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                             Radioisotopes
                           and Life Processes


The Understanding the Atom Series

Nuclear energy is playing a vital role in the life of every man, woman,
and child in the United States today. In the years ahead it will affect
increasingly all the peoples of the earth. It is essential that all
Americans gain an understanding of this vital force if they are to
discharge thoughtfully their responsibilities as citizens and if they
are to realize fully the myriad benefits that nuclear energy offers
them.

The United States Atomic Energy Commission provides this booklet to help
you achieve such understanding.

                  [Illustration: Edward J. Brunenkant]

                                          Edward J. Brunenkant, Director
                                       Division of Technical Information


UNITED STATES ATOMIC ENERGY COMMISSION

  Dr. Glenn T. Seaborg, Chairman
  James T. Ramey
  Wilfrid E. Johnson
  Dr. Theos J. Thompson
  Dr. Clarence E. Larson




                            _Radioisotopes_
                                                      AND LIFE PROCESSES


by Walter E. Kisieleski and
Renato Baserga




                                CONTENTS


  INTRODUCTION                                                          1
  CELL THEORY:
              DNA IS THE SECRET OF LIFE                                 2
  RADIOACTIVE ISOTOPES:
              THE BIOLOGICAL DETECTIVES                                10
  DNA SYNTHESIS:
              THE AUTOBIOGRAPHY OF CELLS                               15
  RNA SYNTHESIS:
              HOW TO TRANSLATE ONE LANGUAGE INTO ANOTHER               25
  PROTEIN SYNTHESIS:
              THE MOLECULES THAT MAKE THE DIFFERENCE                   35
  CELL CYCLE AND GENE ACTION:
              LIFE IS THE SECRET OF DNA                                37
  ISOTOPES IN RESEARCH:
              PROBING THE CANCER PROBLEM                               43
  CONCLUSIONS                                                          45
  SUGGESTED REFERENCES                                                 47


                 United States Atomic Energy Commission
                   Division of Technical Information
           Library of Congress Catalog Card Number: 66-61908
                            1966; 1967(Rev.)


THE COVER

The cover design portrays the inter-relationships suggested by the title
of this booklet: On a trefoil symbolizing radiation are superimposed a
dividing cell, a plant, an animal, and a double helix of a molecule of
deoxyribonucleic acid, a material unique in and fundamental to all
living things.


THE AUTHORS

[Illustration: WALTER E. KISIELESKI is an Associate Scientist in the
Division of Biology and Medicine of the Argonne National Laboratory. He
was formerly associate professor of chemistry at Loyola University in
Chicago. His undergraduate studies were at James Millikin University in
Decatur, Illinois, and his graduate studies were at the University of
Chicago. He received an Honorary Doctor of Science degree from James
Millikin University in 1962. In 1958 he was a delegate to the Second
Atoms for Peace Conference in Geneva, Switzerland. He was visiting
lecturer in the department of biochemistry at the University of Oslo in
Norway in 1963. Dr. Kisieleski is shown operating an automatic
windowless strip counter that scans paper chromatograms and thus locates
labeled substances.]

[Illustration: RENATO BASERGA was born in Milan, Italy, and received a
medical degree from the University of Milan in 1949. He is presently
research professor of pathology at the Fels Research Institute at Temple
University Medical School in Philadelphia, and associate editor of the
journal, _Cancer Research_. Formerly he was associate professor of
pathology at Northwestern Medical School in Chicago, where he was the
recipient of a Research Career Development Award from the National
Institutes of Health.]




                            _Radioisotopes_
                                                      AND LIFE PROCESSES


                                                 By WALTER E. KISIELESKI
                                                      and RENATO BASERGA




                              INTRODUCTION


  _Here and elsewhere we shall not obtain the best insight into things
  until we actually see them growing from the beginning._

                                                               Aristotle

The nature of life has excited the interest of human beings from the
earliest times. Although it is still not known what life is, the
characteristics that set living things apart from lifeless matter are
well known. One feature common to all living things, from one-celled
creatures to complex animals like man, is that they are all composed of
microscopic units known as cells.

The cell is the smallest portion of any organism that exhibits the
properties we associate with living material. In spite of the immense
variety of sizes, shapes, and structures of living things, they all have
this in common: They are composed of cells, and living cells contain
similar components that operate in similar ways. One might say that life
is a single process and that all living things operate on a single plan.

The past few years have been a time of rapid progress in our
understanding of the mechanisms that control the function of living
systems. This progress has been made possible by the development of new
experimental techniques and by the perfection of instruments that detect
what happens in the tiny world of molecules. Prominent among the methods
that have contributed to the explosive growth in our understanding of
biology is the use of radioactive isotopes as laboratory tools.

In this booklet we shall attempt to give an account, in chemical terms,
of the materials from which living matter is made and of some of the
chemical reactions that underlie the manifestations and the maintenance
of life. To accomplish this, we have chosen to describe three types of
molecules that have become the basis of modern biology: deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), and proteins. We will show how
radioactive isotopes can be used to pry into the innermost secrets of
these substances. Before we can understand the function of these
precious molecules, however, it will be necessary to review the
structure of a cell and the physical nature of radioactive isotopes.




                 CELL THEORY: DNA IS THE SECRET OF LIFE


  _We have seen that all organisms are composed of essentially like
  parts, namely cells; that these cells are formed and grow in
  accordance with essentially the same laws; hence that these processes
  must everywhere result from the operation of the same forces._

                                                         Theodor Schwann


Unit of Life

The cell theory, based on the concept that higher organisms consist of
smaller units called cells, was formulated in 1838 by two German
biologists, Mathias-Jacob Schleiden, a botanist, and Theodor Schwann, an
anatomist. The theory had far-reaching effect upon the study of
biological phenomena. It suggested that living things had a common basis
of organization. Appreciation of its full significance, however, had to
await more precise knowledge of the structure and activities of cells.

Some organisms,[1] for instance, amoebae, consist of a single cell each
and are therefore called unicellular organisms. Higher animals are
multicellular, containing aggregations of cells grouped into tissues and
organs. A man, for instance, consists of millions of many different
cells performing a variety of different functions. Cells of higher
animals differ vastly from one another in size, shape, and function;
they are specialized cells.

[Illustration: Figure 1 _One of the earliest photographs of cells taken
with a microscope. This photomicrograph shows cells in the blood of a
pigeon. It was made by J. J. Woodward, U. S. Army surgeon, in 1871.
Woodward had made the first cell micrograph (a graphic reproduction of
the image of an object formed by a microscope) in 1866._]

There is a remarkable similarity, moreover, in the molecular composition
and metabolism[2] of all living things. This similarity has been taken
to mean that life could have originated only once in the past and had a
specific chemical composition on which its metabolic processes depended.
This structure and metabolism were handed down to subsequent living
things by reproduction, and all variations thereafter resulted from
occasional mutation, or changes in the nature of the
heredity-transmitting units. One of the most extraordinary of all the
attributes of life is its ordered complexity, both in function and
structure.

It is agreed among biologists that the functional manifestations of life
include movement, respiration, growth, irritability (reaction to
environmental changes), and reproduction and that these phenomena are
therefore possessed by all cells. The first four of these can be grouped
under a single word: metabolism. We can therefore say that living things
have two common properties: metabolism and reproduction. Therefore, when
we say we are studying life processes, we actually are studying the
metabolism and reproduction of cells. Since metabolism is the sum of the
biochemical reactions taking place in a living organism, it properly
belongs to the field of investigation of biochemists. Cell reproduction
is the concern of both biochemists and morphologists[3] since it can be
studied by either biochemical or morphological techniques.


Cell Structure

[Illustration: Figure 2 _Generalized diagram of a cell, showing the
organelles, or “little organs”, of its internal structure. The
organelles that are labeled are important for this booklet._]

  Cell membrane
  Cytoplasm
  Chromatin
  Mitochondrion
  Nucleolus
  Endoplasmic reticulum
  Nucleus
  Nuclear membrane

The basic structure of a cell is shown in Figure 2. Each cell consists
of a dense inner structure called the nucleus, which is surrounded by a
less dense mass of cytoplasm. The nucleus is separated from the
cytoplasm by a double envelope, called the nuclear membrane, which is
peppered with perforations. The cytoplasm contains a network of
membranes, which form the boundaries of countless canals and vesicles
(or pouches), and is laden with small bodies called ribosomes. This
membranous network is called the endoplasmic reticulum and is distinct
from the mitochondria, which are membranous organelles (little organs)
structurally independent of other components of the cytoplasm. The outer
coat of the cell is called the cell membrane, or plasma membrane, and
forms the cell boundary.

[Illustration: Figure 3 _Electron micrograph of a primary spermatocyte
cell of a grasshopper, showing the nucleus (N), endoplasmic reticulum
(ER), mitochondrion (M), chromatin (C), nuclear membrane or nuclear
envelope (NE), cell membrane (CM), and intercellular space (I). The
magnification is about 25,000 times the actual size._]

The nucleus, which in many cells is the largest and most central body,
is of special importance. It contains a number of threadlike bodies, or
chromosomes, that are the carriers of the cell’s heredity-controlling
system. These contain granules of a material called chromatin, which is
rich in a nucleic acid, DNA (deoxyribonucleic acid). The chromosomes
usually are not readily seen in the nucleus except when the cell, along
with its nucleus, is dividing. When the nucleus is not dividing, a
spherical body, the nucleolus, can be seen. (In some nuclei there may be
more than one.) When the nucleus is dividing, the nucleolus disappears.

