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.