversity by introducing chromosomal rearrangements and changes in gene expression. Some of these transposons are the inserted DNA copies of RNA retroviruses. Another fraction of the extra DNA almost certainly plays a role in maintain- ing the higher-order structure of chromosomes and ensuring their appropriate seg- regation and disposition in the nucleus. The role of extra DNA is an aspect of genetics that is only beginning to be explored.

GENES AND CF:I~ The Physical and Chemical Characterization of Bacteriophages and of Bacterial Genomes Has Revealed a Remarkable Array of Molecular Structures 89 Bacteriophage genomes (the genomes of viruses that attack bacteria) range from single-stranded linear RNA and DNA molecules to small double-stranded linear and circular DNA molecules. By companson, the double-stranded linear or circular molecules that compose the entire genomes of various bacteria are enormous. The quest for correlations between the physical map of the genome and the genetic map obtained by recombinational studies led to the complete sequencing of bacteriophage genomes and is currently the driving force behind the attempt to completely sequence bacterial genomes. The first genome to be sequenced, that of bacteriophage (x-174, resulted in the surprising finding that some of the stretches of nucleic acids encode overlapping genes. Although much has been learned about the DNA of bacteria, less is known about the proteins that are bound to that DNA. Specific proteins have been identified that bind adjacent to and perhaps within the coding region of active genes and are necessary for specific gene transcription and function, but many such proteins are as yet uncharacterized. Existing techniques of genetic, chemi- cal, and physical analyses need to be pushed further, and other techniques will need to be invented to yield a dynamic picture of the changes in the structure of the genome during the cell cycle. Both the overall structure and the disposition of specific proteins and nucleic acids within these structures need to be determined. 200 telephone books 1llllllllllllllllllll t (1000 pages each) t11111111111111111111 111111111111111111111 11111111111111111111 1111111111111111111 111111111111111111111 111111111111111111111 111111111111111111111 111111111111111111111 111111111111111111111 Drosophila, 10 books [| Yeast, 1 book E. colt, 300 pages | Epstein Barr virus, 12 pages (longest sequence now known) FIGURE 4-1 Genome sizes. Shown are the number of telephone books needed to display the DNA sequence of various genanes, ranging from that of a simple virus to the entire human genome.

9o OPPORTUNITIES IN B. OLOGY DNA Replication The study of DNA replication promises at long last to yield information important for understanding both normal proliferative responses, such as those basic to the immune response, differentiation, tissue regeneration, and abnormal processes such as tumongenesis and carcinogenesis. The Major Components of DNA Replication Have Been Determined Although the problem of how a DNA sequence is transmitted in dividing cells was solved conceptually by Watson and Crick in 1953, the elucidation of the actual molecular basis of replication has required considerable time and effort. At many points in the history of the development of the field it appeared that replication was "solved" and that all that was left was to work out the details. For instance, in 1959, the discovery of DNA polymerase I of Escherichia cold led to 10 years of study based on the assumption that replication was catalyzed by this single enzyme. Then, in 1969, it was shown that polymerase I mutants were viable and that DNA polymerase I was, thus, not required for replication. This finding led to the search for and discovery of DNA polymerase III. Polymerase III mutants that were replication-defective identified this polymerase as the au- thentic replicative enzyme. Additional replication mutants showed that more than DNA polymerase was required. What was needed was a strategy to identify the products of the genes defined by the replication mutants. The complexity of replication forced biochemists to fast work out the number of components re- quired in prokalyotes. By using a variety of DNA viruses as model systems, biochemists have been able to identify six essential classes of proteins in addition to DNA polymerase that are required in virtually all replication systems. The Details of DNA Replication Can Now Be Investigated A current model of how the purified proteins interact to carry out movement of the replication fork along both strands of DNA simultaneously is shown in Figure 4-2. Rather than carry out a set of sequential individual reactions with one class of protein carrying out its function, dissociating from the DNA, and the next protein further processing an intermediate, the proteins form a stable complex that remains associated with the DNA during growth. Fork movement of the chain of newly synthesized DNA may be facilitated by ordered conformational changes in the proteins in the complex by the hydrolysis of bound nucleotide triphosphate molecules. Thus the replication complex resembles a machine, with proteins as the moving parts. Good evidence exists that these replication complexes are stable; despite the conceptual attractiveness of the machine model, however, we actually know very

GENES AND CFtil-£ LAGGING STRAND 91 _) _ _ · ~ ' ' ~ ~ ~ ~ ~ ~ 1 ,, ,, j, , 1 ~ I I I I I I I . I I . I I, I I I I I I I I ,,,,i,-~it~' (it I'""""""" Il~l~l ~ LEADING STRAND * ~ i.=_ | TERMINATE *a 0~ 11 lrllllllllllllllllllllllllllllllllllllll 11 ~TIIII~11IIII [~lll IlIIITTI,Tl, 1111!'I 111I''11-111 'TV . . . . . .. . .. . I ~ ~ I I I I I I I I I I I I ~ I · 1 1 1 ~ ~ I I i en' 1 1 1 ,11161111lllllll ~ FIGURE 4-2 lbe trombone model for the propagation of a replication fork of DNA. [Adapted from B. M. Alberts, Cold Spring Harbor Symp. Quant. Biol. 59:1-12 (1984)] little about how the proteins move along the DNA. In the future, many experi- mental disciplines will be brought to bear on this problem. What is the detailed architecture of the complexes? What is the driving force for the set of coordinated protein movements, and what is the inherent timing mechanism of the polymeri- zation cycles? How are error rates minimized? How do accessory proteins increase the time of association of DNA polymerase with the template? How is the size of intermediate fragments determined? Are all of the components of the machines defined or have we missed some by our search methods? The key enzyme remains DNA polymerase, for which an x-ray crystal structure is now available, allowing more precise questions about polymerization mechanisms. Sequencing of cloned polymerase genes and in vitro mutagenesis, based on the

92 OPPORTUNITIES IN BIOLOGY crystal structure, offer fundamental new insights into how these enzymes work, especially with respect to mechanisms of fidelity (ability to copy accurately) and mutagenesis. Comparisons between cellular and viral polymerases are defining differences that can be exploited for antiviral therapy by rational drug design. The original idea that replication was carried out by a single enzyme may seem naive to us now, but it would also be naive to think that DNA replication is the only process mediated by such complex protein machines. Impressive evi- dence has already accumulated that transcription, translation, RNA processing, and muscle contraction are also organized in this way. Yet, only for bacterial DNA replication is the field so advanced as to have all the components not only defined, but purified in abundance, and in a form allowing reconstitution of the entire process in the test tube. Although the current description of the replication apparatus in itself represents an enormous achievement, the most exciting era is just beginning. The basic principles that emerge from future enzymatic replica- tion studies will have a general biological significance far beyond the specific light they shed on DNA synthesis. The Regulation of Chrom~somal Replication Is Poorly Uruierstood The regulation of chromosome replication will be emphasized in research in the coming years because of its important implications for cellular and develop- mental biology. In both prokaryotic and eukaryotic cells, regulation of replication occurs when replication is initiated rather than when the DNA chain is elongated. Therefore, the first step toward understanding regulation is the identification of proteins, genes, and DNA sequences involved in initiation. Apparently only one more protein is necessary for initiation than for elongation in bacteria [although this may not be the case for the replication of simian virus 40 (SV40~. The extra protein is one that selects the sequence of nucleotides in the origin of replication, binds there, and mediates unwinding of the DNA, creating a nucleoprotein struc- ture that directs binding and further unwinding by the enzyme DNA helicase. Interestingly, only months after this simple mechanism was established for bacte- ria, an identical process was reconstituted from partially purified human proteins in the eukaryotic DNA virus SV40. This finding suggests that initiation mecha- nisms may have been conserved over long periods of evolutionary time; although the SV40 system is undoubtedly simpler in detail than euk~uyotic chromosome replication, the basic enzymology of replication may confidently be assumed to be the same in prokaryotes and eukaryotes. Since the systems for the study of initiation of replication are new, much less is known about initiation than elongation. In the future, this recent work on initiation will be extended to address the role of transcription in activating DNA replication. Are there additional proteins? What are the primary, secondary, and tertiary structures of the DNA in specific areas where replication begins? Most importantly, how is the process controlled? The prokalyotic systems will con

GENES AND CF7= 93 tinue to be important for enzymological studies because of the ease of genetic and biochemical analysis and because of their recently proven relevance for higher cells. In addition, new technological advances should permit the testing of principles learned from prokaryotes in the more complicated eukaryotic systems. A second aspect of the regulation of eukaryotic DNA synthesis needs to be understood. The eukaryotic cell apparently retains the ability to initiate new replication units throughout the S phase, but a region once replicated cannot be replicated again in the same cell cycle. This block to reinitiation is essential to ensure that each region of a chromosome is replicated only once per cell cycle. The next few years will see major efforts to try to understand the molecular basis for this aspect of replication regulation. Initial Exhorts Will Emphasize the Further Development of Eukaryotic Experimental Systems for the Stubbly of Replication Two of the systems that seem to offer the most promise for study of eukar- yotic replication are yeast and amphibian eggs. Yeast has been uniquely success- ful in contributing to our knowledge of eukaryotic DNA replication in at least two ways: (1) in the isolation of the DNA sequences essential for chromosomal replication and segregation: origins of replication, telomeres and centromeres; and (2) in the isolation of genes and mutants that have helped characterize several of the proteins involved in replication. Recently it has been found that extracts of amphibian eggs (Xenopus) initiate replication, with two unexpected features: Replication initiation requires the formation of an intact nuclear envelope around the added DNA; and synthesis is periodic, even though there is no periodic Recondensation and condensation of the chromosomes. Intensive study of the proteins that catalyze replication in these eukaryotic systems and others should lead to a complete molecular understanding of the process. Recombination Genetic Recombination Is the Rearrangement of DNA Sequences to Create New Genetic Information Genetic recombination is one of the few ways to introduce variability into the genome. Recombination is most important in the generation of diversity among individuals in a population, although it is also a mechanism for generating antibody diversity in vertebrates (see Chapter 7~. Recombination is also impor- tant in pathological processes such as cancer. Many viruses, including the cancer- causing retroviruses, rely on recombination for an essential part of their life cycle: the joining of the viral genome with the host genome. In addition, many parasites rely on recombination to alter the genes that code for components of their surface coats so as to escape the host's immune defenses. A final area of importance for

94 OPPORTUNITIES IN BIOLOGY genetic recombination centers on the use of recombination by researchers to artificially introduce gene segments into the genome of cells or whole organisms. General Homologous Recombination Is the Simplest and Most Studied Category of Recombination General homologous recombination is the exchange of DNA strands that have a certain degree of complementarily. In homologous recombination the two DNA segments need not be completely identical, but only homologous, to recom- bine. Studies of homologous recombination in bacteria have identified a key protein, RecA, that catalyzes the exchange of genetic information. Many of the essential features of homologous recombination have been reconstituted with purified RecA protein. These biochemical studies have brought researchers to the point of asking two fundamental questions. First, how does RecA locate the homologous segments of DNA and bring them together to initiate the exchange? Second, how does RecA catalyze the extension of the initial point of DNA exchange so that hundreds of thousands of nucleotides of DNA are recombined? We know that this process requires hydrolysis of the high-energy compound adenosine triphosphate. In eukaryotic systems, the stage is set for major research advances. Molecu- lar genetic techniques should expedite the identification, isolation, and characteri- zation of the genes that encode the recombination apparatus. Because of the ease of genetic manipulation, research on the lower eulcalyotes may lead the way in this effort. One hopes, however, that conservation of gene structure will permit identification of related genes in higher euka~yotes. The biochemical study of meiotic components the complex that aligns homologous chromosomes, DNA strand transferases that initiate exchange, and DNA resolvases that terminate exchange-is in its infancy, but recent progress in yeast should attract more workers to the field. In addition to understanding the basic mechanism, we need to learn more about important control features of meiotic recombination. For example, failure of homologues to separate at meiosis is the basis of trisomy diseases like Down's syndrome. A second item for further research is the means by which cells prevent recombination between repeated sequences. Such repetitive DNA is a hallmark of higher eukaryotic cells and presents a potentially devastating opportunity for a homologous recombination system. If left unchecked, recombination between repeated sequences could scramble the genome. We need to know how this is limited and whether breakdowns in this control are responsible for some human disease. A separate issue in the study of general recombination of eukaryotes con- cerns somatic cells. When recombination has been studied in living organisms (usually Drosophila and yeast), somatic recombination is less frequent than recombination in germline cells and seems to be primarily involved in DNA