Not all cells possess all these structures. For instance, the red cells
of the blood do not have a nucleus, and in other cells the endoplasmic
reticulum is at a minimum. The diagram (Figure 2) is valid for a great
majority of the cells of higher organisms.

The cell structures shown in Figure 3 are visible with an electron
microscope. They contain the chemical components of the cell. The chief
classes of these constituents are the carbohydrates (sugars), the lipids
(fats), the proteins, and the nucleic acids. However, a cell also
contains water (about 70% of the cell weight is due to water) and
several other organic and inorganic compounds, such as vitamins and
minerals.

Carbohydrates serve mostly as foodstuff within the cell. They can be
stored in several related forms. Further, they may serve a number of
functions outside the cell, especially as structural units. In this way
structure and function are correlated.

Lipids in the cell occur in a great variety of types: alcohols, fats,
steroids, phospholipids, and aldehydes. They are found in all fractions
of the cell. Their most important functions seem to be to form membranes
and to give these membranes specific permeability. They are also
important as stores of chemical energy, mostly in the form of neutral
fats.

[Illustration: Figure 4 _Scientists using an electron microscope (left)
and an optical microscope (right) in fundamental biochemical research.
Both instruments are important tools in studies of life processes._]

The proteins occur in many cell structures and are of many kinds:
Enzymes, the catalysts for the cell’s metabolic processes, are proteins,
for instance. The nucleic acids are DNA and RNA (ribonucleic acid),
which function together to manufacture the cell’s proteins. Since a
large share of the remaining pages will be devoted to a discussion of
proteins and nucleic acids, at this point we need only emphasize that
these two types of materials are interrelated in their function and that
both are essential.


The Two Nucleic Acids

It is not very fruitful to discuss whether proteins or nucleic acids are
more important. That question is something like the one about the
chicken and the egg. We cannot think of one without thinking of the
other. Although our insight into the mutual dependence of these two
materials has greatly increased in recent years and although we know the
relation between them is a fundamental factor in such events as
reproduction, mutation, and differentiation (or specialization) of
cells, our understanding of their interplay is far from complete. Real
understanding of the relation between them would give us insight into
the essence of growth—both normal and abnormal—or, indeed, one could
almost say, into the complexity of life itself.

[Illustration: Figure 5 _Photomicrograph of Paramecia, one-celled
animals, magnified 1100 times. Many of the same structures that appear
in Figure 3 can be seen here. This photo was taken with an
“interference” microscope designed to permit continuous variation of
contrast in the subject under study._]

Practically all the DNA of most cells is concentrated in the nucleus.
RNA, on the other hand, is distributed throughout the cell. Some RNA is
present in the nucleus, but most of it is associated with minute
particles in the cytoplasm known as microsomes, some of which are
especially rich in RNA and are accordingly named ribosomes. These are
much smaller particles than the mitochondria.

[Illustration: Figure 6 _Stages of the mitotic cycle in a hypothetical
cell with four chromosomes._]

  (a) Interphase
  (b) Early prophase
  (c) Prophase
  (d) Metaphase
  (e) Anaphase
  (f) Telophase
  (g) Interphase, daughter cells


Mitosis

One of the most remarkable characteristics of cells is their ability to
grow and divide. New cells come from preexisting cells. When a cell
reaches a certain stage in its life, it divides into two parts. These
parts, after another period of growth, can in turn divide. In this way
plants and animals grow to their normal size and injured tissues are
repaired. Cell division occurs when some of the contents of the cell
have been doubled by replication, or copying (to be discussed later).
The division of a cell results in two roughly equal new parts, the
daughter cells. The process of cell division is known as mitosis and is
diagrammed in Figure 6.

Mitosis is a continuous process; the following stages of the process are
designated only for convenience. During _interphase_ the cell is busy
metabolizing, synthesizing new cellular materials, and preparing for
self-duplication by synthesizing new chromosomes. In _prophase_ the
chromosomes, each now composed of two identical strands called
chromatids, shorten by coiling, and the nucleolus and nuclear membrane
disappear. During _metaphase_ the chromosomes line up in one plane near
the cell equator. At _anaphase_ the sister chromatids of each chromosome
separate, and each part moves toward the ends, or poles, of the cell.
During _telophase_ the chromosomes uncoil and return to invisibility; a
new nucleus, nucleolus, and nuclear membrane are reconstituted at each
end, and division of the cell body occurs between the new nuclei,
forming the two new cells. Each daughter cell thereby receives a full
set of chromosomes, and, since the genes are in the chromosomes, each
daughter cell has the same genetic complement.

[Illustration: Figure 7 _Photomicrograph of cells of the Trillium plant,
which has five chromosomes, in anaphase. Note the duplicate sets of
chromosomes moving to opposite poles of the cell._]

All life processes use up energy and therefore require fuel. The
mitochondria have a central role in the reactions by which the energy of
sugars is supplied for cellular activity. The importance of this vital
activity is obvious. In this booklet, however, we are concerned with the
processes, involving nucleic acids and proteins, that can be described
as making up “the gene-action system”. The gene-action system is the
series of biochemical events that regulate and direct _all_ life
processes by “transcription” of the genetic “information” contained in
molecules of DNA.




            RADIOACTIVE ISOTOPES: THE BIOLOGICAL DETECTIVES


  _Man ... has found ways to amplify his senses ... and, with a variety
  of instruments and techniques, has added kinds of perception that were
  missing from his original endowment._

                                                        Glenn T. Seaborg


Atomic Structure

Practically everyone nowadays is to some extent familiar with the atomic
structure of matter. Atomic energy, nuclear reactors, and radioisotopes
are terms in everyday usage. However, to appreciate how radioisotopes
can be applied to the study of life processes, we must have at least a
working knowledge of their properties, their preparation, and their
limitations. It is therefore appropriate to examine them in detail so
that the succeeding chapters will be more easily understood.

According to present-day theory, an atom consists of a nucleus[4] that
is made up of protons and neutrons[5] and is surrounded by electrons. In
each atom there is an equal number of protons (positively charged) in
the nucleus and electrons (negatively charged) moving concentrically
around the nucleus; since neutrons have no electrical charge and since
protons and electrons cancel each other’s charges, the whole atom is
electrically neutral, or uncharged. Each atom is identified by an atomic
number and an atomic weight. The atomic number of an element (for
example, carbon, nitrogen, oxygen) is determined by the number of
protons, or positive charges, carried by the nucleus (or by the number
of electrons surrounding the nucleus, which is the same). The atomic
weight is the weight of an atom as compared with that of the atom of
carbon, which is taken as a standard. The weight, or mass, of an atom is
due chiefly to its protons and neutrons because the mass of its
electrons is negligible.


Isotopes

Atoms of the same element, that is, atoms with the same number of
protons and electrons, may vary slightly in mass because of having
different numbers of neutrons. Since the chemical behavior of an element
depends upon its electrons’ electrical charges, extra neutrons (which do
not have an electrical charge) may affect the mass of an atom without
disturbing its chemical properties. Atoms having the same atomic number
but different atomic weights are called isotopes. For example, as shown
in Figure 8, the isotope ¹H, or ordinary hydrogen, consists of a nucleus
containing a proton (charge: +1; mass: 1) around which revolves an
electron (charge: -1; mass: negligible); ²H, known as deuterium,
contains an additional nuclear particle, a neutron (charge: 0; mass: 1);
³H, or tritium, contains two neutrons. Since the chemical behavior of an
element depends upon the number of its electrons, these three atoms,
although differing in weight, behave identically in chemical reactions.
For convenience, the atomic weight is written as a superscript to the
left of the element’s symbol. For instance ¹⁴C is the isotope of carbon
with an atomic weight of 14 (ordinary carbon is the isotope with an
atomic weight of 12, and it is written ¹²C).

[Illustration: Figure 8 _Isotopes of hydrogen._]

  Hydrogen (¹H)
  Deuterium (²H)
  Tritium (³H)

Practically all elements have more than one isotope. There are two
general classes of isotopes, stable and radioactive. Stable isotopes
have no distinguishing characteristic other than their mass; radioactive
isotopes not only differ from their brothers in mass but also are
characterized by unstable nuclei. When the nucleus of an atom is
unstable, because of an unbalanced number of protons and neutrons, a
redistribution occurs sooner or later, and the atom decomposes
spontaneously and emits one of several kinds of radiations. Because of
their common mode of action and effects on living organisms, these
different kinds of radiations are known collectively as ionizing
radiations.

All radioactive elements emit one or more of three types of penetrating
(ionizing) rays. _Alpha rays_ or particles are double-charged helium
nuclei, ⁴He (atomic number: 2; mass: 4). They are emitted by many heavy
radioactive elements, such as radium, uranium, and plutonium. _Beta
rays_ or particles can be either positive or negative. Negative beta
particles are high-speed electrons and are emitted by many radioactive
elements. Positive beta particles are positively charged electrons
(positrons), have only a transitory existence, and are less common.
_Gamma rays_ are electromagnetic radiations, a term that also describes
radiowaves, infrared rays, visible light, ultraviolet light, and X rays.
Gamma rays are usually emitted after the emission of alpha or beta
particles. In our studies of life processes, we are interested only in
the radioactive isotopes that emit gamma rays or beta particles.


