Chapter I: Part 1

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.

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

_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.

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

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.

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.

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.

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.

(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.

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).

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.

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.

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.

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.)

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.

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.

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.

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.

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.

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.

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.

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).

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).

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).

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.

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