GENES AND CF~ 95 repair. Nevertheless, many scientists have demonstrated the efficient recombina- tion of homologous segments of DNA when they are artificially introduced into somatic cells in culture. Although ongoing studies attempt to define the mecha- nism of this recombination, the most exciting possibility is its potential use to manipulate the genetic content of cultured cells. Research will focus on control- ling the frequency and fidelity of recombination so that investigators can intro- duce genes into cultured cells and replace existing genes with modified ones. This sequence of manipulations is readily accomplished in bacteria, yeast, and slime molds, where the technique has been of tremendous value in the genetic analysis of complex loci. Formidable problems arise in extending this technology to higher organisms because of the complexity of their genomes. However, several initial successes in targeting have opened the way to a thorough attack on the problem. Site-Specific Recombination Has Been Studiedfor the Past 20 Years and Many Systems Have Been Studied in Depth In the 1960s it became clear that efficient recombination could take place between segments of DNA that lacked sufficient homology to undergo general homologous recombination. Two features are required for such nonhomologous recombination-the presence of special DNA sites in at least one partner in the recombination process and the activity of specific enzymes (recombinases) that act at these sites. The rearrangements of the genome promoted by site-specif~c recombination are mechanically fascinating. One wants to know how small segments of the genome are identified, brought close together, and rearranged with high efficiency and fidelity. Moreover, site-specific recombination is a critical step in many important biological processes. As a result, the study of site- specific recombination has emerged as a major area of concentration of biological research that will continue to grow rapidly in the next 5 to 10 years. The molecular mechanism of these reactions has been worked out by using the molecules and mutant strains identified by genetic analysis to reconstitute the recombination event in a cell-free (in vitro) system. Site-specific recombination can be divided into broad mechanistic categories according to the degree to which both partners must have specific sites and on the amount of DNA synthesis that accompanies the reaction. In some systems, recombination uses a completely conservative mechanism in which the participating DNAs are simply broken, rearranged, and rejoined. In other systems, complete replicas of the DNA must be synthesized during the recombination event. The integration of the bacterial virus called lambda into the E. cold genome is a reaction with high target specificity and strict conservation of the parental DNA. This complete reaction has been reconsti- tuted in vitro. By combining topological investigations with biochemical and molecular genetic studies, the field is reaching the goal of elaborating plausible models for complex processes such as synapsis, the juxtaposition of DNA seg

96 . OPPORTUNITIES IN BIOLOGY meets that is a prerequisite for efficient, precise recombination. In the future, structural studies using x-ray diffraction and nuclear magnetic resonance tech- niques should be able to add atomic details to the understanding of these pro- cesses that is being deduced from molecular biological studies. In contrast to the conservative reactions (no DNA replication), study of the mechanism of the replicative site-specific recombination (which requires DNA replication) is in its infancy. The transposition of the genome of the bacterial virus mu, is the only example that has been reconstituted in vitro. Several features of the DNA sequence of donors and targets suggest close parallels between mu transposition on the one hand and other rearrangements such as the integration of retroviruses (RNA viruses that replicate by way of a DNA intermediate that is inserted into the host's chromosome) and the recombination of antibody genes. The pioneering work on mu transposition has thus opened the way for rapid progress in the biochemistry of these eukaryotic recombination events. Another important research area, control of the recombination process, has already been extensively studied in proka~yotic and lower eukaryotic systems. Here, the key event seems to be control of the synthesis of a specific recombinase. In some systems, such as the Integration of lambda and sexual differentiation of yeast, such control is elicited through a complex chain of events that couples the synthesis of the recombinase to related events in the life cycle of organisms. In higher eukaryotes, we know almost nothing about the control of recombination. We need to learn whether these events are regulated and, if so, by what mecha- nism. For the immune system, some experiments suggest that accessibility of the recombining sites rather than the availability of the recombinase may be critical. The finding that several human tumors result from an inappropriate rearrange- ment of immune elements highlights the importance of understanding the control of site-specific recombination in higher organisms. DNA Repair and Mutagenesis Organisms Correct Mistakes by DNA Repair Mechanisms It is critical for organisms to maintain and protect the fidelity of the informa- tion encoded in their DNA so they can pass this information on to their descen- dants and so they can access that information during their own lifetimes. How- ever, the integrity of the DNA itself and the information encoded therein are undergoing constant challenges from agents in the environment. Furthermore, mistakes can be introduced into the information by normal cellular metabolic events such as DNA replication and recombination. The study of mechanisms of DNA repair and mutagenesis is of direct relevance to human health, since muta- tions in DNA are responsible for both genetically inherited diseases and the somatic cell diseases known as cancer.

GENES AND CAT [-by The Molecular Mechanisms of DNA Repair and Mutagenesis Need to Be Studied in Prokaryotes, Yeasts, and Mammals 97 Around 1975 it was generally thought that DNA repair end mutagenesis were understood in bacteria and that this knowledge made it possible to approach these issues in eukaryotic organisms. However, new discoveries of a variety of com- plex independent enzymatic systems specializing in repair of different kinds of damage over the past 10 years have revealed that these processes are much more complex, even in bacteria, than was assumed in 1975. These new revelations about the complexity of bacterial systems explain the relatively modest progress in dissecting DNA repair in mammalian cells. It is important that major studies of DNA repair and mutagenesis be continued at several levels at once: studies involving at least prokaryotes, yeasts, and mammals. The main reason for lack of progress in mammalian systems has been the lack of genetic tools similar to the ones used in work on proka~yotes. Opportunities Aboundfor Studies of DNA Repair and Mutagenesis in Prokaryotes such as E. cold Recent studies show that the complexity of the repair systems demand the power of E. cold molecular genetics if we are to obtain a framework within which to understand these processes in all organisms. To begin with, the molecular mechanism by which damaged DNA is processed to give rise to mutated DNA ("error-prone repair") is not understood in any organism, and E. cold is likely to be the first organism in which this fundamental problem will be solved. The exist- ence of regulatory networks of genes induced by DNA damage was discovered in E. colt. Studies of two such gene systems that are induced by DNA damage, the SOS repair system and the adaptive response, have led to the discovery that some proteins (such as the proteins RecA and Ada) have both a regulatory and a mechanistic function in DNA repair. The complexities of the regulation of these systems need further study, as do the functions of all of the induced gene products. Detailed analysis of complex proteins such as these are needed to provide models for systems that lack sophisticated genetics. Major Opportunities Exist to Study DNA Repair in Eukaryotes Yeast can be used to explore uniquely eukaryotic aspects of DNA repair, such as the relationship of DNA repair processes to the cell cycle and the effect of chromatin on DNA repair. In yeast, many genes affecting survival or mutagenesis after DNA damage have been identified, and many of these have been cloned. However, very little is known about the biochemistry of DNA repair in yeast. Moreover, although a few yeast DNA repair genes have been shown to be induced

98 OPPORTUNITIES IN BIOLOGY by DNA damage, virtually nothing is known about the mechanism of their regulation. The study of DNA repair in mammals, especially humans, is a challenging problem of great significance, but one that needs to be approached with greater sophistication. Many studies to date have been primarily descriptive and have allowed only low-resolution inferences concerning the mechanism. Another problem has been that humans with inherited diseases caused by mutations in DNA repair, such as xeroderma pigmentosum and ataxia telangiectasia, have complex phenotypes that make analysis of their deficiencies difficult. Further- more, to date, no one has succeeded in cloning genes that complement the DNA repair deficiencies of these naturally occurring mutants. Problems unique to higher organisms that will need to be addressed include the relative DNA repair capabilities of different types of differentiated cells and the DNA repair capabili- ties of germline cells versus somatic cells. Gene Expression Genes Provide the Information Requiredfor the Formation, Function, and Reproduction of Cells and Organisms The information in the genes specifies the sequences of RNAs and proteins that have the biochemical capacity to synthesize other cellular components (sug- ars and lipids) as well as the DNA, RNA, and proteins themselves. The process that converts information stored in selected genes into RNA and proteins is called gene expression. Specific gene expression leads to the selected transcription copies of appro- priate genes at particular times in the life cycle of the cell or organism. Although we now understand some of the strategies involved, our knowledge of the control of transcription still contains enormous gaps, for both prokaryotic and eukaryotic organisms. An understanding of the regulation of gene expression lies at the heart of understanding how the genome functions to guide the development, growth, and differentiation processes that generate a complex organism from a single- celled fertilized ovum. Gene expression must be properly controlled during the life cycle of any organism. For example, hemoglobin is produced in red blood cells but not in other cells. Yet the hemoglobin gene is present in all cells of the body. Similarly in plants, proteins of the photosynthetic apparatus are expressed in specialized leaf cells but not in cells of the root. By what mechanisms are the genes in individual cells differentially expressed? At least two levels of regulation exist: large-scale activation or inactivation of groups of genes and the precise control of individual genes. Since eukaryotic development is characterized by changes in the expression of complex cohorts of genes, these cohorts may be regulated in parallel. This procedure would be

GENES AND CELLS 99 straightforward if the structure of the nucleus could in some way regulate the activity of the DNA. For example, groups of genes that show coordinate expres- sion might be packaged in discrete domains or comparunents that could be regulated at one or a few key control sites. However, whereas coordinately regulated genes in prokaryotes are frequently linked and controlled by a single promoter (nucleotide sequence to which RNA polymerase initiates transcription), such linkage is not usually found in eukaryotes. In most instances, members of coordinately controlled gene sets in eukaryotes are broadly scattered over the chromosomes, implying that transcription is controlled by trans-acting regulators, which can affect gene regulation at widely separated chromosome sites. However, large-scale gene control is found in several notable exceptions to the general picture of eukaryotic gene regulation. These include inactivation of the X chromosome in mammals and dosage compensation of the X chromosome in insects, in which the transcription of genes on an entire chromosome or a large chromosome segment is regulated as a unit. In this latter case, regulation is associated with a general change in chromatin structure; for example, the inactive X in mammals can be distinguished by its pattern of staining. Genes that are moved onto the inactivated X chromosome are also inactivated: This type of gene inactivation does not generally occur when a gene is simply moved near a member of a coordinately controlled gene set. Formation of Primary RNA Transcripts Depends on Specific DNA Signals For a gene to be transcribed, it must have certain nucleotide sequences arranged in serial order along the DNA chain. The minimal elements are- starting at one end and reading from left to right a promoter sequence, which binds an enzyme (RNA polymerase) that facilitates transcription; a coding region that specifies the mRNA transcript; and terminal sequences that terminate and stabilize the transcript. Experiments in bacteria showed that the promoter region also serves as a binding site for specific gene-regulatory proteins that regulate the transcription activity of the gene. New techniques, especially gene cloning and DNA sequencing, have enabled investigators to demonstrate that a similar system of transcriptional regulation exists in higher organisms, including humans. The promoter region of each gene in a higher organism has sites for several different DNA-binding proteins. Many of the specific proteins are now being character- ized, and their genes cloned, so that the mechanisms by which they work can be studied in detail at the molecular level. One class of binding site that need not be located precisely at the promoter region of genes is called an enhancer. Enhancers can function from distances of nucleotide pairs away from the RNA start site they affect. Enhancers were originally given this designation because gene activity was enhanced when the element was present and diminished when it was absent; we now know that an enhancer can also repress transcription, depending on the exact proteins bound.

