Part 3

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

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.

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.