Radioactive Isotopes

Radioactive isotopes occur as minor constituents in many natural
materials, from which they can be concentrated by fractionation
procedures. In a very limited number of cases, more significant amounts
of a radioactive isotope, for example, radium or radioactive lead, can
be found in nature. Most radioactive isotopes in use today, however, are
prepared artificially by nuclear reactions. When a high-energy particle,
such as a proton, a deuteron, an alpha particle, or a neutron, collides
with an atom, a reaction takes place, leading to the formation of a new,
unstable compound—a man-made radioactive isotope.

The great usefulness of radioactive isotopes, as we shall see later, is
that they can be detected and identified by proper instruments.
Biochemists have long recognized the desirability of “tagging” or
“labeling” a molecule to permit tracing or keeping track of the “label”
and consequently of the molecule as it moves through a reaction or
process. Since the radiations emitted by radioactive isotopes can be
detected and measured, we can readily follow a molecule tagged with a
radioactive atom.

[Illustration: Figure 9 _A laboratory technologist preparing dissolved
biological materials as part of a study of the uptake of radioactive
substances in living organisms. Note the radiation-detection instrument
at right._]

The earliest biochemical studies employing radioactive isotopes go back
to 1924, when George de Hevesy used natural radioactive lead to
investigate a biological process. It was only after World War II,
however, when artificially made radioactive isotopes were readily
available, that the technique of using isotopic tracers became popular.

In our investigations of life processes, we are especially interested in
three radioactive isotopes: ³H, the hydrogen atom of mass 3; ¹⁴C, the
atom of carbon with atomic weight 14; and ³²P, the atom of phosphorus
with atomic weight 32. These radioactive isotopes are important because
the corresponding stable isotopes of hydrogen, carbon, and phosphorus
are present in practically all cellular components that are important in
maintaining life. With the three radioactive isotopes, therefore, we can
tag or label the molecules that participate in life processes.

[Illustration: Figure 10 _A visiting scientist at an AEC laboratory uses
radioactive tritium (³H) to study the effect of radiation on bean
chromosomes. The famous scientist, George de Hevesy, also used beans in
conducting the first biological studies ever made with radioisotopes._]

Hydrogen-3 is a weak beta emitter; that is, it emits beta particles with
a very low energy (0.018 Mev[6]) and therefore with a very short range.
Carbon-14 is also a weak beta emitter (0.154 Mev), although the beta
particles emitted by ¹⁴C have a higher energy and therefore a longer
range than those emitted by ³H. The beta particles emitted by
phosphorus-32 are quite energetic (1.69 Mev) and have a longer range.


Radioactive Isotopes’ Value in Biological Studies

To biologists, then, the essential feature in the use of radioactive
isotopes is the possibility of preparing “labeled” samples of any
organic molecule involved in biological processes. With labeled samples
it is possible to distinguish the behavior and keep track of the course
of molecules involved in a particular biological function.

In this capacity the isotope may be likened to a dynamic and
revolutionary type of “atomic microscope”, which can actually be
incorporated into a living process or a specific cell. Just as a real
microscope permits examination of the structural details of cells,
isotopes permit examination of the chemical _activities_ of molecules,
atoms, and ions as they react within cells. (Neither optical nor
electron microscopes are powerful enough for us to see anything as small
as a molecule clearly.)




               DNA SYNTHESIS: THE AUTOBIOGRAPHY OF CELLS


  _Here, surely, is the prime substance of life itself._

                                                            Isaac Asimov

The many characteristic features of each living species, its complex
architecture, its particular behavior patterns, the ingenious
modifications of structure and function that enable it to compete and
survive—all these must pass, figuratively speaking, through the eye of
an ultramicroscopic needle before they are brought together as a new,
individual organism. The thread that passes through the eye of this
needle is a strand of the filamentous molecule, deoxyribonucleic acid
(DNA). Let us now outline the research that led to these conclusions.


DNA in Somatic and Germinal Cells

One of the fundamental laws of modern biology—which states that the DNA
content of somatic cells is constant for any given species—was first set
forth in a research report of 1948. This finding means that in any given
species, such as a mouse or a man, all cells except the germinal cells
contain the same amount of DNA. Germinal cells, that is, the sperm cells
of the male semen and the female egg, contain exactly half the amount of
DNA of the somatic cells. This must be the case, since DNA is the
hereditary material, and each individual’s heredity is shaped half by
his father and half by his mother. One ten-trillionth of an ounce of DNA
from a father and one ten-trillionth of an ounce of DNA from the mother
together contain all the specifications to produce a new human being.

A large amount of DNA must be manufactured by an individual organism as
it develops from a fertilized egg (one single cell) to an adult
containing several million cells. For instance, a mouse cell contains
about 7 picograms of DNA (one picogram is one millionth of a microgram,
or one millionth of one millionth of a gram). A whole mouse contains in
its body approximately 25 milligrams (25 thousandths of a gram) of DNA,
and all this DNA was synthesized by the cells as the mouse grew to
adulthood. Since the amount of DNA per cell remains constant and since
each cell divides into two cells, it is apparent that each new cell
receives the amount of DNA characteristic of that species.

Once we realize that a cell that is making new DNA (as most cells do)
must divide to keep the amount of DNA per cell constant, it follows that
a cell that is making DNA is one that is soon destined to divide. If we
can now mark newly made DNA with a radioactive isotope, we can actually
mark and thus identify cells that are preparing to divide. The task can
be divided into two parts: (1) to label the newly made DNA and (2) to
detect the newly made, labeled DNA.


Replication of DNA

Figure 11 is a diagram showing the essential structure of the large DNA
molecule. According to the Watson-Crick model,[7] the molecule consists
of two strands of smaller molecules twisted around each other to form a
double helix. Each strand consists of a sequence of the smaller
molecules linked linearly to each other. These smaller molecules are
called nucleotides, and each consists of three still smaller molecules,
a sugar (deoxyribose), phosphoric acid, and a nitrogen base. Each
nucleotide and its nearest neighbor are linked together (between the
sugar of one and the phosphoric acid of the neighbor). This leaves the
nitrogen base free to attach itself, through hydrogen bonding, to
another nitrogen base in the opposite strand of the helix.

In the DNA of higher organisms, there are only four types of nitrogen
bases: adenine, guanine, thymine, and cytosine. Adenine in either strand
of the helix pairs only with thymine in the opposite strand, and vice
versa, and guanine pairs only with cytosine, and vice versa, so that
each strand is complementary in structure to the other strand (see
Figure 12). The full structure resembles a long twisted ladder, with the
sugar and phosphate molecules of the nucleotides forming the uprights
and the linked nitrogen bases forming the rungs. Each upright strand is
essentially a mirror image of the other, although the two ends of any
one rung are dissimilar.

[Illustration: Figure 11 _Diagrammatic structure of the DNA molecule as
proposed by the Watson-Crick model._]

  DNA: Diagrammatic single-strand chemical formula
  DNA: Structural arrangement

When DNA is replicated, or copied, as the organism grows, the two
nucleotide strands separate from each other by disjoining the rungs at
the point where the bases meet, and each strand then makes a new and
similarly complementary strand. The result is two double-stranded DNA
molecules, each of which is identical to the parent molecule and
contains the same genetic material. When the cell divides, each of the
two daughter cells gets one of the new double strands; each new cell
thus always has the same amount of DNA and the same genetic material as
the parent cell.

(All that has been said so far about DNA replication depends upon an
assumption that the DNA molecule is in some way untwisted to allow
separation of two helical strands, but there is no compelling reason to
believe that such an untwisting does indeed take place, nor do we know,
if the untwisting does take place, how it is accomplished. Much that has
been said in the last few paragraphs is therefore purely speculative. It
is, however, based on sound observation and is a more logical
explanation than others that have been advanced.)

[Illustration: Figure 12 _The pairing of the nucleotide bases that make
up DNA._]

[Illustration: Figure 13 _The DNA molecule and how it replicates. (a)
The constituent submolecules. (b) Assembly of subunits in complete DNA
molecule. (c) “Unzipping” of the double nucleotide strand. (d) and (e)
The forming of a new strand by each individual strand. (f) DNA molecule
in twisted double-strand configuration._

Adapted from _Viruses and the Nature of Life_, Wendell M. Stanley and
Evans C. Valens, E. P. Dutton & Co., Inc., 1961, with permission.]


Labeling DNA with a Radioactive Isotope

Of the four bases in DNA, three are also found in the other nucleic
acid, RNA; but the fourth, thymine, is found only in DNA. Therefore, if
thymine could be labeled and introduced into a number of cells,
including a cell in which DNA is being formed, we would specifically
label the newly synthesized DNA, since neither the old DNA nor the RNA
would make use of the thymine. We could in this way mark cells preparing
to divide. (Actually, thymine itself is not taken up in mammalian cells,
but its nucleoside is. A nucleoside is the base plus the sugar, or, in
other words, the nucleotide minus the phosphoric acid.) The nucleoside
of thymine is called thymidine, and we say that thymidine is a specific
component of DNA and can be used, both in laboratory studies and in
living organisms, for labeling DNA.

Thymidine labeled with radioactive compounds is available as
¹⁴C-thymidine (thymidine with a stable carbon atom replaced by a
radioactive carbon atom) and as ³H-thymidine (thymidine in which a
stable hydrogen atom has been replaced by tritium). Thus, when cells
actively making DNA are exposed to radioactive thymidine, they
incorporate it, and the DNA becomes radioactive.