100 OPPORTUNITIES IN BIOLOGY An important property of many enhancers is their tissue-specific action. Se- quences in the promoter region of the human insulin gene, for example, bind proteins found in insulin-producing cells but not in cells that do not produce insulin. Such results indicate that tissue-specific gene expression is at least in part caused by DNA-binding proteins that bind at specific sites next to genes. The demonstration of enhancers helps explain how cells can coordinately express large sets of genes physically separated on different chromosomes. That is, specific DNA-binding proteins may recognize and bind with a similar nucleo- tide sequence present in the promoter region or enhancer of a number of different genes. In this way, a particular set of genes typical of a certain cell type would be activated or inhibited in a coordinated, programmatic fashion by the activation of a specific type of DNA-binding protein. Many such DNA-binding proteins exist, and a catalogue of many of them is needed before we can understand how the network of regulatory proteins affects cell differentiation in higher organisms. These networks are fundamental to the processes of normal cell differentiation and tissue moIphogenesis. Moreover, the inappropriate expression of trans- regulatory proteins could lead to the abnormal gene expression associated with some forms of cancer. Primary RNA Transcripts Are Modified by Additions at Their Ends and Deletions of Internal Segments Modifications of RNA transcripts have an important influence on gene ex- pression. When a transcript is first formed, its free end (the 5' end) is chemically modified, or capped. Such capping is believed to stabilize the transcript. In addition, immediately after completion, most transcripts are modified by having a string of adenylnucleotides added to their other end (the 3' end); this polyadenyl- ated, or poly(A), modification is believed to influence their life expectancy. Most transcripts are also extensively modified by the precise deletion of internal segments, a process called mRNA splicing. The splicing mechanism must proceed with high fidelity, since an error of even a single nucleotide could change the reading frame (nucleotide sequence that codes for a specific protein), leading to translation of an altered protein. Variations in splicing may lead to greater flexibility in gene formation and in the expression of genes. RNA splicing is required because of the existence of introns in eukaryotic genes. Introns are segments of DNA that are not used to specify protein se- quences but are interspersed among the coding sequences. Their presence ex- plains why long nuclear RNAs are produced as precursors for shorter cytoplasmic mRNAs. The intron sequences are excised from the precursor by RNA splicing, and the mature mRNA is transported to the cytoplasm. The length of an intron can vary from 60 or 70 to more than 100,000 nucleotides, and a typical vertebrate or plant gene might contain several introns. Since the coding sequences used for translation of a protein are formed during splicing, variations in the pattern of splicing of a precursor RNA permit

GENES AND char r-s 101 one gene to specify the formation of multiple proteins. Not only does such variation in splicing exist, but more importantly, the splicing pattern of a precur- sor can vary from one cell type to another. This means that splicing is regulated by factors specific to cell type. Several questions are central to the study of RNA splicing. What specifies the set of intron sequences to be excised by splicing? Small nuclear ribonucleo- protein (snRNP3 particles are crucial to sequence recognition during splicing. The splicing of the shortest intron requires the activities of at least four different particles, whereas longer introns may require even more. Changes in such factors could regulate different splicing patterns in different cell types. Another important question concerns the chemistry of the splicing reactions. What are the mechanisms of the endonucleolytic cleavage and ligation steps, and are these reactions catalyzed by RNA or protein components? The study of splicing is a new frontier in molecular biology. The most startling finding from this research has been that of the self-splicing of particular types of RNA sequences. As the name suggests, this splicing reaction occurs in the absence of protein and is catalyzed by conserved sequences within the intron. When analyzed in detail, the RNA sequences within this intron have a catalytic activity similar to that commonly associated with several different types of protein enzymes. Catalytic RNAs are now thought to have been common in primitive biological systems and still to be integral to many contemporary pro- cesses (such as protein synthesis). In the past few years, they have been discov- ered in viruses, bacteria, fungi, protozoa, animals, and plants. It is even possible to imagine that the first cells on earth may have contained only RNA enzymes, and that cells existed before there were proteins. Precursors to mRNAs are transcribed at many sites in the nucleus, processed by splicing and polyadenylation, and then transported through nuclear pores to the cytoplasm. Nothing is known of the mechanism of transport. These precursors are bound to a highly conserved set of basic proteins. In the cell, these basic proteins are retained in the nucleus and may play an important role in both splicing and polyadenylation. The complex steps of RNA processing offer several points at which gene expression could be modulated, such as by differen- tial splicing to yield different mRNAs. The field of RNA processing is young and has only begun to be explored. Specific DNA Modification May Influence Gene Expression and Corfer Heritable Traits Specific DNA base pairs are modified most frequently by methylation (the addition of methyl groups to DNA). In prokaryotic organisms it was thought that modification was required only to prevent DNA degradation by restriction en- zymes. Recently, however, direct effects of methylation on the regulation of gene expression have been found. In mammalian genomes it has long been thought that methylation might correlate with the heritable control of specific gene ex

102 OPPORTUNITIES IN BIOLOGY pression. A variety of indirect experiments suggests that demethylation can derepress gene activity (activate a gene from a repressed state). In mammalian embryos, the expression of certain genes depends on whether the genes have been transmitted by the egg or by the sperm (see Chapter 5~. The basis of this "imprinting" is not known, but data suggest that specific methylation changes that occur during the formation of egg and sperm may be involved. Although DNA methylation could explain the heritable patterns of gene expression displayed by various cell lineages in multicellular organisms, we do not know how large a role methylation plays, nor do we know what controls the patterns of DNA modification. In some complex multicellular organisms (for example, Drosophila), the absence of evidence of DNA methylation suggests that it evolved rather late in evolution as a back-up system. Clearly we have much to learn about the role of methylation and other heritable modifications in the control or gene expression. The Study of the Regulation of Gene Expression Offers Great Potentialfor New Research Opportunities and Practical Applications At each level-DNA structure, transcription, RNA processing, mRNA translo- cation, translation, and the determination of the three-dimensional structure of the protein product and its introduction to the appropriate cellular compartment new genetic techniques can be used to probe and understand the controlling processes. It is these processes that constitute "gene action" and determine cellular function. We know very little about how RNA polymerase reads the various promoter and enhancer sequences that determine the genes to be tran- scribed. It is not at all clear that the presence of transcription factors is sufficient to generate a regulatory system that can persist even after cells replicate, yet we know that cell determination results in patterns of gene expression that are inherited by daughter cells. We need to know the details of these and associated processes to understand the mechanisms that control cellular differentiation. The ability to intervene and affect any of these processes specifically would make it feasible to design a host of new therapeutic drugs. For example, if we understood how genes such as those that control the production of interferon are induced, we might be able to mimic such a process selectively to enhance resistance to viral infections. The same kind of arguments apply to the regulation of growth in cancer cells and of immune responses. Gene Transfers Provide Many Opportunities to Study in Detail How Genes Function Using gene transfer technology, one can remove the control regions (en- hancer or promoter) of a gene a bit at a time to identify segments that are critical for normal function. One can also switch control regions from one gene to

GENES AND CFr.~.s 103 another, so as to cause genes to be expressed under circumstances in which they would normally be silent. Although such studies have been possible for only a short time, they have provided much information (and some surprises) about gene regulation. In many organisms it is now possible to insert the modified gene directly into germline cells so that entire organisms carrying the new DNA are obtained. Studies of transgenic animals are now providing important information on both the mechanisms that regulate tissue-speciD~c gene expression and those that cause cancer (for a discussion of transgenic animals, see Chapter 2~. Genome Organization The Organization of Complex Genomes Can Be Studied by Gene Mapping Every cell of the human body contains an identical set of an estimated 60,000 to 100,000 genes. Most genes are present only once (single-copy genes). Each gene can be assigned to a particular chromosome site. This activity, called gene mapping, is improving our understanding of cells. Gene mapping in humans and selected mammalian species has advanced impressively during the past decade. In humans, approximately 1,400 genes of known function have been mapped to some degree of precision, and another 1,000 DNA markers of unknown function have been localized. A preliminary low- resolution genetic map of the human genome has already been constructed. Restriction Fragment Length Polymorphism DNA Markers Have Increased the Power of Mendelian Techniques for Mapping Genes RFLPs are naturally occurring variants in the nucleotide sequence of DNA that can be used as genetic markers. They are transmitted from one generation to the next in the same way as the genes that govern eye or flower color. The advantage of RFLPs is that they can be detected through the use of only a few cells of the organism by the techniques of molecular biology. In addition, RFLPs relate the genetic linkage map to the physical (DNA) map. In essence, RFLPs add a large set of new markers for mapping any genetic locus of interest and for obtaining a high-resolution genetic map of any chromosomal region of particular interest. The Value of a Human Gene Map Has Become Apparent in the Past Several Years The map serves as a body of data that can be used to address a broad range of biomedical questions. It becomes more useful in generating and resolving hy- potheses as the data base grows. For example, He map has been useful in establishing the connection between chromosome rearrangement and proto~nco

104 OPPORTUNITIES IN BIOLOGY gene activation. Proto-oncogenes are normal genes generally involved in cellular growth control, but they have the potential to cause cells to become cancerous when inappropriately expressed. In some cases inappropriate activation has been associated with rearrangement of the chromosome bearing the proto-oncogene. The gene map has also enabled investigators to detect linkage relationships between RFLP markers and specific genes that carry a risk for genetic disease. This can now be accomplished for many genetic diseases, such as various ane- mias, phenyLketonuria, hemophilia, and Huntington's disease. The identification of linkage relationships between genes causing genetic disease and RF1LP markers is an important first step in physically isolating genes whose mutants cause genetic disease. A start in this direction has been made for a number of human heriditary diseases, such as Duchenne's muscular dystrophy, Huntington's disease, and cystic fibrosis. Isolation of these disease-causing genes will be the first step in analyzing the molecular basis of these diseases. The use of this approach to isolate genes and determine their products can reveal the underly- ing molecular basis of more than 3,000 human genetic conditions and provide crucial insights into human developmental processes. If the number of human genes is approximately 100,000, only 1 percent of them have been mapped to date. Currently, new genes are being mapped at the rate of one every day. Two important new mapping activities are just now beginning: the formulation of maps based on overlapping cloned DNA segments and the DNA sequencing of the nucleotides that constitute these segments (see Chapter 3~. CYTOPLASM: ORGANELLES AND FUNCTIONS In a eukaryotic cell, the nucleus is surrounded by the cytoplasm, which in animal cells usually accounts for nine-tenths of the cell's volume; the cytoplasm is surrounded in turn by a cell membrane, or plasmalemma. The cytoplasm contains within a protein polymer matrix several types of minute, functionally specialized cell organs, or organelles, most of them present in multiple copies. In prokaryotic cells (bacteria), by contrast, the genome is not separated from the cytoplasm, there are no membrane-bounded organelles, and the plasma mem- brane subserves many of the specialized functions of eukaryotic organelles. Prokaryotic and Eukaryotic Cells Use Related Strategies to Compartmentalize Their Biosynthetic Reactions Prokaryotic cells have only one membrane, located at the cell surface (al- though some bacteria have a double membrane). In contrast, eukaryotic cells, such as those of animals and plants, have, in addition to their plasmalemma, at least a dozen different types of chemically specific membranes that create sepa- rate intracellular compartments with different microenvironments required by

