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FRONTIERS OF BIOLOGY of mammalian cells of various cell types and to observe some of the chem- ical events associated with each of the four major phases of cell life. Discovery of the consequences of cell~ell interactions has been par- ticularlv significant. The free-livine mammalian cell is motile and proceeds ~ ;, ~ through round alter round ot the cell cycle, out wnen cells In culture nnc~ each other and establish physical contact, motility abruptly ceases and the cells are likely to remain in phase Go. Whatever may initiate the cancerous transformation, the consequences are exactly the reverse, regaining of motility and re-entry into Stage S. Cell-cell interactions are of immense significance in cell life, but the molecular bases for such events remain totally obscure. DEVELOPMENT OF AN ORGANISM Sexual reproduction, the addition of two sets of genes, makes possible the great variety of individuals that constitute a species, and also underlies the processes that have made possible the origin of new species. No phenom- enon within the experience of man is more wondrous than the events follow- ing the union of sperm and egg to form a complete zygote. The fertilized egg divides again and again. The embryo grows and proceeds through an invariant series of shapes. Early in the process, all cells appear outwardly to be virtually identical, although in fact this is never true. With the passage of time, however, some cells begin to take on the characteristics of the specialized cells of adult forms. Primitive organs develop; here and there cells die while other cells migrate from relatively distant portions of the embryo to take new places. In time, there is a fully formed organism, with each cell seemingly in a foreordained place and exhibiting the special properties of the cells of muscle, nerve, liver, kidney, bone marrow, connective tissue, etc. The heart of the mystery lies in the fact that all the information directing this process must, necessarily, have been encoded in the nucleus of the zygote. Moreover, all the genetic infor- mation of the zygote is later to be found in the nuclei of all the cells of the fully formed organism. How then does the process of differentiation occur? In the main, understanding of the basis for differentiation remains woe- fully meager. Two models are already before us. The first comes from the understanding. oriainallv pained from studies of bacteria, that, at any _ ~ in, ~ ~ ~ . time in the life of a cell, only a portion of its genetic information is being expressed; much of the genome remains repressed until some specific event can achieve derepression. Similarly, in each of the four major phases of the life of any dividing cell in the normal cell cycle, different aspects of the

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THE LIFE SCIENCES genome find expression. We can only assume that essentially equivalent events are operative in the process of differentiation. In the adult form, only cells that become erythrocytes engage in the synthesis of hemoglobin, but the genes for hemoglobin synthesis are to be found in all cells. Only cells destined to be muscle synthesize the contractile protein rnyosin; only fibro- blasts make collagen; only cells in the retina elaborate and organize the visual pigments; and so on. This is a thoroughly satisfying concept, but it provides no insight into the actual nature of the repressors and derepres- sors; it fails to account for the events that give direction to the future of any specific cell. An intense effort is currently under way to ascertain the intrinsic nature of these events now that a suitable set of models permits rational questions. Patently, each cell-in the developing embryo is sensitive to its environ- ment. Long suspected, the definitive evidence came from studies of bits of embryo in tissue culture. Tiny embryo sections taken from an area that, from past experience, should go on to become working muscle have been grown in tissue culture; they go through repeated cell divisions, and, at a characteristic time, the cells gather together to form a primitive "myotube." Cell membranes become obscure as the cells blend into a single syncytium, which then begins to contract spontaneously. But the initial cell samples were found to have contained two types of cells: One type normally goes on to become muscle, and one becomes the fibroblasts responsible for the synthesis of the connective tissue protein, collagen, as well as the variety of carbohydrate polymers that are secreted into the medium. If the same experiment is repeated, starting with only a single cell capable of becoming muscle, the cells do indeed proliferate but the myotube does not form and contractions are not evident. If, however, the flask in which the experiment is conducted is coated with some pure collagen, then the ensuing events are much as might have occurred were fibroblasts present in the culture. Thus, only when the potential muscle cells "sense" collagen in their environ- ment can they complete their normal development. Moreover, such experiments need not be conducted by starting with relatively primitive cells. Fully differentiated cells lens or retina cells of the eye, primitive pancreas cells, or those of the kidney~an be employed in such experiments and will divide as many as 50 consecutive times, each division yielding differentiated cells resembling the one cell with which the experiment began. But again, such experiments are feasible only when conducted in a medium that provides fluid from the area originally sur- rounding the cell with which the experiment began. Indeed, that cell can be separated from the fluid by membranes of varying porosities, and the experiment can be successfully repeated. It is clear that the dividing cells are receiving "information" from their environment, but in no instance,

