Part 2

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.)

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