GENIES AIMED CELLS 105 various processes, such as respiration, photosynthesis, protein synthesis, and intracellular digestion. In proka~yotic cells, the plasmalemma is the site of a number of important biosynthetic activities, including the synthesis of membrane lipids, adenosine triphosphate, and cell-wall components. This membrane also contains a multi- tude of transporters, receptors, and sensors for chemotactic movements, which create the interface of the bacterium with its extracellular environment. In eukaryotic cells, comparable activities are differently distributed. Some of them (such as those of the transporters and receptors) remain in the plasmalemma, but others are relocated to different intracellular membrane systems. Lipid synthesis, for instance, in animal cells occurs only in the endoplasmic reticulum (ER), a network of membrane-bounded channels that pervades the cytoplasm. In plant cells, lipid synthesis occurs in plastics. From the ER or plastics, newly synthesized lipids are distributed to all other cellular membranes. The ER also contains a complex set of enzymes that modify lipid-soluble aromatic compounds imported by the cell from the environment. The modifications increase the water solubility of these compounds, thereby facilitating their elimination. Since ster- ols, drugs, herbicides, toxins, and chemical carcinogens are among these com- pounds, the relevant enzymes constitute an intracellular detoxifying system. The ER membrane is also the site of important steps in protein traffic regulation. Issues awaiting resolution include the means by which proteins and lipids are transported from their site of synthesis in the ER to a multiplicity of destinations and the way differences in lipid chemistry are established and main- tained in different membranes. We must also advance our understanding of the ER detoxifying system to shed light on problems related to chemically induced cancers and toxic effects of chemical pollutants in the environment. Protein Synthesis and Regulation Protein Synthesis and Regulation of Protein Traffic Take Place in the Cytoplasm Protein Synthesis. For all cells, protein synthesis is a major, continuous activity needed for the production of intracellular enzymes, contractile and cy- toskeletal assemblies, membranes, ribosomes, chromosomes, and many other functionally important macromolecular assemblies. In more complex multicellu- lar organisms, it is also needed for the production of proteins destined for export out of the cell, such as enzymes, hormones, growth factors, blood proteins, antibodies, or components of the extracellular matrix. In all cells, proteins are synthesized on ribosomes, which translate into amino acid sequences the instructions received from active genes in the form of mRNAs. The ribosomes themselves are macromolecular assemblies of ribosomal RNA and protein molecules. The two sets of components are produced separately in the cytoplasm (proteins) and in the nucleus (ribosomal RNAs) and are modified and

106 OPPORTUNITIES IN BIOLOGY assembled in a special compartment in the nucleus (the nucleolus) before being transferred to the cytoplasm as part of a functioning ribosome. In both prokaryotic and eukaryotic cells, ribosomes are basically similar and protein synthesis proceeds by similar steps. In eukaryotic cells, however, the ribosomes are larger and require more factors for their activity. These differences are probably related to the emergence of more versatile regulatory processes in euk~uyotes. Protein Traffic Control. In eukaryotic cells, ribosomes and protein synthesis occur primarily in the cytosol, to which mRNAs have direct access from the nucleus and in which the pool of amino acids and all ancillary factors required for protein synthesis are located. Only 2 percent of the total protein production is accounted for by small mitochondrial ribosomes whose products are used exclu- sively in mitochondria. In plant cells a somewhat larger fraction of the cell's protein is produced by chloroplasts (or other differentiated forms of plastics). From the cytosol, proteins are accurately directed to more than 20 different sites of final functional residence. These sites are membranes or compartment contents, each endowed with chemical specificity. ER Targeting System. Many proteins are directed to the ER membrane as they are being synthesized. The selectivity is based on mutual recognition between signals (called signal sequences) within the amino acid sequence of the protein to be transported and a signal recognition complex. This complex in- volves a ribonucleoprotein particle (called a signal recognition particle) and at least one transmembrane protein (its receptor) in the target ER membrane. The ER targeting system recognizes and processes proteins destined for secretion and lysosomes (storage compartments for degradative enzymes), as well as membrane proteins for many intracellular compartments. Secretory and lysosomal proteins are fully translocated across the ER membrane into the ER cisternal space. Membrane proteins are partially translocated and remain an- chored in the membrane. Recent experiments indicate that a membrane protein can be converted into a secreted polypeptide, and conversely, a secretory protein can be converted into a membrane protein by deleting or adding the information for the membrane anchoring sequences from their mRNAs. The mode of operation of the ER targeting system has been elucidated in reconstituted in vitro systems in which ribosomes are allowed to translate into proteins the genetic information encoded in specific mRNAs in the presence or absence of ER-membrane vesicles in vitro. The results show that many compo- nents of the systems are functionally equivalent in different species, phyla, and even kingdoms, which implies that this part of the traffic regulation system originated early in evoluiion and has been conserved. Post-ER Steps in Traffic Control. Once past the ER, proteins are moved within the cell through a specialized membrane-bounded compartment called the Golgi complex, where they are further modified by glycosylation, sulfation, and

GElVES AND CF1 r c 107 proteolysis; they are sorted while in transit to lysosomes, secretion vacuoles, or different membranes. The Golgi complex itself contains at least three subcom- partments. Transport from the ER to Golgi subcompartments, from one subcom- partment to another in the Golgi complex, and finally, from the last Golgi elements to the plasmalemma requires energy and is effected by vesicular carri- ers; thus, past the ER, protein traffic can be regulated at least in part by controlling the movements of vesicular carriers. These carriers apparently recycle continu- ously among compartments. The best known among these vesicular carriers are the secretion granules or secretion vacuoles of various glandular cells. They transport products to the cell surface and discharge them into the extracellular medium by a process known as exocytosis. Sorting of the proteins to their ultimate destinations probably involves mutual recognition between a signal and a receptor, but so far only the signal for lysosomal proteins has been chemically defined. Its receptor has been isolated and partially characterized and its gene sequenced. Reactants involved in the sorting of other proteins remain unknown, as are the signals and receptors that regulate the traffic of vesicular carriers. Studies to identify them are being actively pursued. Other Tra~c-Control Systems. The ER targeting system (which includes the Golgi complex) is undoubtedly the most complex component of the overall protein ~affic-control system of the cell. The other components are simpler, and most of them probably transport the protein in a single step: from the cytosol directly to the final destination. The amino acid sequence of the signal that directs certain proteins to the nucleus is known in a few cases, but the corresponding receptor remains to be identified. A substantial body of information already exists about traffic regulation of proteins targeted to mitochondrial and chloro- plast membranes. Among the protein products of plant nuclear genes are some that function in the mitochondria and some in chloroplasts; it is not known how the systems differ. In a simpler form, protein traffic regulation occurs in proka~yotic cells and has been studied extensively in gram-negative bacteria, which are provided with two concentric membranes at the cell surface. The number of final destinations is considerably fewer the two membranes and the intervening space and perhaps the outside of the cell. A signal, generally comparable to that found in eukaryotic proteins targeted to the ER membrane, has been identified and analyzed in detail by sequencing and by extensive genetic modifications. This line of work has led to the recognition of functionally critical residues in the signal sequence, but the other components of the system are still unknown. We can anticipate considerable activity in this fertile and exciting area, especially in relation to the identification and characterization of signals and their receptor partners and to the intracellular location of receptors and sorters (mole- cules that control the selection and movement of proteins from one compartment to another). Although the picture is already rich in detail, many uncertainties remain to be resolved by further research. The reasons for removing the signal sequence are not clear, nor are the reasons for He complexity of the enzyme that

108 OPPORTUNITIES IN BIOLOGY effects the removal. The enzyme may have additional functions since it consists of six different proteins. The translocation process itself is still a mystery. Structural biology is likely to yield three~imensional information on such interactions if large enough quantities of relevant proteins can be obtained. The main goal is to understand how cells process their many proteins and how they achieve and maintain the chemical specificity of their membranes. Another basic process that remains to be understood in molecular terms is membrane fusion. The process is critical for cell division, cell fusion in egg fertilization, and vesicle fusion along different pathways of intracellular transport. Membrane fluidity is a prerequisite for membrane fusion. It is also a prerequisite for membrane growth, which appears to proceed by expanding preexisting mem- branes. At the same time, intact diffusion barriers need to be maintained in highly dynamic membrane systems. Much remains to be discussed about how these processes are controlled. MITOCHONDRIA: FUNCTION AND BIOGENESIS Mitochondria Produce Most of the Cell's Main Energy Source, ATP The mitochondrion is characterized as the power plant of the cell, because it performs the enzyme-catalyzed, stepwise oxidation of nutrients (such as sugars, fats, and amino acids) in a process called respiration. The most interesting product of this process is ATP, the direct source of the energy required for most of the chemical work the cell must perform to power its growth, movement, synthe- sis of new components, and other functions. A relatively small amount of ATP is produced in the cytosol (luring sugar catabolism (glycolysis), but by far the largest amount is generated in mitochondria. The mechanism of mitochondrial ATP synthesis has stubbornly resisted full elucidation, but progress continues to be made as a result of our insistent probing into this fundamental energy-transducing process. Mitochondria are also of interest because they contain their own complement of DNA, which cooperates with the DNA of the nucleus in the control of mitochondrial formation. The origin and evolution of the mitochondrion are linked to the origin and evolution of eukaryotic cells. Understanding of mito- chondrial function in turn sheds light on a wide array of fundamental and practical issues, ranging from certain metabolic and genetic diseases to evolution itself. New Mitochondria Arise from Existing Ones, and They Are Characterized by Unique Functions Mitochondria have two membranes (inner and outer) that define two concen- tric separate spaces. The inner space houses hundreds of enzymes, including those involved in the oxidative reactions that supply the energy needed for cell function. The inner mitochondrial membrane contains the energy-conversion

GENES AND CFr.r ~ 109 apparatus. The majority of the mitochondrial proteins are specified by nuclear genes; they are synthesized on ribosomes in the cytoplasm and then imported into the organelle. A limited set of proteins of the inner mitochondrial membrane, namely, some protein subunits of the oxidative phosphorylation apparatus, are encoded in DNA molecules located within the organelle itself and are synthesized by an organelle-specific translation system. The distinctive structural RNA components of this system RNAs and transfer RNAs~re also encoded in mitochondrial DNA. Mitochondria do not arise de nova in the cell by self- assembly of their constituent molecules, but are formed by growth and division of existing mitochondria. The mitochondrial DNA from several organisms, including humans, has been completely sequenced, and much of its informational content has been elucidated. Furthermore, all mitochondrial gene products in humans have been functionally identified. A dramatic outcome of these studies has been the discov- ery that the mitochondrial genetic system in the organisms studied, except plants, uses a code that differs in several respects from the universal code and, in addition, utilizes for reading the code a novel mechanism, which requires only a restricted set of transfer RNAs. Excellent Opportunities Exist to Study the Mechanisms of Expression and Replication of Mitochondrial DNA Studies of the enzymes and ancillary proteins responsible for mitochondrial DNA replication, DNA transcription into RNA, and RNA processing to mature RNA species are making rapid progress, aided by the development of soluble in vitro preparations derived from broken mitochondria and by the use of recombi- nant DNA technologies. Specific proteins have already been identified and, in some cases, isolated in partially or completely pure form. As in the case of many nuclear gene transcripts, the coding sequences of several mitochondrial gene transcripts in lower eukaryotes, especially yeast and filamentous fungi, are interrupted by nonfunctional segments, or introns. These introns must be removed to produce the mature RNA. The transcripts of some mitochondrial genes have the remarkable capacity to excise their own introns in vitro in the absence of protein; that is, they function as enzymes acting on themselves. The discovery of mitochondrial introns has opened an active field of research, which is expected to provide deep insights into the mechanisms of RNA splicing in general and into the origin and evolution of introns. The Formation of New Mitochondria Is Under the Control of Both the Nucleus and Mitochondria The dual control of mitochondrial formation is most dramatically illustrated by the chimeric structure of nearly all the enzyme complexes of the oxidative phosphorylation system: Each such complex contains some subunits encoded in