We have thus found a way to complete the first part of the task, the
labeling of new DNA. We still must find out how to distinguish labeled
DNA among the many components of the cell. We might do it with a system
based on measuring the amount of radioactivity incorporated into the DNA
of cells exposed to radioactive thymidine, as an approximation of the
frequency of cell division in the group of cells. However, a better
method for studying cells synthesizing DNA, and thus preparing to
divide, is the use of high-resolution autoradiography.


Detecting DNA with Autoradiography

Autoradiography is based on the same principle as photography. Just as
photons of light impinging on a photographic emulsion produce an image,
so do beta particles (or alpha particles) emitted by decomposing
radioactive atoms. A photographic emulsion is a suspension of crystals
of a silver halide (usually silver bromide) embedded in gelatin. When
crystals of silver bromide are struck by beta particles, the silver
atoms are ionized and form a latent image, so called because it is
invisible to our eyes. After the emulsion is developed and fixed, each
little aggregate of reduced silver atoms becomes a visible black speck
on the emulsion. The distribution and combination of the specks make up
the photographic image (see Figure 14). In ordinary photography such an
image is a negative, which has to be converted into the positive
photograph by printing. In autoradiography we are satisfied to look at
the negative image since the clusters of developed silver atoms,
appearing under a light microscope as black dots, supply all the
information we need.

[Illustration: Figure 14 _Schematic diagram of a radioautograph._]

The distinction of having made the first autoradiograph belongs to the
French physicist, Antoine Henri Becquerel; and to another Frenchman, A.
Lacassagne, goes the credit for having introduced this technique into
biological studies. Lacassagne used autoradiography to study
distribution of radioactive polonium in animal organs. After World War
II, when radioactive isotopes were first available in appreciable
quantities, autoradiography was further perfected through the efforts of
such scientists as C. P. Leblond in Canada, S. R. Pelc in England, and
P. R. Fitzgerald in the United States.

Today autoradiography is sufficiently precise to locate radioactively
labeled substances in individual cells and even in chromosomes and other
structures within the cell. Two conditions must be met to achieve this
high resolution: (1) The radiation from the radioactive element in the
cells must be of very short range. (2) The cells must remain in close
contact with the photographic emulsion throughout the various
experimental manipulations. When these conditions are met, the black
dots will appear in the emulsion directly above the cell or cell part
from which the radiation came (see Figure 14).

Shortness of range is satisfied by use of tritium, since its beta
particles travel only about 1 micron (one thousandth of a millimeter)
and the diameters of mammalian cells range from 15 to 40 or more
microns. A mammalian-cell nucleus is at least 7 to 8 microns in
diameter.

[Illustration: Figure 15 _Cells being prepared for autoradiography. (a)
Cells being coated with a photographic emulsion. (b) Coated cells being
exposed to produce a latent image._]

The condition of close contact between cells and emulsion is achieved by
the technique of dip-coating autoradiography. In this process the glass
slide on which the cells are carried is dipped into a melted
photographic emulsion (see Figure 15a), a thin film of which clings to
the slide. After it has been dried, the slide is placed in a lighttight
box and kept in a refrigerator for the desired period of exposure,
usually several days or weeks. During this period disintegrating
radioactive atoms within the cells continue to emit beta particles,
which, in turn, produce a latent image in the overlying emulsion. After
the exposure is complete, the slide is developed and fixed like a
photographic plate, and a stain is applied which penetrates the emulsion
so that the outlines of the cells and their internal structures can be
seen. The fixing process removes all silver bromide that has not been
ionized so that the emulsion is reduced to a thin, transparent film of
gelatin covering the stained cells and containing only the clusters of
silver grains that were struck by the beta particles.

[Illustration: Figure 16 _Radioautographs of tumor cells. Above, tumor
cells and blood cells. Below, magnification of tumor cells._]

When the finished autoradiograph is examined under the microscope, it
will look like the radioautographs of tumor cells in Figure 16. In the
upper micrograph the tumor cells are the larger ones and the smaller
ones are blood cells. The dense structures in the center of the tumor
cells are nuclei. The cells were exposed to tritium-labeled thymidine,
and those synthesizing DNA at the time of exposure took up the thymidine
and became radioactive. They can be identified by the black dots
overlying the nuclei; the dots are the aggregates of silver grains
struck by the beta particles.

Notice that only the nuclei contain radioactivity; the reason for this
is that radioactive thymidine is incorporated only into DNA localized in
the nuclei of cells. This picture identifies not only the cells that
were making DNA at the time the label was administered but also the
cells that were destined to divide in the immediate future, since cells
synthesize DNA in preparation for cell division.

If we want to compare two populations of cells to find out which is
proliferating (dividing) more actively, counting the fraction of cells
labeled will give the number of cells synthesizing DNA in preparation
for cell division. Of course, a rough approximation of the proliferating
activity can be obtained by simply counting the number of cells actually
dividing. But with tritiated thymidine we can obtain not only much more
accurate measurements but also considerable information that cannot be
obtained by simply counting the number of cells in mitosis. We shall
discuss the cell cycle later on, but for the moment we should emphasize
that much of our knowledge of the cell cycle stems from the use of
high-resolution autoradiography.

It is clear that autoradiography enables us to find out _which_ cells
are dividing in a cell population and _how many_ of them do so. For
instance, in a given tissue or organ, not all cells are capable of
dividing into two daughter cells. In the epidermis, which is the thin
outer layer of the skin, only cells in the deepest portion can divide.
The other cells, although originating from cells in the deep layer, have
lost the capacity to divide, and eventually die without further
division. If we take a bit of skin, expose it to tritiated thymidine,
and determine the amount of radioactivity incorporated into the skin
cells’ DNA, we obtain a fair measurement of the amount of DNA being
synthesized. However, this purely biochemical investigation cannot
possibly give any information on which specific cells are synthesizing
DNA. For this, autoradiography provides the information we need.




       RNA SYNTHESIS: HOW TO TRANSLATE ONE LANGUAGE INTO ANOTHER


  _Mathematicians are like Frenchmen: whatever you say to them they
  translate into their own language, and forthwith it is something
  entirely different._

                                                     Wolfgang von Goethe

We have mentioned previously that there are two main types of nucleic
acids: DNA, the genetic material itself, and RNA, the molecule that
translates the genetic message from DNA into terms the cell can use as
“instructions” for making protein. Cells differ from each other on the
basis of kinds of proteins they contain, and, since differences among
cells determine differences among organisms, it follows that differences
in the composition of DNA serve to explain the variety in living
organisms populating the world. However, if differences between two
organisms can be explained by differences in the chemical composition of
their respective DNA’s, how can we explain differences between cells of
the same organism? How can we explain that cells of the human pancreas
secrete insulin, whereas other cells in man produce no insulin? Or how
can we explain that certain cells make bone and others make fat? If
indeed all cells in the same organism contain the same amount and kind
of DNA (since all DNA in an organism derives from the duplication of the
DNA of the fertilized egg cell and its descendants), it would seem, at
first glance, that DNA is not the molecule responsible for differences
among the cells. The clarification of this apparent contradiction is
found in the remarkable properties of the other nucleic acid, the
translator molecule, RNA.


Three Kinds of RNA

In the first place, there are at least three different kinds of RNA. The
largest quantity is a special kind called ribosomal RNA, or r-RNA. It is
found in close conjunction with proteins and makes up the structural
frame upon which the protein-synthesizing machinery is built. The r-RNA
and the proteins to which it is firmly bound form the ribosomes, the
RNA-rich microsomes that are attached to the endoplasmic reticulum.
Proteins are synthesized on ribosomes. We shall see later what
determines the differences among proteins and how these differences are
dictated directly by RNA and indirectly by DNA.

Besides r-RNA, there is a kind of RNA called soluble RNA, or transfer
RNA, or s-RNA. It combines with r-RNA to complete the sequence of events
that synthesizes the proteins. A bond between r-RNA and s-RNA is
established by a third RNA molecule called messenger RNA, or template
RNA, or m-RNA. This m-RNA molecule is truly the messenger that carries
the genetic message from DNA to the protein-synthesizing apparatus.

Dr. Michael Shimkin, a Temple University scientist, in his analogy has
compared the DNA → RNA → protein sequence to the activities of a
newspaper staff. DNA is the editor; m-RNA molecules are copyboys who
carry the editorials to the typesetters, the r-RNA and s-RNA, who then
take the “letters” of nucleic acid and set them into slots in accordance
with the editor’s directions. There are also workers who melt down
outworn letters and still other workers who make new letters for further
use; these are the enzymes, special kinds of proteins. If we wish to
continue the analogy, we may say that each kind of cell in the organism
has a different subeditor, who writes that cell’s own editorial.
Actually we might say that all cells have the same board of editors in
common, but only one editor functions in any given type of cell. In
biological terms this means that only a portion of all the cellular DNA
is active in each cell.

The active DNA is the DNA that makes m-RNA that will carry instructions
to the protein-synthesizing machinery of that type of cell. Cells of the
same organism therefore differ from each other on the basis of the
segment of DNA that is active in making m-RNA. Let us now see how we can
use radioactive isotopes to investigate the synthesis of RNA.