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FRONTIERS OF BIOLOGY 73 except in the requirement of dividing muscle cells for collagen, is there understanding of the nature of that chemical message, much less how that message affects the genetic apparatus. Cell~ell interaction apparently lies at the heart of the differentiation process, but again its nature is obscure. The chemical aspects of the cell surface remain to be established. Examination by the techniques of im- munochemistry shows that each differentiated cell type has a defined chemical surface specific to that type. More than that. within a riven type. .. . . . .. is. .. .. .. . there must be subtle distinctions that account for the mechanism by which a given nerve cell takes up residence in such a position as always to form a synapse with a specific other nerve cell or to innervate a specific structure. That the latter does indeed occur is shown by a few relatively simple ex- periments. For example, if the nerve to an area of skin is sectioned and the patch of skin is rotated by 180 degrees- e.g., the skin that used to be on the abdomen now lies on the back-the nerve grows out new endings to the skin. But if now one lightly touches the animal's back, he scratches his abdomen. As the nerve grew back, it found the original types of cells with which it was supposed to make contact, but the brain records those as having been in their original position. In certain lower forms like the frog, one can successfully section the optic nerve and then rotate the globe of the eye by 180 degrees. The optic nerve regenerates, and each nerve sends out endings until it finds the same portion of the eye to which it had origi- nally been attached. In consequence, when a fly appears overhead, the frog's tongue darts down instead of up. The nerves regenerated and found the original portions of the eye to which they had always been attached, and hence in the frog's forebrain his entire optical field has been inverted with respect to reality. What chemical attraction leads the growing nerve cell to the portion of the retina to which it was "foreordained"? If man is to understand himself, to find new bases for contraceptive techniques, to find diagnostic procedures that can detect improperly fash- ioned fetuses early in pregnancy, perhaps one day to undertake surgical repair of such fetuses, it is imperative that an intensive effort be made to understand the fundamentals of the process of differentiation. Only a beginning has been made in this direction. Perhaps the experimental limi- tation has been failure to identify the most suitable biological study object. Much of earlier embryology rested on studies of the development of sea urchins, frogs, salamanders, and chicks. None has proved quite as reward- ing as the use of E. cold for genetic studies or use of the squid axon in neurophysiology studies. Many investigators have been seeking simple models of the differentiation process-for example, spore formation in bacteria and fungi or flagellum formation in motile bacteria-systems that show great promise but that have not yet revealed new generalities. Rather,

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74 THE LIFE SCIENCES have they simply afforded additional examples of the validity of the concept of genetic repression and derepression as the underlying mechanism of differentiation. Consider, for example, the initial event of fusion of sperm and egg. This event has been studied for generations; electron microscopy has now re- vealed the process in great detail, but, withal, the process remains mysteri- ous. The developed egg, before fertilization, is unique among cells. It is extremely large, as cells go, and is provided not only with storage carbohy- drate and fat, but also with an abundance of nucleoli, the small bodies within the nucleus within which are made the specific forms of RNA that serve as the structural, and perhaps functional, material of ribosomes. The mature egg already has a very considerable complement of ribosomes on which RNA messages are lodged, but these are not finding expression- protein synthesis is not occurring. Upon the approach of the sperm, its "acrosome," the bulge at the leading edge, burrows through the egg cell membrane, the sperm nucleus seeks and finds the egg cell nucleus, and these fuse. Shortly thereafter, the ribosomes become "activated," the nucleoli begin to disappear, more ribosomes appear in their stead, and the preformed messages begin to be read. Thus, the initial events in embryo formation are turned on by the introduction of the sperm nucleus, but the messages which are then read were prepared in the developing egg before ever the sperm entered. The