110 OPPORTUNITIES IN BIOLOGY the nucleus and some encoded in mitochondrial DNA. Because of this dual control, the formation of functional mitochondria requires a quantitative and temporal coordination of expression of the relevant nuclear and mitochondrial genes. Two main classes of nuclear genes are the object of intensive investigation based on recombinant DNA techniques and on structural and functional ap- proaches: (1) genes coding for subunits of the enzyme complexes of the oxidative phosphorylation system and for mitochondrial carriers used in metabolite trans- port and (2) genes coding for proteins involved in the expression and replication of the mitochondrial genome. Most of the latter genes are probably distinct from those that code for the homologous components of the nuclear-cytoplasmic appa- ratus, although interesting exceptions have recently been reported. They concern the possible existence of common nuclear genes for cytoplasmic and mitochon- drial components, which could account for at least some of the reported influ- ences of the mitochondrial genome on the remainder of the cell. Research now under way promises to elucidate the mechanisms and signals involved in the interactions between the nuclear and mitochondrial genomes in the formation of functional mitochondria. Furthermore, research in the area of nuclear-mitochondrial interactions should help us understand how the assembly and function of mitochondria are integrated with those of the rest of the cell and how these processes can be modified in relation to cell respiratory demands, cell differentiation, and senescence. Proteins Are Imported into Mitochondria The hundreds of distinct polypeptides that make up a mitochondrion are distributed in a specific way in four compartments: the outer membrane, inter- membrane space, the inner membrane, and inner mitochondrial space. After their synthesis on cytoplasmic ribosomes, the nuclear gene-coded polypeptides are imported to their correct location within the mitochondrion. Biochemical studies and the application of recombinant DNA technology have shown that proteins destined for one of the three innermost compartments are usually made from precursors with extensions at the amino-terminal end; these extensions can be as long as 100 amino acids and function as signals directing the proteins to the proper location. Still unanswered questions include: Which molecules are involved in the import of polypeptides into the mitochondrial What is the role of cytosolic factors in the import process? How do mitochondrial signal sequences perform their function? Why does translocation of proteins across the mitochondrial inner membrane require a gradient of electrical potential across that membrane? Do contact or fusion points between the two membranes function as ports of entry for protein import?

GENES AND CFI-~-£ 111 Crystallographic Studies Should Reveal the Tertiary Structure of the Oxidative Phosphorylation Apparatus The subunit composition of the enzyme complexes of the oxidative phosphorylation system is largely known, as is the location nuclear or mito- chondrial-of the genes specifying these subunits. From the nucleotide sequence of these genes, the amino acid sequence of the subunits can be inferred. Defini- tive knowledge about their tertiary structure should eventually come from crystal- lographic studies now in progress. This information, together with data derived from other approaches to studies of the spatial relations of the subunits in each enzyme complex, is likely to provide useful models of the three-dimensional structure of each complex and of its topology in the inner mitochondrial membrane. These models will help in interpreting the results of ongoing functional studies on the individual complexes in intact mitochondria and in reconstructed systems. CELL MOTILITY AND THE CYTOSKELETON An Understanding of the Basis of Cellular Motility Is Central to Our Understanding of the Functioning of All Organisms Cell motility is necessary for the survival of virtually all living species. For example, the egg would not be fertilized without a motile sperm, and every cell division that occurs thereafter requires a degree of motility in some cell parts. Without active changes in cell shape and cellular migrations, embryos would not form. Without cellular motility, white blood cells would neither accumulate at sites of inflammation nor ingest invading microorganisms. Without active and rapid movements of organelles in axons and large plant cells, the peripheral parts of these cells would not be nourished. Cell Structure Three Types of Protein Polymers Constitute the Cytoskeleton and Interact with Force-Producing Enzymes to Cause Cells to Move Cells of both animals and plants contain three different types of fibers-actin filaments, intermediate filaments, and microtubules each of which is formed by the polymerization of distinct protein molecules. Together these fibers provide mechanical support for the cell and thus are considered to be a cytoskeleton. The actin filaments and microtubules also participate in cellular movements, including locomotion of whole cells, cell division, and movement of subcellular compo- nents. This combined ability to maintain form against mechanical forces and to

112 OPPORTUNITIES IN BIOLOGY produce and transmit force means that this cytoskeletal motility system can determine cell shape and hence the structure of both tissues and whole organisms. A clear understanding of this system will be essential for unlocking the secrets of embryology. This field is still in an explosive growth phase during which investigators have isolated and started to characterize the major molecular components of these systems. The inventory includes not only the protein subunits of the polymers themselves but also a surprising number of regulatory proteins. For example, in the actin system alone, one cell has already been shown to have almost 20 accessory proteins, which together with the actin constitute 25 percent of the total cell protein. In the developing brain, the microtubule system may include a similarly large proportion of the total cell protein. In skin, the keratin molecules that form the intermediate filaments constitute the major protein in the cells. The Cytoskeleton Provides Form to Cells and Therefore to Organisms The polymeric nature and intracellular distributions of the filaments and microtubules suggest that they may mechanically stabilize the cytoplasmic ma- trix. Recent physical studies on purified cytoskeletal fibers and analysis of the mechanical properties of live cells support this conclusion. Other work has shown that some of the glycolytic enzymes bind to actin filaments and that polyribosomes are associated with isolated cytoskeletons. Thus, beyond impart- ing mechanical integrity, the cytoskeleton may provide scaffolding for enzymes that participate in cellular metabolism and protein biosynthesis. In this way, the cytoskeleton, like membranes, may be an essential integrator of cellular pro- cesses. It is now possible to describe, in broad outline, how these protein polymers assemble and how some of the steps in the assembly process may be regulated, at least for actin and microtubules. To a large extent, the construction of the system of cytoplasmic fibers can be explained by the process of self-assembly, in which the protein subunits are driven by chemical attraction for each other to polymerize without external direction. This spontaneous process is controlled by a variety of regulatory proteins, some of which must react to signals from the external envi- ronment that direct the organization of the cytoskeleton. Cells also contain organelles, such as the centrosome, that help to organize the cytoskeleton. The centrosome is the site where the assembly of microtubules is initiated. Biochemical and cellular experiments indicate that the mechanisms control- ling the assembly and organization of these fibers in the cell are both complex and subtle, as befits a system with such an important influence on cell architecture. To gain a better understanding of how form is determined in biology, considerable new work will be required (1) at the molecular level to elucidate the molecular composition, regulation, and dynamics of the cytoskeleton and (2) at the cellular level to determine how external stimuli affect the assembly of the cytoskeleton.

GENES AND char red Cell Movement Research on the Mechanisms of Cell Movements Is Progressing on a New Frontier 113 In parallel with studies on the structural elements of the cytoplasm, work on mechanisms of cell movements has pushed forward rapidly during the past 15 years on two main frontiers; during recent years, a third and possibly a fourth frontier have begun to open. In each case a specific motor protein is responsible for movement. The first frontier involves the microtubule-dynein system found in cilia and flagella- whiplike organelles (such as sperm tails) that beat rapidly. Cilia are found in groups on the surface of epithelial cells such as those lining the air passages in our lungs, where they are responsible for sweeping mucus and inhaled foreign material out of the lungs. If the cilia are not active, serious infection is inevitable. Flagella form the tails of sperm and propel them toward their meeting with the egg. In cilia and flagella, microtubules interact with a giant enzyme molecule called dynein to convert the chemical energy stored in ATP into a force that bends the cilia and flagella. Since the chemical steps in this process have now been identified, studies on the molecular mechanism that produces the motion can now be pursued vigorously. Dynein is also present in the cytoplasm, outside cilia and flagella where it can move particles along microtubules in the direction from the cell periphery toward the cell center. The second frontier is the characterization of myosin the force-producing enzyme long known to cause contraction in highly specialized muscle cells and more recently recognized to exist along with actin in most other cells, even those not specialized for contraction. Superficially most myosins are similar, and it seems likely that all myosins produce force by interacting with actin filaments and ATP in the same fundamental way. The steps in this process have been studied in detail in muscle (an especially favorable test system). Investigations using spectroscopy, x-ray diffraction, electron microscopy, and biochemical methods are also under way to locate the site in the myosin molecule where motion is produced. Myosin and actin are widely believed to be responsible for many forms of cell movements in addition to muscle contraction. For cytokinesis (the final step in cell division), direct experimental evidence validates this hypothesis. Other types of movements required similar experiments in order to explain their molecular basis. In the past, most cell biologists suspected that either dynein or myosin was responsible for most cell movements, including the ubiquitous rapid movements of cellular organelles in the cytoplasm, but it has recently been discovered that a new type of motor protein called kinesin moves particles along microtubules in the opposite direction from dynein. Together these two motors provide a two- way rapid-transit system for organelles through the viscoelastic cytoplasm. This

114 OPPORTUNITIES IN BIOLOGY mnesin-dynein-microtubule system can shuttle a vesicle manufactured in a nerve cell body in the spinal cord to the nerve endings in the big toe (and back) in a few days! Even newer evidence suggests that an unusual form of myosin may pull some organelles along actin filaments. Breakthroughs of this type have raised the hope that we may soon understand how the mitotic apparatus works and how the traffic of organelles is directed to the proper destinations in the cell. Each of these motile systems operates under exquisite controls that allow cells to respond to internal or external stimuli and to produce a coordinated motile response. In skeletal muscles and heart, the contractile proteins are turned on by calcium, which activates regulatory proteins bound to the actin filaments. In the smooth muscle cells found in internal organs and in nonmuscle cells, the myosin is activated chemically by the attachment of phosphate to the protein. It is not yet understood how these chemical reactions are coordinated in the living cell to produce the complex patterns of movement that are essential for life. The purse- string-like contraction that splits two daughter cells apart at cell division is an example of a movement in response to an internal stimulus arising from the poles of the mitotic spindle. The rapid locomotion of white blood cells to sites of infection and their ingestion of bacteria are examples of complex movements in response to external signals. In these examples, the stimuli and the responses are well documented, but little is known about the mechanisms that convert the stimulus into the response. Regulation of microtubule-based movements presents a similar challenge. If work on cell motility continues with its current momentum, progress is likely in the following areas. Molecular Inventory and Characterization. The complete catalog of the molecular components of the cytoskeletal motility system should be completed for a few cell types that are particularly favorable for use as model systems. These include the slime molds Dictyostelium discoideum and Physarum, the protozoan Acanthamneba, sea urchin eggs, macrophages, platelets, and the intestinal epithe- lial cell. Completion of this molecular inventory may require new functional assays for proteins that have yet to be discovered. The primary structures of the major components need to be determined by sequencing cloned DNA, and the three-dimensional structures of the major components must be determined by x- ray crystallography. The first atomic resolution structure of a cytoskeletal protein (the actin-binding protein called profiling has been completed, and good progress is being made on actin and myosin. Cellular Organization and Dynarrucs. Electron microscopy should lead to more precise localization of the components of these systems inside whole cells. Vastly improved probes consisting of antibodies coupled to colloidal gold, to- gether with better methods to prepare cells, should give us a clearer picture of macromolecular architecture. Furthermore, it should be possible to characterize

GEIVES AND CF-r r ~ 115 the dynamics of the cytoskeleton in live cells by using new fluorescence tech- niques (see Chapter 2~. Purified protein molecules can be tagged with fluorescent dyes and then injected into live cells. Functions and Regulation of the Cytoskeletal Motility System. Perhaps the major challenges in the field will be to determine the functions of the various components inside living cells and to learn how these functions are regulated by the cell. One approach is the use of in vitro assays with purified components. It is remarkable that functions as complex as the contraction of actin and myosin, the movement of an organelle on a microtubule, or the growth of microtubules from the centrosome to the kinetochore of a chromosome can all be reproduced today in vitro. These assays should become more sophisticated, enabling cell biologists to test for the ability of purified components to carry out complex functions outside living cells. Producing mutant cells with defects in single components will also be valuable in demonstrating functions and identifying regulatory mecha- nisms. This may be a laborious process because there are multiple genes for many components, and even where there is a single gene, the protein itself may be part of a highly redundant system that will function nearly normally without any given component. The microinjection of inhibitory antibodies to inactivate a single component inside a cell and the inactivation of relevant genes are also promising approaches. These and other creative new approaches will be necessary to test current ideas regarding the physiological functions of the cytoskeletal motility system. A long-term challenge will be to characterize the mechanisms by which an external stimulus, such as a chemoattractant, causes a cell to move in a particular direction. Mechanical Properties. Analysis of the mechanical properties of the cy- toskeleton and its component molecules is essential, but has only begun, in part because few cell biologists are familiar with the engineering techniques required for the work. This is an area of potential collaboration between cell biologists and engineers. CELL MEMBRANE The Cell Membrane Not Only Forms a Protective Surface But Also Receives Chemical Messages from the Environment The outer cell membrane is an extremely thin, sheetlike assembly of lipid and protein molecules that provides a boundary to the cell's body. This exquisitely delicate skin, called plasmalemma, is a diffusion barrier for water-soluble sub- stances. In the plasmalemma, the cell assembles all the molecular equipment needed for its exchanges and interactions with the environment.

