Part 6

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, 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.

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.