chemical nature of this activation process is totally unknown. In mammals, the egg then travels down the horn of the uterus and almost invariably lodges in the same spot on the thin wall of the uterus. Neither the mechanism nor the virtue of this "choice" of location is known. But it is there that placenta formation is to occur. Cell division occurs all the while the fertilized egg travels down the uterus to its location and may exist in the 100-cell stage of the blastocyst at the time of nidation attachment to the uterine wall. In species that prepare more than one egg at a time, the eggs then space themselves equidistantly in the uterus. The basis for this spacing is unclear. Thus description of the early stages of mammalian life has become much more detailed, but understanding of the process is surprisingly meager. Development of the Nervous System Through interactions of embryonic cells and tissues, some cells on the dorsal surface of the early embryo are induced to organize themselves into a thickened plate, which rolls into a tube and sinks beneath the surface. From this tube and from associated cellular masses, the entire nervous system arises. The essential events during this key phase of development

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FRONTIERS OF BIOLOGY involve individual cells that, typically, form elongated cytoplasmic strands that become the adult nerve fibers. Before the embryonic nerve cells first send out their processes toward the periphery or to some station within the nerve centers, four interrelated events may be discerned: cell divisions; migrations of cells; changes in the shape, size, and content of individual cells; and the death and disappearance of partly differentiated cells. Closely timed and precisely interlocking, these events produce all the major features that finally characterize the adult nervous system-e.g., a recognizable neural axis with appropriately segregated parts, rudiments of cranial and spinal nerves, and enlargements, foldings, and outpocketings that foretell both the final gross form and the inner detail of localized cell groupings. The key point is that the final unity emerges from the coordinated activity of thousands of individual cells, each engaging in one or more of the four activities mentioned, each behaving as if it knew its place in the final structure, yet each subordinating itself to that structure. Just as these developmental events are to be understood best in cellular terms, so the function of the completed nervous system demands expres- sion in the same terms. The human brain and spinal cord consist of billions of cells, all arranged in an orderly manner with precise intercon- nections, structural and functional. Communication among nerve cells depends on the passage of impulses. These arise in individual cells and are effectively transferred from cell to cell either by one cell directly exciting another or by an intermediate step involving a chemical transmitter. Small groups or very large numbers of nerve cells are thus brought into activity in highly selective patterns. Out- of this complex of neural events emerges the coordinated product we call behavior. In the early embryo the young neurons develop sharp affinities and dis- affinities for one another and for peripheral tissues. They reveal these properties both by entering selectively into specific cellular associations that become the interknit pathways and centers of the adult nervous sys- tem and by selectively re-establishing some of these associations during nerve regeneration. The capacity of developing cells generally to form intercomplementary groupings lies at the basis of essentially all embryonic events at cellular and higher levels, but the origin of these abilities and their nature are largely unknown. Plant Embryogenesis The developmental process in plants presents a unique set of puzzles. One attribute of this process-the photoperiodic control of flowering is par- ticularly fascinating. In many plants the length of the daily exposure to

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THE LIFE SCIENCES light determines whether flowers and their associated structures can de- velop and thus, in effect, controls sexual reproduction. This process is often quite striking. For example, some varieties of soybeans are "short-day" plants, which flower only when the daily period of illumination is shorter than some critical day length. In consequence, although plant sowings may have been made over the spring and summer and the plants are thus of varying size, when the days begin to grow short, all flower at once. If kept under artificial illumination, they may never Dower at all. Mysteriously, in many such short-day plants the effect of a few days' exposure to an appropriate day length may persist long afterward. For example, in the cocklebur, a com- mon weed that will never flower if kept under continuous light, flowering will be initiated if the plant is exposed only once to a dark period more than nine hours long, even if it is immediately replaced in continuous light. No anatomical or biochemical test has yet distinguished between induced and noninduced plants immediately after the inducing dark period. Yet clearly, some basic change has been brought about, some self-main- taining