116 OPPORTUNITIES IN BIOLOGY The Plasma Membrane Shares Many Basic Structural Features with Other Types of Cell Membranes Membrane structure relies on the use of a continuous bimolecular layer of lipids, the diffusion barrier, which is fluid at the temperature of the environment in which the cell lives. The barrier is traversed by transmembrane proteins that subserve a variety of functions, and it is reinforced by a fibrillar infrastructure made up of other different proteins. Depending on cell type, these infrastructures are built either for imparting tensile strength to a delicate membrane or for controlling the lateral mobility of transmembrane proteins, which if not restrained would move rapidly in the membrane because of the fluidity of the lipid bilayer. The first type of infrastructure has been studied extensively in the red blood cells of humans, and its molecular components and their mode of assembly are well known. Their function is to reinforce the membrane and to give~the cell its characteristic shape. More recent work shows that the same or related proteins are used by many other cells to solve similar problems. Most of the studies on the infrastructure that controls lateral protein mobility have focused on the miniature geodetic cages formed by the protein called clathrin and associated proteins. These clathrins are found on structures called coated pits and coated vesicles associated with the plasmalemma as well as with certain intracellular membrane systems. Coated pits trap functionally important molecules from the environment or from intracellular compartments. Permeability Modifiers Are Transmembrane Proteins That Facilitate the Transport of Water-Sotuble Molecules Across the Lipid Bilayer Many permeability-modifier proteins transport nutrients such as glucose and amino acids. Others are channels for ions, and still others are energy-driven pumps that move sodium, potassium, hydrogen, or calcium ions in or out of the cells against concentration gradients. Many of these molecular pumps are called ATPases because they obtain the energy needed for their work by splitting ATP. The main function of the pumps is to maintain stable intracellular ionic concentra- tions at optimal levels for the cell's activities. During the last few years, many transporters, channels, and pumps have been moved from their previous status as hypothesized physiological entities to that of well-defined protein molecules. Moreover, the amino acid sequence of many of them has been deduced from the nucleotide sequence of the cognate complemen- tary DNAs. Knowledge of the amino acid sequence of these proteins is needed as a first step toward understanding their function and the way they fit into mem- branes. Channels and pumps generate differences in molecule and ion concentrations (chemical and electrochemical gradients) as well as electrical charge separations (membrane potentials) across the corresponding membranes. These gradients and potentials are used by cells to propagate signals along the cell surface, as in nerve

GENES AND CF~S 117 and muscle cells, and to drive the transport of other molecules and ions across the membrane, as in the cells of the intestine and the kidney. THE EXTRACELLULAR MATRIX OF ANIMALS The Cells of Multicellular Animals Are Supported and Organized by a Continuous Extracellular Matrix Composed of Fibrous Proteins and Complex Polysaccharides In specialized connective tissues, such as bone, cartilage, and tendon, the extracellular matrix is predominant, but even in tissues such as muscle, liver, and brain, each cell is surrounded by a fine matrix. Connective tissues provide the avenues through which blood vessels pass to nourish every organ and serve as homes for the body's defensive cells, including phagocytes and lymphocytes. Consequently, most inflammatory diseases such as arthritis occur in the connec- tive tissues. The extracellular matrix is produced primarily by cells called fibroblasts, but also by epithelial and muscle cells. For years we have known that collagen (the most abundant protein in our bodies) and elastin form the major fibers in the extracellular matrix. During the past 10 years biochemists have identified more than 10 different types of collagen that are specialized for forming bone, cartilage, and basement membrane (a ruglike structure that all epithelia lining and cover- ing tissues-stand on). Some collagens form flexible fibers with tensile strength similar to that of steel, while others form three-dimensional networks. The regular arrangement of collagen in tendons has been known for some time, but the elucidation of the molecular organization of less-regular collagen structures such as basement membranes opens a fascinating research opportunity. Rubberlike elastic fibers are responsible for the ability of large blood vessels and skin to recoil when stretched. Elastic elements allow blood vessels to modulate the pulsatile flow produced by beats of the heart The mysterious loss of elastic fibers during aging has generated a multimillion dollar cosmetic industry to combat wrinkles. The amino acid sequence of elastin in known, but its molecular structure and its association with other molecules in elastic fibers are major research challenges that, when solved, should help explain and perhaps prevent some cardiovascular diseases and effects of aging. Connective tissues are also rich in a variety of organ-specific complex sugar polymers, some of which are chemically linked to proteins. They are called, as a group, glycosaminoglycans, or GAGs for short. The name derives from their repeating component sugars. Together with collagen fibrils they are responsible for making the cartilage covering most joint surfaces tough and elastic. GAGs also fill the eye and are major components of skin, blood vessels, and other organs. There is now active research on a variety of proteins that confer biological specificity on the extracellular matrix. For example, an elongated protein called

118 OPPORTUNITIES IN BIOLOGY fibronectin binds cells to the matrix. It has binding sites for a receptor protein of the plasma membrane of connective tissue cells, collagen, and GAGs, so it can link them all together. The bond to the cell is relatively weak, so that a cell can gain traction on the matrix but still move through it. Another protein, laminin, binds epithelial cells to the basement membrane. Perhaps the most remarkable feature of these and other adhesive molecules is that they recognize and bind to a very small site (as few as three amino acids-arginine-glycine-aspartic-acid) on collagen and other matrix molecules. The attachments of both fibronectin and laminin can be altered in tumors, and this change is thought to contribute to the ability of tumor cells to invade some tissues-these invasive tumors are the major cause of death from cancer. Future research should reveal ways in which these adhesive interactions can be modified in beneficial ways in tumor therapy. Evi- dence is also growing that binding to the matrix modulates cellular physiology. Active work is also being done on specific growth factors that promote the formation of specialized connective tissue such as bone and on other proteins that initiate the formation of the calcium-phosphate crystals that make bone hard. It has long been appreciated that physical forces on bones control their formation and that inactivity of the elderly contributes to the thinning of bones in osteoporo- sis. Here is a major opportunity for multidisciplinary research by cell biologists, biochemists, and engineers to learn how physical forces are transduced into the cellular activities that maintain normal bone and that fail in osteoporosis. Equally fascinating are the questions of how the information specifying the shape of the skeleton is laid down in the genetic code, how the cells of the connective tissue translate this information, and how matrix molecules influence the development of adjacent tissues. CELL REGULATION Cellular Activities Are Regulated by a Combination of Information Provided by the Genes and by Extracellular Signals The timing of major decisions made by cells, such as whether to grow, to divide, to move to one location or another, or even to die, is determined by genetic programs and also by environmental clues, such as hormones and contacts with other cells. To understand cell regulation, one must study the production and effects of signals coming from the nucleus as well as the mechanisms responsible for transducing extracellular signals into cellular actions. Cell Division The Cell Division Cycle Is the Master Program of Cell Regulation That Organizes a Variety of Subroutines In a very broad sense, to understand cell division, we need to understand more than its component parts: how membranes are assembled and disassembled

GENES AND CFr-r ~ 119 during mitosis and cytokinesis, how DNA is replicated and organized, and how the mitotic spindle is assembled and disassembled. Beyond this, we need to understand how controls integrate the behavior of the spindle with the replication and segregation of the chromosome, as well as integrate growth and differentia- tion with cell division. Most importantly, we need to understand how cells "decide" whether or not to leave their normal resting state in tissues and go on to grow, replicate their DNA, and divide. An identification of these controls should lead to a real understanding of several important diseases-most notably prolif- erative diseases such as cancer and degenerative diseases-some of which are likely to result from a failure of normal proliferation. Controls that integrate growth with division occur in the first growth phase of the cell cycle, called G1 in yeast and animal cells. Although many of the components of this control system have been identified in yeast, the links between nutrition, protein synthesis, and the apparatus for cell division remain unknown even for this unicellular organism. After a century of study, the mechanisms that move chromosomes during cell division in both somatic cells (mitosis) and germ cells (meiosis) are finally becoming clear. The main elements are well known: the mitotic spindle com- posed largely of two arrays of microtubules. One set runs from the centrioles at the spindle poles to the centromeres (kinetochores) of the chromosome. The chromosomes are pulled to the poles as the kinetochore moves along these microtubules toward the poles. Remarkably, this movement seems to be powered by energy stored in the microtubules, whose depolymerization at the kinetochore determines the rate of movement. The second set of microtubules runs from one pole toward the other. These microtubules are slid past each other by an ATP- requiring motor to push the poles apart, which helps to separate the two sets of chromosomes. A centriole is located at each pole and remains one of the most poorly charac- tenzed elements from a molecular standpoint. An understanding of the molecular organization of the centriole will be essential in defining its role in chromosome movement, spindle and aster formation, and its other function as the basal body for cilia and flagella. Ultimately, the mechanical problems of chromosome movement will need to be placed in the overall context of cell-cycle control, including (before the actual separation of chromatics at mitosis) DNA replication, shutdown of RNA tran- scription, chromosome condensation, and breakdown of the nuclear envelope; and (after mitosis) nuclear envelope reformation, chromosome condensation, and reinitiation of transcription. Specific protein phosphorylations help drive a cell into mitosis, and the recent development of in vitro systems in which some of these processes occur outside the cell holds promise for a detailed molecular analysis in the near future. Remarkably, some of the central components of the control process are nearly identical in cells as disparate as those of humans and yeast; thus, many of the details of the human cell cycle can be worked on in simpler cells such as yeast, which are readily amenable to a combined molecular and genetic analysis.