Labeling RNA with a Radioactive Isotope

RNA synthesis is investigated with radioactive tracers in the same way
as DNA synthesis. If we can mark, with a radioactive atom, a small
molecule that is incorporated into newly formed RNA, we can then trace
the course of the labeled RNA molecule with a radiation-detection
device. DNA had one advantage in this regard—the fact that one compound,
thymidine, was a precursor of DNA, a specific material that could be
incorporated only into DNA. We do not know similar specific precursors
of RNA. But we know several precursors that are predominantly
incorporated into RNA; the most common of these are the nucleosides
adenine, cytidine, and uridine, and the smaller molecule, orotic acid.
All these precursors can be labeled with either ³H or ¹⁴C, and their
incorporation into RNA can be measured.


Detecting RNA with Autoradiography

As in DNA synthesis, we can use autoradiography to follow the
incorporation of precursors into RNA. By proper treatment of the
tissues, we can make sure that all the radioactivity visible by
autoradiography is due to labeled RNA, even though some of the precursor
also enters DNA molecules. Even so, the kind of information obtained
from autoradiographs of tissues exposed to RNA precursors is different
from that obtained with DNA precursors. The advantage of high-resolution
autoradiography in DNA studies is the possibility of identifying
particular cells that are synthesizing nucleic acid. This advantage is
apparently lost in the case of RNA. The reason is that, at any given
time, only a few cells are making DNA, whereas practically all cells are
synthesizing RNA constantly. The only exceptions are cells in the
midpoint of mitosis. At the beginning (prophase) and at the end of cell
division (telophase), RNA is synthesized. If we want a quantitative
measurement of RNA synthesis, other methods, to be examined presently,
are considerably more precise. But autoradiography can still give us
valuable information.


Other Methods of Detecting RNA

If we look at cells soon after they have been exposed to an RNA
precursor, we find that the radioactivity detectable by autoradiography
is only in the nuclei of the cells. No radioactivity can be detected in
the cytoplasm, although we know that the cytoplasm of living cells
contains large amounts of r-RNA and s-RNA. One or two hours later,
however, radioactive RNA appears in the cytoplasm as well as in the
nucleus. What autoradiography is telling us is that RNA is made in the
nucleus and then is slowly transferred to the cytoplasm.

Autoradiography cannot tell us whether the RNA that has been newly
synthesized in the nuclei of cells is m-RNA, s-RNA, or r-RNA. The
methods necessary to make this distinction are based on the chemical
fractionation of the tissue, isolation of RNA, determination of its
amount by quantitative analysis, and determination of the amount of
radioactivity by physical methods. Let us examine these steps
separately.

[Illustration: Figure 17 _Injecting a mouse with a radioactive
solution._]

Chemical Fractionation of Tissue

After an animal has been injected with a radioactive precursor of RNA,
some of it will be incorporated into DNA as well as into RNA (remember
that the precursors of RNA lack specificity), and part of the precursor
will be broken down into smaller molecules. The injected animal can be
sacrificed, and an organ or another tissue, for instance, the liver, can
be removed. Then the liver is homogenized, that is, ground to a pulp
with a modern version of the mortar and pestle. The homogenate (pulp) is
treated with cold (weak) acid. Proteins and nucleic acids are insoluble
in cold acids and therefore precipitate to the bottom of the test tube.
All molecules that are soluble in a cold acid are left in the
supernatant (the remaining liquid); among these are small molecules,
like those of the RNA precursor. The precipitate (the solid material
that settles to the bottom), now containing proteins and nucleic acids,
is then treated with a strong alkali, for instance, sodium hydroxide.
Alkali will digest RNA into smaller molecules but does not affect DNA.
If we now add acid to the solution, DNA, being insoluble in acid, will
precipitate again; RNA, having been broken down into small molecules,
will remain in the supernatant. DNA can then be extracted from the
precipitate by boiling in strong acid. Proteins from the tissue remain
in the final residue.

We have now fractionated the tissue into four portions: the acid-soluble
fraction (containing small molecules), RNA, DNA, and proteins. (The
cell’s lipids and sugars come out during alcohol rinses between the weak
acid and the alkali steps.) Chemical analysis allows us to measure
precisely the amount of RNA or DNA in its respective fraction and
therefore in the tissue or organ. The amount of radioactivity in the RNA
fraction can then be determined by a technique known as liquid
scintillation counting.

Liquid Scintillation Counting

Liquid scintillation counting is the preferred method for the
measurement of low-energy beta-emitting radioisotopes commonly used in
cell-fractionation studies (see Figure 18). It is convenient, sensitive,
and rapid for routine measurement of radiation in hydrocarbons, other
organic compounds, and aqueous solutions containing such isotopes as ³H,
¹⁴C, and ³²P.

Liquid scintillation solutions share with other scintillating materials
the property of converting into visible light the energy deposited in
them by ionizing radiation. In theory, if a sample of a beta emitter is
dissolved in a liquid scintillator solution, every beta particle emitted
will be absorbed completely because the range of penetration of beta
particles in liquids is quite short (ranging from 0.008 millimicron for
³H to 7.9 millimicrons for ³²P in a medium of unit density). The kinetic
energy of the beta particles is largely used up in the ionization and
excitation of the most abundant molecular species present, the solvent
in which the scintillating material was dissolved. A fraction of the
energy thus expended by each beta particle is transferred from excited
solvent molecules to scintillator molecules; thus the electrons in the
atoms of the scintillator molecules are raised to an excited (higher
energy) state. When these electrons return to the ground, or unexcited,
state, a fraction of them emit a photon of light. Thus each beta
particle produces a burst of photons.

[Illustration: Figure 18 _Technician placing a tray of samples in a
liquid scintillation counter. The radioactivity of each sample is
recorded as the trays revolve._]

If a vessel containing the liquid scintillator and the radioactive
sample is placed near a suitably sensitive instrument known as a
photomultiplier tube, each burst of scintillator photons activates this
device and causes it to release a burst of photoelectrons. Each burst of
photoelectrons is multiplied successively in a series of electronic
steps; as a result, there is a suitably large electrical-output pulse to
be recorded.

One of the principal advantages of the liquid scintillation method is
the ease of sample preparation. We need only transfer a known volume of
a liquid sample or weigh a given mass of a solid sample into a sample
bottle, add a known amount of the liquid scintillator solution, and stir
until there is a homogeneous solution. Samples thus prepared are placed
in a refrigerated counting apparatus. After a short waiting period to
allow time for the samples to cool and for a natural, short-lived
phosphorescence (due to exposure to room light) to subside, the samples
are ready to be measured.

[Illustration: Figure 19 _Placing radioactive samples in a refrigerated
unit for liquid scintillation counting._]

One disadvantage of liquid scintillation counting is that different
compounds show different degrees of quenching (loss of emitted photons),
and the effect must be checked for each class of compounds in each
concentration range. This checking is usually done with an internal
standard technique, the sample being counted before and after a
standard, or known, emitter is added.

Another difficulty is that the best scintillating solvents are not the
best chemical solvents for most biological materials. The solubility
problem is also aggravated by the low temperatures at which liquid
scintillation counters are usually operated for more effective
instrument performance.

With the method we have described, we can obtain a fairly accurate idea
of the rate of RNA synthesis in a given tissue. There are other things
we would like to know about RNA. The first of these is the kind of RNA
being synthesized. During alkaline digestion all kinds of RNA are broken
down into their component nucleotides; we must therefore use other
methods if we wish to know the kind of RNA in which the radioactivity of
the precursor has been incorporated.

Isolation of RNA

Native RNA, that is, RNA not broken down into its smaller constituents,
can be obtained in a variety of ways, but the most popular one makes use
of phenol extraction, which removes DNA and proteins and leaves RNA in
solution. If this phenol-purified RNA is dissolved in a concentrated
sugar solution and spun in a centrifuge at a very high velocity, it will
separate into three major components. These components separate because
they have different molecular weights, and the larger the molecule, the
faster it forms a sediment in the centrifugal field. Two of these
components are s-RNA, the lightest of all, and r-RNA, which is divided
into two subfractions. We can also identify a third component, m-RNA,
with the centrifuge system but only with some difficulty and only after
labeling it with a radioactive precursor, because the amount of m-RNA in
a cell is very small.

[Illustration: Figure 20 _Diagram of ascending paper chromatography._]

Quantitative Analysis

Another important feature of RNA (or DNA, for that matter) is its base
composition, that is, the percentage of each of the nucleotides that
make it up. The four bases that, with ribose and phosphoric acid,
comprise the RNA molecule are guanine, adenine, cytosine, and uracil. It
will be noted that three of the four—guanine, adenine, and cytosine—are
the same as those in DNA, but thymidine in DNA has been replaced by
another base, uracil. To determine the percentage of each base in a
given RNA molecule, we must digest RNA with alkali to produce
mononucleotides, which are smaller molecules, each consisting of a base,
ribose, and phosphoric acid. We can now separate the four nucleotides by
using paper chromatography (see Figure 20).

[Illustration: Figure 21 _A paper chromatography showing separation of
amino acids in two directions. Radioactivity in samples then produced
this record by radioautography._]

In this technique a mixture of compounds is deposited on the edge of a
special type of paper. This edge is then immersed in a solvent that
slowly permeates the paper (at a constant speed) by capillary action. As
the solvent moves from the immersed edge toward the other edge, which is
hanging freely, it carries the mixture of nucleotides with it. Each of
the compounds in the mixture travels at a different speed, however;
thus, as the solvent front moves along the paper, the dissolved
compounds are separated from each other and appear as distinct spots on
the paper. To locate the nucleotides on the paper and to determine the
percentage composition, we can use a chromatogram scanner, a device that
scans the paper chromatograms, measures the radiation from them, and
thus locates the labeled substances (see Figure 22).