or steady-state condition that governs the entire subsequent devel- opment without additional external stimulus. Obviously, insight into the nature of this induced state would be valuable to commercial plant growers. Even more mysterious is the fact that the effects of the continuous dark period can be utterly abolished if it is interrupted by only one minute of exposure to light! Red light is the most effective region of the spectrum, and even this can be annulled if the brief exposure to red light is followed immediately by exposure to light of a wavelength in the "far red." Discovery of these properties led to a search for a pigment with an appropriate absorption spectrum that might account for those effects. Such a material, a protein called "phytochrome," has indeed been isolated. But the function of this material in plant physiology continues to remain obscure. One other facet of photoperiodism the fact that it can be translocated- is of interest. For short-day plants, flowering can be induced if only a portion, indeed if only one leaf, of the plant is maintained in the dark for a sufficient period. Presumably, this implies the formation of some com- pound that can serve as a plant hormone and, leaving the site of formation, will trigger the events that lead to the initiation of flowering. All attempts to find such a hormone have thus far failed, yet such a compound must exist. For example, if a branch of a plant maintained in continuous light is grafted onto a plant that has been maintained for a sufficient period in the dark, to induce its own flowering, flowering is also initiated in the grafted branch, which has never been out of the daylight. These events, of obvious commercial interest to plant growers, are also of profound significance to the whole field of developmental biology because they may

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FRONTIERS OF BIOLOGY 77 well serve as clues to the nature of the chemical messages that induce dif- ferentiation in all developing embryonic forms. Animal Viruses An understanding of the cellular processes induced by infection with animal viruses is important for progress in several areas of biological and medical sciences. About 60 percent of all illnesses are estimated to be caused by viral infection. The meager results of over 20 years of empirical searching for clinically useful antiviral agents indicate that further knowledge of the fine structure of viral components and the molecular events involved in viral replication and virus-induced cell damage is required in order to design effective agents for the treatment of viral diseases. Understanding of the cancer cell and the neoplastic process in molecular terms, a hopeless pursuit several years ago, is now a realistic research goal for those studying tumorigenic viruses and the mechanism of virus-induced cell transformation. Because tumor-producing viruses can now be obtained in highly purified form and suitable "normal" cells in culture can be trans- formed into "neoplastic" cells, experimental systems are becoming avail- able for a rational analysis of the molecular basis of neoplasia. Animal viruses provide unique experimental footholds for attacking these complex problems of mammalian cell function for which other ap- proaches may be virtually nonexistent. Virus infection is the only experi- mental procedure available by which a defined segment of genetic material can be introduced into a mammalian cell. Since viruses contain only a limited number of genes, from five to several hundred, it is technically feasible to analyze in detail the factors governing the synthesis of specific viral macromolecules employing the virus-infected cell for experimental analysis. More than 500 animal viruses of various sizes and degrees of chemical complexity have been discovered, each containing either DNA or RNA and multiplying or maturing in different parts of the host cells; over 300 of these can infect man. Animal viruses have been tentatively classified into eight groups on the basis of biochemical and biophysical properties of the virion (the extracellular mature virus particle): four DNA-contain- ing groups, the papovaviruses, adenoviruses, herpesviruses, and poxviruses; and four RNA-containing groups, the picornaviruses, reoviruses, arbo- viruses, and myxoviruses. Viral DNA's range from 3 million to 160 million in molecular weight, are double-stranded, and either circular (papova- viruses) or linear. Viral RNA's have molecular weights ranging from 2 million for the picornaviruses to over 10 million for the reoviruses, the