120 OPPORTUNITIES IN BIOLOGY Mitosis in somatic cells and meiosis in germ cells resemble each other in such respects as the formation of the spindle apparatus and the general breakdown and reformation of the nucleus. However, details of chromosome behavior differ markedly. Numerous questions need addressing. What causes homologous chromosomes to pair before meiosis? How are the molecular events of crossing over controlled? What causes the unique behavior of the centromeres during meiosis? How is DNA synthesis suppressed before the second meiotic division? These special problems of meiosis remain largely unexplored from a molecular standpoint. Again, important information will come from organisms such as yeast. Cell-to-Cell Communication Cells Have Developed Mechanisms for Interacting with Other Cells Cell-cell interactions are important in simple organisms for such functions as sexual reproduction, colony formation, and attachment to various substrates. In multicellular organisms, cell-cell interactions have become much more complex, since they are essential for the integration of large cell populations into structur- ally coherent and functionally controlled tissues, organs, and organisms. Short-range communication depends on direct contact between cells and their neighbors or the surrounding environment. Long-range communication requires the movement of informational molecules (such as hormones and growth factors) from one cell to another through the blood or other extracellular spaces and the binding of these molecules to specific receptors on the surface of the target cell. Sh~rt-Range Corr~nunication Requires Plasma Membrane Specializations The critical importance of cell-cell interactions is illustrated by testing the developmental sequence of multicellular organisms. As the one-celled embryo begins to divide and cells begin to differentiate, mechanisms of short-range cell- cell communications emerge. They consist of gap (or communicating) junctions that link a cell to its neighbors through common transmembrane channels. These junctions create common intracellular environments in cell subpopulations and ensure rapid cell-to-cell propagation of membrane permeability changes and intracellular messengers. Short-range interaction mechanisms also include junc- tional complexes between cells, which allow the developing organism to build walls of cells, called epithelia, that separate the different parts of its body. In addition, cells in general and epithelial cells in particular participate in short- range interactions with the newly formed extracellular matrix. These interactions are mediated by mutual recognition between cell membrane receptors and spe- cific parts of matrix proteins. Cell membranes have multiple receptors for many matrix proteins, which are large monomeric or polymeric protein molecules with

GEAlES AND CFl.r ~ 121 specific sites for binding to the plasmalemma as well as to other matrix proteins. The result is the progressive construction of a mechanically coherent body in which the cells are kept in place by their attachment to one another as well as to structural differentiations (for example, basement membranes and collagen fi- bers) of the extracellular matrix. These attachments are effected through rigid plates on the plasmalemma that connect bundles of fibrils from the extracellular matrix to actin filaments or intermediate filaments in the cytoplasm. The rigid plates are maintained in relatively fixed positions by the f~brillar cables, which are under tension because they generally follow the lines of stress propagation within the entire system. This type of construction allows the cells to retain their shape, resist pressure, and recover from deformations. During embryonic development, the production of matrix proteins follows a sequential program presumably matched by the production of plasmalemmal receptors for specific matrix proteins. Certain cell migrations are controlled by cell-matrix interactions and can be experimentally blocked by antibodies to (or small peptides derived from) relevant matrix proteins. Cell migration is thought to be controlled by a process that activates secretion of matrix proteins and concomitant production of cognate receptors. The cells move along tracks laid down by themselves or by their predecessors. In the adult organism, gap junctions control the propagation of contraction waves in the heart muscle and in the smooth muscles of the intestine and uterus. Modulations in the construction of junctional complexes also control the permea- bility of epithelia in the intestine, lung, and kidney as well as the permeability of the vascular endothelium. Long-Range Communication Requires Messenger Molecules and Receptors As embryonic development progresses, mechanisms of short-range commu- nication are extended and diversified, but long-range interactions through hormones and growth factors become progressively more important. Long-range communi- cation requires the production of chemical signals, such as hormones and growth factors that react with target cells and modify their activities. The essential elements of any long-range communication system are a chemical messenger molecule, a cellular receptor, and a mechanism that transduces the binding of the messenger molecule to the receptor into a biochemical change in the target cell. The biochemical change then modifies the physiological behavior of the cell. Messenger Molecules Have a Wide Variety of Chemical Compositions The chemicals that carry messages from one cell to another are extraordinar- ily diverse. The classical hormones include derivatives of cholesterol (cortisol, testosterone, estrogens), derivatives of amino acids (thyroid hormone), small peptides (growth hormone), and other types of molecules (epinephnne). More

122 OPPORTUNITIES IN BIOLOGY recently, attention has turned to a growing list of protein and polypeptide messen- gers that regulate the cell cycle of target cells and are grouped together as growth factors. Growth Factors Growth Factors Are Ligands That Bind to Receptors and Cause DNA Synthesis to Begin Major advances in purification and characterization of the growth factors during the past decade have facilitated an understanding of their modes of action. Until the advent of genetic engineering, studies were limited to those factors available in sufficient quantity from biological sources. Much early work was done with three growth factors: erythropoietin, a glycoprotein from the kidney that stimulates red blood cell production from a common stem cell progenitor of both red and white blood cells; nerve growth factor (NGF), which stimulates the development of neurons; and epidermal growth factor HUGE), which was discov- ered in part because it induces premature eyelid opening and tooth eruption in neonatal mice by stimulating epidermal cell proliferation. The initial discoveries of these three hormones were made in animal models, which were later used to assay the progress of their purification. Research on these hormones was accelerated greatly during the past decade through advances in animal cell culture technology. One principal limitation of that technology was a requirement for serum. This was overcome through the realization that serum is a rich source of growth factors, especially those required by mesenchymal cells. That realization led to the identification of platelet-derived growth factor (PDGF), a powerful mitogen for fibroblastic cells that is wide- spread in nature and that can play multiple roles. Other hormones are present in platelets, including tumor growth factor beta, which is a potent modulator of mesenchymal cell proliferation as well as a powerful growth inhibitor for other cells such as epidermal cells. These observations helped explain the well- documented selectivity for preferred growth of mesenchymal cells in cell culture medium containing serum. Once this major roadblock was overcome, progress in identifying requirements for growing epithelial cells in culture was rapid. Much interest in controlling growth of epithelial cells results from their being the origin of approximately 90 percent of human tumors. The suspected role of growth factors, growth modulators, and their receptors in cancer has created intense interest in an understanding of their actions and synthesis. Unlike hormones produced by endocrine organs, such as insulin and growth hormone, growth factors are not secreted into the blood. Instead, these paracrine hormones are released at or near their target cells. A functional homer rogue of EGF, termed tumor growth factor alpha, and growth factors of the insulin-related families are secreted by a variety of cells lines derived from tu- mors. The normal progenitors of these tumor cells themselves are responsive to

GE: VES AND CELLS 123 these hormones from which the tumor cells have become independent. This class of mechanism, in which a growth factor is produced and utilized by the same cells, has been termed autocrine hormone function. Although uncontrolled auto- crine behavior could contribute to tumor progression, it is unlikely that acquisi- tion of a single uncontrolled autocrine mechanism would cause cancer. The stimulation of reproduction of epithelial or mesenchymal cells probably requires the synergistic action of several growth factors acting in a specific temporal manner. Immunomodulators, such as tumor necrosis factor and interleukins 1 and 2 (IL-1 and IL-2), are substances that influence the expansion of immune cell populations. They are produced and utilized by white blood cells at sites of inflammation, including areas of tumor growth, by a paracrine process. Thus the effective dose of a hormone normally develops only in the area of the cells that release it. A number of paracrine-acting immunoregulatory agents are currently undergoing clinical trials, even though extreme toxicity has frequently been observed at doses sufficiently high to achieve pharmacological effectiveness. Thus the development of drug delivery systems that mimic the local delivery specificity of a physiological paracrine process represents major challenges and new opportunities in current attempts to apply immunomodulators in cancer therapy. Receptors There May Be Even More Receptor Proteins Than Messenger Molecules Receptors are protein molecules that bind specific hormones or growth factors and relay a signal that converts an extracellular message into a biochemi- cal action inside the cell. There may be more kinds of receptors than messenger molecules because the receptors for a single messenger molecule can be different on different types of cells. The receptors handle their information-transduction functions in a variety of ways. During the past 20 years, our perception of receptors as molecular entities has changed dramatically. Our thinking has evolved from a picture of relatively inert structures able to bind specific ligands that then induce a signaling function to the concept that these proteins contain structural information that enables more diverse functions. We now understand that receptor systems contain structural elements that enable them to bind ligands, participate in signal transduction, and respond to regulation by various cellular mechanisms. In the simplest case (S1 in Figure 4-3) the hormone (for example, coriisol, sex steroids, vitamin D, or thyroid hormones) is sufficiently soluble in lipids to diffuse across the plasma membrane and act on an intracellular receptor. Alterna- tively, the receptor protein in the plasma membrane is oriented toward the exterior of the cell, where it can bind the extracellular ligand (S2) and carry its signal across the membrane into the cell (as low-density lipoprotein receptor and trans

124 DOOC ~ ~0_ D S1 S2 S3 S4 S5 OPPORTUNITIES INBlOWGY X Z Cytoplasm Extracellular fluid SO FIGURE 4-3 Strategies for transmembrane signaling. [Adapted from H. R. Boume and A. L. De- Franco, in The Oncogenes, R. Weinberg and M. Wigler, eds. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., in press), chapter 3] ferrin). Some receptors are transmembrane proteins with an extracellular portion that binds a ligand such as insulin, EGF, or PDGF (Sib; a transmembrane seg- ment; and an intracellular part with enzyme activity. Binding of ligands to the extracellular site can stimulate the phosphorylation of protein tyrosine groups by the kinase activity of the intracellular domain. Still other extracellular signals (S4), including acetylcholine and y-aminobutyric acid), act by binding to a trans- membrane ion channel; opening of the channel in response to binding of the ligand allows specific ions to cross the membrane and alter the electrical potential across the membrane. Finally, a large number of distinct extracellular signals (SS) are detected by receptors that act through a family of proteins (G proteins) that bind guanosine triphosphate (GTP) to regulate production of intracellular mediators of hormone action, known as second messengers [or signaling mecha- nisms, which include calcium and cyclic adenosine monophosphate (cyclic Abed. These second messengers then diffuse through the cell's interior and initiate various biochemical processes. These multiple receptor pathways allow a diverse approach to the control of receptor function and cell metabolism, permitting rational approaches in the design of therapeutic drugs to correct altered cell functions. Transmembrane Signaling Ion Channels Mediate the Cell's Electrical Communication with Its Environment It has been long been known that "excitable" cells in the neuromuscular system communicate rapidly by means of electric signals generated by the selec

GENES AND CFIal ~ 125 live passage of ions through protein channels in their plasma membranes. Now it is clear that virtually all cells use related membrane channels as part of their signal transduction systems. Membrane proteins, called ion channels, form tiny water- f~lled pores across the plasma membrane that are narrow enough to permit diffusion of one or another of small ions such as sodium, potassium, and calcium between the internal and external solutions. Passage of charged ions through the pore forms an electric current that generates potential changes for signaling. The nervous system generates many kinds of electrical signals by using many types of ion channels, each specific for a few common ions and each opened and closed under the control of specific stimuli, such as membrane potential changes, neuro- transmitters, light, or mechanical stimuli. The study of ion channels has advanced greatly during the past 10 years because of spectacular technical improvements in electrical recording methods and in molecular biology. A new recording method, called the patch clamp, permits routine observations of the opening and closing of single ion channels on a submillisecond time scale. Hardly anywhere else in science has it been possible to monitor conformational changes of one molecule. With the patch clamp, channels have been shown to click abruptly between open and closed conforma- tions. We are now faced with a wealth of recordings of the transitions that need to be understood in kinetic and ultimately molecular terms. Different channel types can be recognized by their patch-clamp signatures- different ionic preference, different absolute conductance, and different rules for opening and closing. At least 30 types have been recognized in the past 8 years, and new ones are being discovered every month. The patch clamp reveals that ion channels are present in the plasma mem- brane of all eukaryotic cells, not only excitable cells like nerves and muscle. Because this ubiquity was not previously suspected, an important task is to determine what roles these signaling molecules play in the housekeeping func- tions and daily life of cells outside the nervous system. By far the best-understood ion channel to date is the acetylcholine (ACh) receptor channel that mediates the transmission of signals from nerves to muscles (Figure 44~. The ACh receptor opens a pore in the muscle membrane when the neurotransmitter ACh is released by the nerve impulse in a nearby nerve terminal. The protein subunits of this pent~neric receptor molecule have been sequenced. DNA cloning and sequencing have revealed related ACh receptors in other cells. The cloned messages for various ion channels have been injected into frog oocytes, which then make the proteins and incorporate them into their plasma membranes, where they can be studied by patch clamp-methods. Several groups are selectively mutating these messages to test which parts of the sequence are responsible for each feature of the overall function. The three-dimensional structures of selected channels that should become available in the near future, along with results of ongoing molecular biological and electrophysiological stud- ies, will elucidate the operation of these channels at the molecular level.