[Illustration: Figure 22 _Recording of radioactivity in a sample by
radioautography and paper chromatography. The peaks of the trace
prepared by a chromatogram scanner coincide with the areas of separated
components on the same chromatogram, as revealed by radioautography. The
radioautograph is superimposed on the chromatogram recording._]

Another technique used to separate the nucleotides of RNA is column
chromatography. In this method mixtures of nucleotides are separated as
they pass down a column of chemicals (see Figure 23).

[Illustration: Figure 23 _Students visiting Argonne National Laboratory
listen to a scientist explain the column chromatography process, in
which mixtures of nucleotides are separated as they pass down a column
of chemicals._]

We have now learned how to use radioisotopes to investigate the
synthesis of RNA, the molecule that translates the DNA message into the
language of proteins. Let us now see what we can learn about the
synthesis and function of proteins.




       PROTEIN SYNTHESIS: THE MOLECULES THAT MAKE THE DIFFERENCE


  _If a man will begin with certainties he shall end in doubts; but if
  he will be content to begin with doubts he shall end in certainties._

                                                           Francis Bacon

Proteins occupy a central position in the structure and functioning of
living matter and are intimately connected with all the metabolic
reactions that maintain life. Some proteins serve as structural elements
of the body, for instance, hair, wool, and the scleroproteins of bone
and collagen, the latter an important constituent of connective tissue.
Other proteins are enzymes, which are extremely important since they
regulate all metabolic reactions. Most of the proteins in the tissues of
actively functioning organs, such as the liver and the kidney, are
enzymes. Other proteins participate in muscular contraction, and still
others are hormones or oxygen carriers. Special proteins called histones
are associated with gene function, and the antibodies that an organism
produces to defend itself from bacteria are also proteins.

The differences in proteins, especially in enzymes, account for
differences among cells. It is now appropriate to ask what makes one
protein different from another. We know that the structure of a protein
depends upon several factors, such as the molecular weight. But the main
differences among proteins depend upon the sequence, or order, of the
amino acids that are linked together in the protein molecules.


Amino Acids and Protein Structure

Amino acids are the fundamental structural units of proteins. There are
20 amino acids found frequently in mammalian proteins, and these
molecules may be linked to one another to form a chain called a
polypeptide chain. The structure of a protein then depends on: (1) the
quantity of each amino acid present; (2) the sequence of amino acids in
the polypeptide chain; (3) the length of the polypeptide chain, that is,
the molecular weight; and (4) the folding and the side (nonlinear)
arrangement of the polypeptide chain molecules, that is, the secondary
and tertiary structures.

How can we investigate protein synthesis by using radioactive isotopes?
Since proteins are made up of amino acids, the logical conclusion, after
what we have learned about DNA synthesis and RNA synthesis, is that the
best way would be to mark an amino acid and follow its incorporation
into a molecule of protein. We could label a mixture of several amino
acids, but, for the sake of clarity, we will describe the incorporation
of a single labeled amino acid.


Labeling an Amino Acid with a Radioactive Isotope

Suppose we have the amino acid leucine labeled with ¹⁴C and we inject a
solution containing it into an experimental animal. Since leucine is
incorporated into proteins, if we isolate the proteins and determine
both the amount of proteins and the amount of radioactivity, we can
measure fairly accurately the rate of protein synthesis.
Autoradiography, by the way, is of little help in studying most protein
synthesis because all cells are always synthesizing proteins and so are
all labeled after a single exposure to a radioactive amino acid. With
RNA precursors autoradiography at least told us where RNA was being
made, but with amino acids we do not even get this information because
proteins are synthesized both in the nucleus and in the cytoplasm.

Under these circumstances radiochemical methods are better for studying
protein synthesis. Proteins are isolated from the residue left after a
nucleic-acid extraction process similar to that described previously,
and the amount of protein is determined by a simple colorimetric
analysis based on comparison of the color of the solution with a
standard color. The amount of radioactivity (remember that we are now
using a precursor labeled with ¹⁴C) can be determined with a gas-flow
counter, which is probably more widely used at present than any other
instrument for counting beta emitters, chiefly because of its
reliability and low cost.

[Illustration: Figure 24 _A college chemistry major analyzing a sample
of radioactive materials with an instrument known as a proportional beta
counter._]




         CELL CYCLE AND GENE ACTION: LIFE IS THE SECRET OF DNA


  _Some circumstantial evidence is very strong, as when you find a trout
  in the milk._

                                                     Henry David Thoreau

For a biologist interested in the mechanism of cell proliferation, the
most important event in the life of a cell was, until very recently,
cell division. As we mentioned, when a cell divides into two daughter
cells, it undergoes a process called mitosis; mitosis itself is
subdivided into four stages called prophase, metaphase, anaphase, and
telophase. Mitosis in most cells takes less than one hour. Between one
mitosis and the next, there can be an interval, from a few hours to
several days in length, during which a cell is said to be in interphase.
The entire period between the midpoints of two successive mitoses is
called the cell cycle.


Interphase

Until a few years ago, we knew very little about interphase. In fact, in
one classic book on histology,[8] while a description of mitosis
required almost 12 pages, interphase was dismissed in less than six
lines! The reason for this lack of interest was, of course, the fact
that no adequate methods were available for studying metabolic
activities of cells in interphase. The methods of high-resolution
autoradiography and radiochemical analysis of synchronized cell
populations have become available only in the past few years.

We now know that metabolic activities during interphase are of primary
importance in understanding the mechanism of cell division. It is, in
fact, the orderly sequence of metabolic events occurring in interphase
that leads from one mitosis to the next.


The Cell Cycle

Figure 25 is a diagram of the cell cycle. Try to imagine the cell cycle
as a race track and individual cells as cars that race around it. You
are sitting at the finish wire, which is mitosis (we chose mitosis
because it is easy to recognize when the cell is observed with the aid
of a microscope). At a certain time during the race, all the cars in a
portion of the track, say a 200-yard sector of the backstretch, are
sprayed with a blue dye as they race by. These cars are now marked, just
as cells synthesizing DNA are marked if briefly exposed to tritiated
thymidine, the common radioactive precursor of DNA. As soon as these
cars have been sprayed, you observe all the cars as they pass the finish
line in front of you. At first, you will see cars that were nearest the
wire and were not sprayed; then the dye-marked cars will pass; and
finally more unmarked cars, those that had passed the finish line but
had not reached the spray area when the marking was done, will come by.
If you replace the words spray, cars, and wire with the words
radioactivity, cells, and mitosis, you have described the cell cycle and
the flow of cells in the cycle.

Now, if all cars were going at the same speed, you could calculate with
great accuracy the time taken for any one car to go around the track, or
from the finish line to the backstretch, or through the spray sector,
and so on. However, since cars move at different speeds, you can only
obtain an average time for all sprayed cars. Similarly, since individual
cells behave differently, you can only obtain averages of the times
these cells spend in the various portions of the cell cycle.

[Illustration: Figure 25
THE CELL CYCLE]

These cell-cycle portions are four in number, according to nomenclature
originated by A. Howard and S. R. Pelc, two English investigators who
first described the cycle: (1) mitosis; (2) G₁, which is the period
between mitosis and DNA synthesis; (3) S phase, which is the period
during which DNA is replicated; and (4) G₂, which is the period between
DNA synthesis and the next mitosis. Only cells in the S phase (DNA
synthesis) are marked when exposed to a radioactive precursor of DNA.


DNA Synthesis and the Cell Cycle

Because it has several important implications in biology and medicine,
it is important to remember that DNA synthesis occurs only during the
short, well-defined S period of the cell cycle. Other synthetic
processes go on throughout the cycle. We mentioned, for instance, that
all cells can be labeled by a brief exposure to a radioactive amino
acid, a precursor of proteins; this means that protein synthesis occurs
throughout the entire cell cycle, including mitosis. When we use a
radioactive RNA precursor, all cells except those in anaphase and
metaphase are labeled; this means that RNA synthesis occurs throughout
the entire cycle except during anaphase and metaphase. But a radioactive
tag on a DNA precursor reveals that only during the S phase is there DNA
synthesis.[9]

It is also important to remember that a cell that has synthesized DNA is
a cell that, with a few exceptions, will divide in the very near future.
Thus, for an understanding of the mechanisms that control cellular
proliferation, it is important to investigate the factors that control
DNA synthesis. Our recent knowledge of the cell cycle has therefore led
to a shift in the focus of investigation from mitosis to DNA synthesis.

Another point to remember is that not all cells keep going through the
cell cycle indefinitely. As shown in Figure 25, when a cell divides, the
daughter cells have two alternatives, either to go through another cycle
or to leave it altogether. Cells that leave the cycle are called
differentiated cells and will eventually die without any further
division. Many cells in an adult organism also have lost the capacity to
make DNA and therefore the capacity to divide. These cells often have
other specialized functions in the body; examples are nerve cells and
muscle cells.

The synthesis of other macromolecules (giant molecules, like DNA)
connected with the gene-action system is another field of active
investigation. We have described how we can investigate the synthesis of
proteins and RNA with radioactive isotopes, and we have given some
information on the gene-action system, which is also shown in Figure 26.

