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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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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
Representative terms from entire chapter:
viral dna
