Chapter II: Part 2

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

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

The Cell Cycle

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

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

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

DNA Synthesis and the Cell Cycle

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

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

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

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

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

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

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

Translation of the Genetic Message

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

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

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

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

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

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

ISOTOPES IN RESEARCH: PROBING THE CANCER PROBLEM

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

Winston Churchill

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

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

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

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

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

CONCLUSIONS

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

Arthur Schopenhauer

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

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

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

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

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

SUGGESTED REFERENCES

Books

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Articles

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

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

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

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

Reports

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

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

Booklets

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

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

Motion Pictures

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

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

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

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

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

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

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

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

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

FOOTNOTES

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

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

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

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

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

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

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

[8]The study of tissues.

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

PHOTO CREDITS

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

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

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

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

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

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

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