The genetic material of a cell is DNA. The DNA molecule is in the form
of a double-stranded helix that is supported by a protein backbone.
Genes are often described as simply segments of DNA. They differ from
each other only in the order in which the four nucleotide bases that
make up DNA are arranged. (Look at Figure 13 again.) Since a single gene
is usually made up of several hundred bases, it is easy to imagine the
infinite variety of genes that could exist by simply changing the order
of the four bases several hundred times.

[Illustration: Figure 26
THE GENE-ACTION SYSTEM]

Not all genes in the cells of a living organism are active. In fact,
most of them are inactive, or, as geneticists say, repressed. What
represses genes to make them inactive is not known, but many
investigators believe the activity, or lack of it, is regulated by
proteins called histones. If a gene is repressed, nothing happens; it
remains inactive, presumably until something removes the repressing
factor. But an active gene sets in motion a train of events that results
in activation of one of the processes of life: The gene’s DNA directs
the manufacture of RNA, which in turn brings about the synthesis of a
specific protein to carry out a specific metabolic process. In other
words, all the activities of the cell are dictated by active genes (the
DNA molecules) through the mediation of RNA and are executed by
proteins.

Here is what happens as nearly as scientists can reconstruct it:


Translation of the Genetic Message

The DNA of a particular active gene manufactures a molecule of m-RNA by
the same kind of replication that it uses for making more DNA. In m-RNA
the sequence of bases is the same as in the parent DNA segment; for this
reason, m-RNA is also called DNA-like RNA. As shown in Figure 12, a
cytosine molecule in m-RNA corresponds to a cytosine molecule in DNA, a
guanine to a guanine, and so on, except that the m-RNA has uracil in all
the places where thymine occurs in DNA. The order of the nucleotides in
the m-RNA is the same as that in the DNA, so the m-RNA carries the
genetic code of the gene that made it. This process, all of which occurs
in the cell nucleus, is one of copying, or transcription, rather than
translation, since the same “codewords” (the nucleic-acid bases) are
reproduced.

The new m-RNA molecule then travels from the nucleus to the cytoplasm
and attaches itself to an unoccupied ribosome (see Figure 27). Here it
fits to a molecule of r-RNA and blends its shape geometrically, or
spatially, with the shape of the r-RNA in lock-and-key, or
jigsaw-puzzle, fashion. The combined new RNA molecule is now capable of
manufacturing a specific protein.

[Illustration: Figure 27 _Protein synthesis in a ribosome (microsome),
and its control by DNA in the nucleus, using RNA as an intermediary._

 Adapted from _Principles of Biology_, Neal D. Buffaloe, Prentice-Hall,
Inc., 1962, with permission.]

At this point an s-RNA molecule arrives, bringing with it one amino-acid
molecule, which then combines with other amino acids in the specific
order dictated by the RNA to form a specific protein. After the amino
acids have been formed into the protein molecule, they detach themselves
from the s-RNA molecule. The s-RNA molecule has two recognition sites by
which it matches up to its neighbors: One recognizes, or “fits”, the
amino acid, and the other recognizes a corresponding triplet of bases on
m-RNA. There is thus a particular s-RNA molecule for each amino acid and
a particular triplet of bases on the m-RNA molecule for each triplet of
bases that is specific to the s-RNA molecule.

In this process the machinery has translated the nucleic-acid code into
the protein code; that is, it has translated a sequence of the bases
into a sequence of amino acids. This process is therefore called
translation of the genetic message. Once the protein has been
synthesized, it will become active in performing some of the cell’s
metabolic activities.

The gene-action system actually is somewhat more elaborate than this.
There are feedback mechanisms, genes that control the activity of other
genes, either directly or through the production of specific proteins,
and so on. However, the scheme just outlined gives a fair, if
simplified, idea of how the genetic message is carried to the entire
cell and how it is translated into actual life processes.




            ISOTOPES IN RESEARCH: PROBING THE CANCER PROBLEM


  _... a riddle wrapped in a mystery inside an enigma._

                                                       Winston Churchill

The various procedures in which radioactive isotopes play a major role
have been applied to many studies and investigations in the fields of
biology and medicine. In fact, most of the concepts of modern biology
that we have been discussing in this booklet owe their discovery to the
judicious use of radioisotopes. To illustrate how radioisotopes can be
used to solve a practical problem, we have chosen a typical example, the
investigation, at a molecular level, of the effectiveness of an
anti-cancer drug.

Several drugs that exert a beneficial effect, at least temporarily, on
the course of certain cancers have been used by doctors for several
years. Most of them were discovered empirically, that is, by accident,
during routine trials against cancers. Doctors know they work but do not
always know how. They would also like to know the mechanism of the
drugs’ action at the molecular level so that the knowledge might open
the way to the discovery of other drugs more effective against cancer
and less toxic against normal cells. The following experiment shows how
the molecular effect of an anti-cancer drug is studied.

[Illustration: Figure 28 _Technician preparing tissues for comparative
studies._]

Cells growing in tissue cultures are often used to test anti-cancer
drugs (see Figure 28). These cells, derived from human cell lines, are
grown in glass or plastic bottles as a suspension in a nutrient medium.
To begin, a culture is divided into halves. To one half is added the
anti-cancer drug Actinomycin D. The other half will continue to grow
without addition of other substances and will serve as a control, or
comparison. After a suitable time has elapsed for the drug to act on the
cultured cells, similar portions of the drug-treated cells and the
control cells will be tested in several ways. One portion of each kind
of cells is incubated with ³H-thymidine to determine the effect of the
drug on DNA synthesis. Two other portions are incubated with ³H-cytidine
to study the effect on RNA synthesis. Another pair will be tested with
¹⁴C-leucine to investigate protein synthesis. The effect of the drug, of
course, is determined by comparing the untreated control with the
drug-treated culture.

The biochemical, autoradiographic, and counting techniques that we
described previously are all used to determine the uptake of the
radioisotopes into the cell’s components. Chromatography is used to
ascertain if the drug has changed the concentration of precursors
(thymidine, cytidine, or leucine) in the nutrient medium, since a change
in these could produce misleading results. Finally, if the drug is found
to have an effect on RNA, we can investigate the type of RNA that is
affected by centrifuging phenol-purified RNA.

The results will disclose the primary site (DNA, RNA, or proteins) of
the drug action on cell metabolism. More elaborate experiments can
pinpoint more intimately the mechanism of action. By studying the life
processes of cells, we can advance toward a common denominator in
anti-cancer drugs that will lead to an effective anti-cancer treatment.




                              CONCLUSIONS


  _Thus, the task is, not so much to see what no one has seen yet; but
  to think what nobody has thought yet, about what everybody sees._

                                                     Arthur Schopenhauer

The use of radioactive isotopes in the study of life processes is of
importance in understanding them. With the use of autoradiographic and
radiochemical techniques, it is possible to obtain valuable information
regarding the life of cells and the intimate mechanisms by which life
processes determine the fate of the entire organism.

Our knowledge of the cell cycle and of the gene-action system has been
useful in determining how organisms grow and how cancer cells behave. It
has been determined that certain normal adult cells divide more
frequently than some cancer cells and that the growth of cancers depends
not so much on the speed of cellular proliferation as on the number of
cells actually dividing.

[Illustration: Figure 29 _Radioautograph showing DNA synthesis during
chromosome replication. Chromosomes from cells in the root tip of the
Tradescantia plant were labeled with ³H-thymidine. In A and B, the
midportion of DNA synthesis, the radioisotope is distributed throughout
the chromosome arms; in C, near the end of DNA synthesis, it is confined
mainly to the end of the arms._]

Knowledge of the cell cycle has also brought new insight to the control
of cell division, as in studies related to the therapy of cancer. The
most important problem now is, not the control of cell division, but the
control of the synthesis of DNA.

Our information on the gene-action system provides broad new opportunity
for the investigation of many life processes. Hormone action, processes
by which the body develops immunity to disease, and even cell division
itself are apparently regulated through the gene-action system. This, in
turn, offers possibilities for investigations meant to control these
processes.

It is difficult to chart the future course of modern molecular biology,
but it is not difficult to predict that the next few years will bring to
biology the same kind of sweeping advances that revolutionized physics a
few decades ago. The DNA molecule has been called the atom of life. When
we have harnessed it, the harnessing of the uranium atom will seem, in
comparison, a result of scientific adolescence. When man has mastered
the genetic code, he’ll hold a vast power in his hands—power over the
nature of coming generations.




                          SUGGESTED REFERENCES


Books

_The Cell_, Carl P. Swanson, Prentice-Hall, Inc., Englewood Cliffs, New
  Jersey, 1964, 114 pp., $1.75.

_Inside the Living Cell_, J. A. V. Butler, Basic Books, Inc., New York,
  1959, 174 pp., $3.95.

_Life and Energy_, Isaac Asimov, Doubleday & Company, Inc., Garden City,
  New York, 1962, 380 pp., $4.95.

_Applied Nuclear Physics_, Ernest C. Pollard and William L. Davidson,
  John Wiley & Sons, Inc., New York, 1956, 352 pp., $6.00.

_Adventures in Radioisotope Research_, the collected works, with recent
  annotations, of George de Hevesy, Pergamon Press, Inc., New York,
  1961, 1047 pp. (2 volumes), $30.00.