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78 THE LIFE SCIENCES latter uniquely being double-stranded. Their great variety of chemical compositions, structures, and sites of replication, presumably also reflecting differences in replicative patterns and induced cellular modifications, makes animal viruses unique tools in experimentally dissecting cellular function in molecular terms. By infecting homogeneous cell cultures, one can insert viral genetic material of different types and sizes into defined intracellular regions and study the ensuing biosynthetic events. Studies with representative members of the four DNA virus groups, during the past five years, have revealed the following series of events during an infectious cycle: (1) attachment of virus to specific receptor sites on the host cell; (2) uptake of intact virus into phagocytic vesicles and transport to cytoplasmic or, possibly, nuclear sites; (3) intracellular un- coating of viral DNA; (4) transcription of specific regions of viral DNA; (5) attachment of the transcribed product, viral mRNA, to cytoplasmic or, possibly, nuclear ribosomes; (6) synthesis of viral-specific enzymes and other "early" proteins, utilizing the viral message on cellular ribosomes; (7) replication of viral DNA by enzymes of uncertain origin; (8) a second wave of transcription involving parental or progeny viral DNA or both; (9) translation of these viral mRNA's to viral-structural proteins and other viral-specific proteins, some of which engage in regulatory functions such as "switching off" the synthesis of virus-induced "early" enzymes, pre- sumably as repressors; and ( 10~ the final construction of the virion, presumably by self-assembly. While the overall replication patterns of the various DNA viruses are similar, the individual biosynthetic steps can differ considerably. Three interesting general patterns involved in the replication of most DNA viruses are the early inhibition imposed upon host-cell macromolecular synthesis, the mechanism of which is unknown; stimulation of the activity of DNA- synthesizing enzymes; and the regulatory role of "late" viral gene products on "early" viral gene functions. Further analysis of these phenomena should provide insight into the mechanisms that regulate cellular and viral macromolecule synthesis. Hopefully, information concerning regulatory mechanisms that operate specifically in viral infections may provide clues to the control of virus disease. The biosynthesis of RNA animal viruses, especially poliovirus, has been studied intensively during the past few years and, although similar to that of DNA viruses in many respects, it differs in several significant ways. RNA viruses attach to cells, penetrate, and are then uncoated. But the DNA-RNA transcription step, characteristic of DNA viruses, is bypassed. The parental viral RNA strand itself serves as a messenger RNA. It forms viral polyribosomes and directs the synthesis of viral-specific proteins, among which are: (1) regulatory proteins that, in some manner, inhibit

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FRONTIERS OF BIOLOGY normal host-cell synthesis of RNA and protein, (2) a unique RNA-de- pendent RNA polymerase that catalyzes synthesis of new viral RNA on the surface of the invading RNA, and (3) viral-structural proteins. RNAase-resistant RNA structures, thought to be double-stranded inter- mediates in viral RNA replication, have been demonstrated with many RNA viruses. Interferon is the name given to a cell-coded protein formed in response to infection with most DNA and RNA animal viruses. In sufficient quantity, interferon inhibits virus multiplication and, thus, may play a role in recov- ery from viral disease. Recent studies indicate that interferon acts by inducing the synthesis of a second cell protein that, in turn, somehow pre- vents the association of viral mRNA with ribosomes to form viral poly- ribosomes. Clearly, if a sufficient supply were available, interferon would have great therapeutic potential. Current research is directed toward means of eliciting maximal interferon synthesis in cell cultures. A double-stranded, synthetic RNA has proved quite effective in this regard and has afforded protection against inoculation of mice with large doses of hoof-and-mouth .. . alsease virus. Conversion of a normal cell to a cancer cell is thought to involve a permanent genetic alteration in the affected somatic cells. But the mam- malian cell contains at least a million genes, and identification of the specific genes that are altered, in terms of their products (i.e., proteins and enzymes), without some suggestive clues has posed insurmountable tech- nical difficulties. However, the analysis of viral carcinogenesis greatly simplifies this problem. Cancer-producing viruses contain only between 7 and 50 genes, yet one or several of these genes can induce cancer in animals and transform normal cells to malignant cells in culture. Under- standing of the functions of these relatively few viral genes should greatly facilitate elucidation of the carcinogenic process. Indeed, significant progress has been made in understanding the mech- anism of tumor induction and cell transformation by DNA and RNA oncogenic viruses. Shortly after they invade their host cells, the DNA C7 ~ tumor viruses, including eight human adenoviruses (31 human adeno- viruses are known), virus SV40, and polyoma virus, are no longer demonstrable as infectious in virus-induced tumor cells. However, the presence in and on these cells of viral-specific proteins ("tumor antigens") distinct from virion structural proteins argues for the persistence and func- tioning of at least part of the viral genome in the tumor cell. The direct demonstration of viral DNA in virus-induced tumor cells is a technically formidable problem since a single viral gene would represent a very small portion of total cellular DNA, about one part in a million. However, it has recently been shown that tumor cells, induced in animals by polyoma 79