Renato Baserga

“ 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 2 in our understanding of biology is the use of radioactive isotopes as laboratory tools. ”

“ 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. ”

“ 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.) ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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 4 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ Figure 6 Stages of the mitotic cycle in a hypothetical cell with four chromosomes.
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) . ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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.)
15 DNA SYNTHESIS: THE AUTOBIOGRAPHY OF CELLS
Here, surely, is the prime substance of life itself. ”

“ 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 ”

“ 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. ”

“ 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.
Figure 26
THE GENE-ACTION SYSTEM
Not all genes in the cells of a living organism are active. ”

“ 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.
Figure 18 Technician placing a tray of samples in a liquid scintillation counter. The radioactivity of each sample is recorded as the trays revolve. ”

“ 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.
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. ”