_The Biochemistry of Nucleic Acids_, J. N. Davidson, John Wiley & Sons,
  Inc., New York, 4th edition, 1960, 287 pp., $4.25.

_The Machinery of the Body_, A. J. Carlson and C. Johnson, The
  University of Chicago Press, Chicago, Illinois, 1961, 752 pp., $6.50.

_Life: An Introduction to Biology_, George G. Simpson and William S.
  Beck, Harcourt, Brace & World, Inc., New York, 2nd edition, 1965, 869
  pp., $8.95.

_From Cell to Test Tube_, Robert W. Chambers and Alma Payne, Charles
  Scribner’s Sons, New York, 1962, 216 pp., $1.45.

_Isotopic Tracers in Biology_, M. D. Kamen, Academic Press Inc., New
  York, 3rd edition, 1957, 474 pp., $9.50.

_Autoradiography in Biology and Medicine_, G. A. Boyd, Academic Press
  Inc., New York, 1955, 399 pp., $10.00.

_A Tracer Experiment: Tracing Biochemical Reactions with Radioisotopes_,
  Martin D. Kamen, Holt, Rinehart & Winston, Inc., New York, 1964, 127
  pp., $1.28.

_Molecular Biology: Genes and the Chemical Control of Living Cells_, J.
  M. Barry, Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 1964, 139
  pp., $3.35.

_Elementary Biophysics: Selected Topics_, Herman T. Epstein,
  Addison-Wesley Publishing Company, Inc., Reading, Massachusetts, 1963,
  122 pp., $2.95 (hardback), $1.75 (paperback).


Articles

Autobiographies of Cells, R. Baserga and W. Kisieleski, _Scientific
  American_, 209: 103 (August 1963).

Electrons, Enzymes, and Energy, Michael G. Del Duca and John M. Fuscoe,
  _International Science and Technology_, 39: 56 (March 1965).

_Scientific American_, 205 (September 1961). This is a special issue on
  the living cell. The two articles cited below are of particular
  interest:

  How Cells Divide, Daniel Mazia, 205: 101.
  The Living Cell, Jean Brachet, 205: 50.


Reports

_Liquid Scintillation Counting: Proceedings of a Conference Held at
  Northwestern University, August 20-22, 1957_, C. G. Bell, Jr. and F.
  N. Hayes (Eds.), Pergamon Press, Inc., New York, 1957, 292 pp.,
  $10.00.

_Atomic Energy Research: Life and Physical Sciences; Reactor
  Development; and Waste Management_, A Special Report of the U. S.
  Atomic Energy Commission (December 1961), Superintendent of Documents,
  U. S. Government Printing Office, Washington, D. C. 20402, 333 pp.,
  $2.25.


Booklets

_Radioisotopes in the Service of Man_, Fernand Lot, National Agency for
  International Publications, 317 East 34th Street, New York 10016,
  1958, 82 pp., $1.00.

_Science and Cancer_, M. B. Shimkin, Public Health Service Publication
  No. 1162, Superintendent of Documents, U. S. Government Printing
  Office, Washington, D. C. 20402, 1964, 137 pp., $0.60.


Motion Pictures

_The Cell: Structural Unit of Life_, 10 minutes, sound, color or black
  and white, 1949, Coronet Films, Inc., 65 E. South Water Street,
  Chicago, Illinois 60601.

_Continuity of Life: Characteristics of Plants and Animals_, 11 minutes,
  sound, color or black and white, 1954, Audio-Visual Center, Indiana
  University, Bloomington, Indiana 47405.

_DNA: Molecule of Heredity_, 16 minutes, sound, color (No. 1825), black
  and white (No. 1826). 1960, Encyclopaedia Britannica Films, Inc.,
  Wilmette, Illinois 60091.

_The Science of Genetics_, AIBS Secondary School Film Series, No. 13280,
  25 minutes, sound, color, 1962, McGraw-Hill Book Company, Inc., 330
  West 42nd Street, New York 10036.


Available for loan without charge from the AEC Headquarters Film
  Library, Division of Public Information, U. S. Atomic Energy
  Commission, Washington, D. C. 20545, and other AEC film libraries:

_Tracing Living Cells_, Challenge Film No. 11, 29 minutes, sound, black
  and white, 1962. Produced by Ross-McElroy Productions for the National
  Educational Television and Radio Center under a grant from Argonne
  National Laboratory. This nontechnical film demonstrates some of the
  uses of radioisotopes in the study of cell division and in medical
  therapy.

_The Eternal Cycle_, 12½ minutes, sound, black and white, 1954. Produced
  by the Handel Film Corporation. This nontechnical film illustrates the
  use of radioisotope tracers in biological research and is suitable for
  intermediate- through college-level audiences.

_Chromosome Labeling by Tritium_, 15 minutes, sound, color, 1958.
  Produced by the Jam Handy Organization for the U. S. Atomic Energy
  Commission. This technical film discusses the advantages of tritium
  over other radioisotopes as labeling material in autoradiography.

_A is for Atom_, 15 minutes, sound, color, 1953. Produced by the General
  Electric Company. This nontechnical film explains the structure of the
  atom, natural and artificially produced elements, stable and unstable
  atoms, principles and applications of nuclear reactors, and the
  benefits of atomic radiation to biology, medicine, industry, and
  agriculture. It is suitable for elementary- through high-school
  audiences.




                               FOOTNOTES


[1]An organism is a complete living plant or animal.

[2]Metabolism is the sum of the life-sustaining activities in a living
    organism, including nutrition, production of energy, and synthesis
    (building) of new living material.

[3]Morphologists are biologists specializing in the structure of
    organisms or in the study of whole organisms. Biochemists, by
    contrast, study chemical reactions of biological materials.

[4]This is not to be confused with a cell nucleus. This word was
    borrowed from biology for atomic theory, however.

[5]An exception is the hydrogen atom, which has no neutron in its
    nucleus.

[6]Mev is the abbreviation for million electron volts.

[7]A concept for which James D. Watson of the United States and Francis
    H. C. Crick of England shared a Nobel Prize in 1962.

[8]The study of tissues.

[9]There are additional, more subtle metabolic events that lead to the
    synthesis of DNA, but they are not important in this discussion.


PHOTO CREDITS

  Figure 1  Armed Forces Institute of Pathology Negative No. 4156
  Figure 3  Dr. T. Tahmisian, Argonne National Laboratory
  Figure 4  Oak Ridge National Laboratory (photo on right)
  Figure 5  Oscar W. Richards, American Optical Company
  Figure 7  Brookhaven National Laboratory
  Figure 9  Battelle-Northwest Laboratory
  Figure 10 Oak Ridge National Laboratory
  Figure 19 Argonne National Laboratory
  Figure 23 Argonne National Laboratory
  Figure 24 Argonne National Laboratory
  Figure 28 Argonne National Laboratory
  Figure 29 Brookhaven National Laboratory


This booklet is one of the “Understanding the Atom” Series. Comments are
invited on this booklet and others in the series; please send them to
the Division of Technical Information, U. S. Atomic Energy Commission,
Washington, D. C. 20545.

Published as part of the AEC’s educational assistance program, the
series includes these titles:

  _Accelerators_
  _Animals in Atomic Research_
  _Atomic Fuel_
  _Atomic Power Safety_
  _Atoms at the Science Fair_
  _Atoms in Agriculture_
  _Atoms, Nature, and Man_
  _Books on Atomic Energy for Adults and Children_
  _Careers in Atomic Energy_
  _Computers_
  _Controlled Nuclear Fusion_
  _Cryogenics, The Uncommon Cold_
  _Direct Conversion of Energy_
  _Fallout From Nuclear Tests_
  _Food Preservation by Irradiation_
  _Genetic Effects of Radiation_
  _Index to the UAS Series_
  _Lasers_
  _Microstructure of Matter_
  _Neutron Activation Analysis_
  _Nondestructive Testing_
  _Nuclear Clocks_
  _Nuclear Energy for Desalting_
  _Nuclear Power and Merchant Shipping_
  _Nuclear Power Plants_
  _Nuclear Propulsion for Space_
  _Nuclear Reactors_
  _Nuclear Terms, A Brief Glossary_
  _Our Atomic World_
  _Plowshare_
  _Plutonium_
  _Power from Radioisotopes_
  _Power Reactors in Small Packages_
  _Radioactive Wastes_
  _Radioisotopes and Life Processes_
  _Radioisotopes in Industry_
  _Radioisotopes in Medicine_
  _Rare Earths_
  _Research Reactors_
  _SNAP, Nuclear Space Reactors_
  _Sources of Nuclear Fuel_
  _Space Radiation_
  _Spectroscopy_
  _Synthetic Transuranium Elements_
  _The Atom and the Ocean_
  _The Chemistry of the Noble Gases_
  _The Elusive Neutrino_
  _The First Reactor_
  _The Natural Radiation Environment_
  _Whole Body Counters_
  _Your Body and Radiation_

A single copy of any one booklet, or of no more than three different
booklets, may be obtained free by writing to:

           USAEC, P. O. BOX 62, OAK RIDGE, TENNESSEE    37830

Complete sets of the series are available to school and public
librarians, and to teachers who can make them available for reference or
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nuclear science, or references to other reading material, may also write
to the Oak Ridge address. Requests should state the topic of interest
exactly, and the use intended.

In all requests, include “Zip Code” in return address.


                Printed in the United Slates of America
USAEC Division of Technical Information Extension, Oak Ridge, Tennessee




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