126 OPPORTUNITIES INBIOLOGY ~VrE~RY~ ~ ~:~?:~: I ?he~ n?Ig?~ti~specit~icibr~hibit-by::-l~l~sUr~e~eptors~Ior~ho~rmon~es~:~ ~ ~ :~ ~ ~ ~ :~.~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~:~?~:~a nd~? ~athe~r~?:~g~ands ~ Nimbi new th~?~the~::~abil~ ty~of~::~recepto~rs Rio: ~ ~l~nte~n~al'~::~: ~ : ~ ~ ~ ~ ~ ~. T~ ~ ~ ~ ~ ~: ~ ~ :~ ~ ~ ~ ~ :~ :~ ~ ~ ~: ~ ~ ~ ~ ~ ~ ~: ~ ~ .- ~ :~ ~ ~:~?~?~?~n~o ~ mesa Ant ye ~specn~receplor~ Iga~nt :S At ~?u~s~promot~lng~spe~-~ ~:~ ~ ~ ~ ~ ~ ~.~ .~ In: At. ~ ~ ~ ~ ~ ~ ~ ~. ~ ~ ~ ~ ~ ~ ~ ~,~ ~ ~ ~: ~ ~ ~ ~ ~ ~ ~ ~ :: ~ :. ~ ~ ~?~c~c~'nternal~:lmt~on?: of?to~?n~w~?resu~lta:nt? - ll~death.~pe?rtm~ents:~w~th Intact I: ~:~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ T~ ~ ~in:~hav~u~nei? ui ~albr _ (i~ ~ ~ A ~ider~mal~ ~?~ ?9?rD.it?~a - t ~or~transfarrin~ :: ~pto~rs:~:~:~lntern~al~e~em:~?an~d ~:ld~ce~ll~deat~h~: :::: ~ .. ~ ~.~ ~.~ ~:~ ..: ~,.~. ~ ~ ~ ~ :~ ~.~ ~ ~ ~:~ ~ ~ ~:~ ~ ~.~ .~ ~ ~ I? resu?lis.~l~ln~oM~er forts ~techn~qus:~to <~be~u:s~l:~i~n~ vim~:~?tissue~ ~specif icity of ~:~ ~ ::~ _~ _ : :: ?~d~ : :: :::::: :~:: A: i:: I:::: ::: ::::: :::::: :::: .~ :: :: ~:::~: ::::.::: :::::~:: i:: :: :::: :~: :::~:: :~: :::: ::::::: :~:::::~:::: Sea: i:: ::: ~:::~:::~ ::: : ~ ::::: :::::: ~ ~ce ~ln~s~reGeplo~r-~-at~ateo~:ena~oc:ybos~are~:~oase£ ~on~a~n~re~neas~:~ ~ ~ ~:~ ~ ~ ~ ~ ~ ~ ~ ~'~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~. :~ ~ ~ :~ If: ~ ~ ~:~ ~ If:: ~ ~ ~ ~ ~ ~ ~::~::~betweenn~:~cel:ls~:~n~:~:~re~sp - Tore - ~p~r~ - ,~ss~lon~.~ ~O~u~s~tu~mo~r~:lls~Q~ : :::: ~ : ::~:: : : :~::: : ~ ::: :: :: ::: : I: ::: :: ::: : : : i:: ~ : :: : :::: :: If: :~: ~ ~ :~ :: : : : ::: ~ : : : i: :: ::::: : ::::: i: :: : : : :: : ::: :: ::::: : ~ : : : ::: ~ ::: :: :: :: ~l~uch~high6~r~n:u~mb~em~:~^ertair:~r~no~rmal c~s~from~ ~:~ : :~ ::: :~ ::::::: :~::~:~:: ~ ::::: ~ :: :: ~ : ::: : :: ::: :: : : ~ :::: ~ :~: : if:: :~:: ~ : ~ : ~ ~ ~ : :: ~ ~ ~ :: ~ :: ~e~d~ed .~: fir ~ta~dth6::~result:~:~ :::::::::::: :.:: :~:: :: :~: :::::::::: ~::~:: :::: :::::: If: :: :: ~ :::::::: .: ::: ~ :: :::: ::::::: :.::::: : :: I:: ::::: :::::::::::: ~:~:~: ::::: :::: :~:: :~::: ::::::::: :: :::: ::: ~ ::::: ~ :::::: : :::::::: ~ ::: :: ::::::: I: ~ : :::~:: :: ~ U ~u~: ~:~::~tumor~ce~lls~to~the kllil~9 act:lon~of~th~tox~In~-spe~le~llg~d~con)~ugates.~:~Alter-~:~::~:~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ :: ~ ~ ~.~ ~ ~ .:~y~:~some~:~:roor~:tells:~m~express:~un~s~6nti¢Qn~s~.~For~:~ ,~. A, ~ ~ ~ ~ ~: :~ ply ~ ~exp~res~s~n~ Woof ~:~:~th~e~fetal For m: ~:~a:~::~pto~r :~ so:m~lm es ~ occurs in ~ if: ~: :: :: ~ ~ ~therape~utlc:~ap~oach.~ ~ Bus ~ ~we~::~:h~a9Q~ New ~oppo:~r tu r~ies~to~:~test~:~th:es~e~: Hi- ~:~:~ :~:: I::::: :::::::::: ::: :::::: :: :: ::: :::: ::: :: ::: :: ::::::: : : ~:::::::: I: : :: :::::::: :: :: :::: : ::::::: : : ~ ~ poll yeses Do ~ op~:~ette~r:~straten'es~:'or~u I~ Nice ~:~s~u~rtace ~:~:~Iow~Tarcetlrl~:~soeenic~Is~Io~r:~orua~aellPvew.:~:~ :~ ::: :~ :::: if: ~ ::: : Captors to: ~:~ : ~ ::: ~: : ~ : ~ :: ~:~ :~:: : ~ :: I: :~ :: ::: : :::: ::~: :~ ~::::~:::: : : :: :: :~ ~ ~ I:::: ~ :::: : : :: ~ ~ :: : : : :~ ~ ~ Because ion channels are large, exposed macromolecules having important functions, they have become the target of many classes of natural toxins and of many clinically active drugs. Toxins from cobras, scorpions, cone shells, dinoflagel lates, puffer fish, frogs, sea anemones, and many plants act directly on channel molecules. The animal toxins are useful specific labels for the biochemical identification and purification of channel macromolecules. Exploration of such neurotoxic molecules continues to be a rewarding approach to obtaining new reagents for research.

GENES AND CF~ ACh in r~ir~ette ~:~.~-.' TV EM = -140 mV 1 ~ 100 msec 127 Embryonic muscle ~1 7 pA = 42 x 1 o6 ions/see ~ 1 ~ ~1 ~ ~ r O FIGURE 44 Acetylcholine opens receptor channels in muscle, a process that can be measured by the patch clamp technique. [Benil Hille, University of Washington] Plant neurotoxins have already been the inspiration for systematic develop- ment of standard clinical agents. Thus curare led to neuromuscular blocking agents, cocaine led to local anesthetics and a class of antiarrhythmics, and papav- erine led to another class of antiarrhythmics. These agents act on ACh receptor channels, sodium channels, and calcium channels, respectively, by mechanisms that are still only partially understood. Many widely used neuroleptics, tranquil- izers, and antipsychotics act directly on channels. There is some hope now that forthcoming knowledge of the three-dimensional structure of channels can lead to the rational design of major new classes of clinical agents with specific actions. Channel Modulation. All organs of the body are innervated by the two major branches of the autonomic nervous system-the sympathetic and the parasympa- thetic. In classical terms, signals in the sympathetic nervous system prepare each organ for times of stress-fight or flight whereas signals in the parasympathetic prepare for more vegetative functions such as digestion. The molecular details of how the body responds to these signals are emerging. The sympathetic neuro- mnsmitter (norepinephrine) and the parasympathetic neurotransmitter (ACh) act on several classes of membrane receptors to produce several intracellular second messengers, which in turn modulate the activities of a variety of ion channels. The receptors, the G proteins activated by the receptors, and effecter enzymes and channels are being identified, purified, and sequenced.

128 OPPORTUNITIES IN BIOLOGY Modulation by a cyclic AMP-dependent phosphorylation plays interesting roles in disparate activities. Stimulation of the sympathetic nerves to the heart releases norepinephrine, which speeds the heart rate and strengthens the stroke in each beat the familar response of the heart to exercise. We believe that in the pacemaker cells of the heart, the rate of depolarization in each cycle is set by the rate of opening of voltage-gated calcium channels and that in the ventricle the force of the pump stroke is set by the number of calcium ions entering per beat. Much of the response to sympathetic stimulation can be attributed to the phosphorylation of voltage-gated calcium channels, which increases the probabil- ity of their opening. Each step, from activation of the ,3-adrenergic receptor by norepinephrine to phosphorylation of the channel, has been carefully documented. Another example is a learninglike response called sensitization in the sea slug Aplysia. Here serotonin released by action of one set of sensory nerve fibers increases the neurotransmitter released by another. The cyclic AMP-dependent phosphorylation of a potassium channel, which here shuts the channel off, seems to account for this sensitization. A major recent triumph of visual physiology was the discovery of how the light signal modulates the operation of a channel in rods and cones of the retina to initiate the electrical signals leading to vision. The coupling of rhodopsin to transducin to a phosphodiesterase enzyme is described in Chapter 6. The impor- tant point here is that the result of a cascade of reactions is the eventual change of the concentration of cyclic guanosine monophosphate (cyclic GMP), which is the direct regulator of the ionic channel. This final stage of transduction was demon- strated with the patch-clamp technique: The channel opened whenever cyclic GMP was applied to the cytoplasmic side of a patch of membrane pulled off the photoreceptor. The possibility that olfactory or taste transduction also requires a cascade of intermediate intracellular messengers offers opportunities for study in the coming years. G Proteins Are Crucial in Many Kinds of Signal Transduction Cyclic AMP was discovered as an intracellular second messenger for epi- nephrine and glucagon more than 25 years ago. Cyclic AMP is synthesized by hormone-sensitive adenyl cyclase. It mediates the cellular effects of a host of polypeptides, biogenic amines, and lipids that regulate mobilization of stored energy (carbohydrates in liver, triglycerides in fat cells), conservation of water by the kidney, homeostasis of calcium ions, contractility of heart muscle, production of adrenal and sex steroids, and many other endocrine and neural functions. Studies indicate that odorant stimuli activate adenyl cyclase in the olfactory epithelium of the nose, suggesting that cyclic AMP is also the intracellular second messenger that mediates the sense of smell. More recently, biochemical and genetic studies of the regulation of cyclic AMP synthesis led to the discovery of Gc, a membrane protein that couples hormone receptors to stimulation of adenyl cyclase.

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]

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

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.

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.

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

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]

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.

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.

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,

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

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.

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Biology has entered an era in which interdisciplinary cooperation is at an all-time high, practical applications follow basic discoveries more quickly than ever before, and new technologies—recombinant DNA, scanning tunneling microscopes, and more—are revolutionizing the way science is conducted. The potential for scientific breakthroughs with significant implications for society has never been greater.

Opportunities in Biology reports on the state of the new biology, taking a detailed look at the disciplines of biology; examining the advances made in medicine, agriculture, and other fields; and pointing out promising research opportunities. Authored by an expert panel representing a variety of viewpoints, this volume also offers recommendations on how to meet the infrastructure needs—for funding, effective information systems, and other support—of future biology research.

Exploring what has been accomplished and what is on the horizon, Opportunities in Biology is an indispensable resource for students, teachers, and researchers in all subdisciplines of biology as well as for research administrators and those in funding agencies.

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