Part 5

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

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.