by Isaac Asimov
The next step was taken by the American biochemist Mahlon Bush Hoagland, who had also been active in working out the notion of mRNA. He showed that in the cytoplasm are a variety of small RNA molecules, which might be called soluble-RNA or sRNA, because their small size enables them to dissolve freely in the cytoplasmic fluid.
At one end of each sRNA molecule was a particular triplet of nucIeotides that just fitted a complementary triplet somewhere on the mRNA chain: that is, if the sRNA triplet were AcC, it would fit tightly to a UCG triplet on the mRNA and only there. At the other end of the sRNA molecule was a spot where it would combine with one particular amino acid and none other. On each sRNA molecule, the triplet at one end meant a particular amino acid on. the other. Therefore, a complementary triplet on the mRNA meant that only a certain sRNA molecule carrying a certain amino acid molecule would affix itself there. A large number of sRNA molecules would affix themselves one after the other, right down the line, to the triplets making up the mRNA structure (triplets that had been molded right on the DNA molecule of a particular gene). All the amino acids properly lined up could then easily be hooked together to form an enzyme molecule.
Because the information from the messenger-RNA is, in this way, transferred to the protein molecule of the enzyme, sRNA has come to be called transfer-RNA, and this name is now well established.
In 1964, the molecule of alanine-transfer-RNA (the transfer-RNA that attaches itself to the amino acid alanine) was completely analyzed by a team headed by the American biochemist, Robert William Holley. This analysis was done by the Sanger-method of breaking down the molecule into small fragments by appropriate enzymes, then analyzing the fragments and deducing how they must fit together. The alanine-transfer-RNA, the first naturally occurring nucleic acid to be completely analyzed, was found to be made up of a chain of seventy-seven nucleotides. These include not only the four nucleotides generally found in RNA (A, G, C, and U) but also several of seven others closely allied to them.
It had been supposed at first that the single chain of a transfer-RNA would be bent like a hairpin at the middle and the two ends would twine about each other in a double helix. The structure of alanine theory transfer-RNA did not lend itself to this theory. Instead, it seemed to consist of three loops, so that it looked rather like a lopsided three-leaf clover. In subsequent years, other transfer-RNA molecules were analyzed in detail, and all seemed to have the same three-leaf-clover structure. For his work, Holley received a share of the 1968 Nobel Prize for medicine and physiology.
In this way, the structure of a gene controls the synthesis of a specific enzyme. Much, of course, remained to be worked out, for genes do not simply organize the production of enzymes at top speed at all times. The gene may be working efficiently now, slowly at another time, and not at all at still another time. Some cells manufacture protein at great rates, with an ultimate capacity of combining some 15 million amino acids per chromosome per minute; some only slowly;some scarcely at all—yet all the cells in a given organism have the same genic organization. Then, too, each type of cell in the body is highly specialized, with characteristic functions and chemical behavior of its own. An individual cell may synthesize a given protein rapidly at one time, slowly at another. And, again, all have the same genic organization all the time.
It is clear that cells have methods for blocking and unblocking the DNA molecules of the chromosomes. Through the pattern of blocking and unblocking, different cells with identical gene patterns can produce different combinations of proteins, while a particular cell with an unchanging gene pattern can produce different combinations from time to time.
In 1961, Jacob and Monod suggested that each gene has its own repressor, coded by a regulator gene. This repressor—depending on its geometry, which can be altered by delicate changes in circumstances within the cell—will block or release the gene. In 1967, such a repressor was isolated and found to be a small protein. Jacob and Monod, together with a co-worker, Andre Michael Lwoff, received the 1965 Nobel Prize for medicine and physiology as a result.
Through laborious work since 1973, it would seem that the long double helix of the DNA twists to form a second helix (a superhelix) about a core of a string of histone molecules, so that there is a succession of units called nucleosomes. In such nucleosomes, depending upon the detailed structure, some genes may be repressed, and others active; and the histones may have something to do with which active gene becomes repressed from time to time or is activated. (As usual, biological systems always seem more complex than expected once one probes deeply into the details.)
Nor is the flow of information entirely one way, from gene to enzyme. There is “feedback” as well. Thus, there is a gene that brings about the formation of an enzyme that catalyzes a reaction that converts the amino acid threonine to another amino acid, isoleucine. Isoleucine, by its presence, somehow serves to activate the repressor, which begins to shut down the very gene that produces the particular enzyme that led to that presence. In other words, as isoleucine concentration goes up, less is formed; if the concentration declines, the gene is unblocked, and more isoleucine is formed. The chemical machinery within the cell—genes, repressors, enzymes, end-products—is enormously complex and intricately interrelated. The complete unraveling of the pattern is not likely to take place rapidly.
But meanwhile, what of the other question: Which codon goes along with which amino acid? The beginning of an answer came in 1961, thanks to the work of the American biochemists Marshall Warren Nirenberg and J. Heinrich Matthaei. They began by making use of a synthetic nucleic acid, built up according to Ochoa’s system from uracil nucleotides only. This polyuridylic acid was made up of a long chain of …UUUUUUUU… and could only possess one codon, UUU.
Nirenberg and Matthaei added this polyuridylic acid to a system that contained various amino acids, enzymes, ribosomes, and all the other components necessary to synthesize proteins. Out of the mixture tumbled a protein made up only of the amino acid phenylalanine. This meant that UUU was equivalent to phenylalanine. The first item in the codon dictionary was worked out.
The next step was to prepare a nucleotide made out of a preponderance of uridine nucleotides with a small quantity of adenine nucleotides added; thus, along with the UUU codon, an occasional UUA, or AUU, or UAU codon might appear. Ochoa and Nirenberg showed that, in such a case, the protein formed is mainly phenylalanine but also contains an occasional leucine, isoleucine, and tyrosine, three other amino acids.
Slowly, by methods such as these, the dictionary was extended. It was found that the code is indeed degenerate, and that GAU and GAC might each stand for aspartic acid, for instance, and that GUU, GAU, GUC, GUA, and GUG, all stand for glycine. In addition, there was some punctuation. The codon AUG not only stood for the amino acid methionine but apparently signified the beginning of a chain. It was a capital letter, so to speak. Then, too, UAA and UAG signaled the end of a chain: they were periods, or full stops.
By 1967, the dictionary was complete (see table 13.1). Nirenberg and his collaborator, the Indian-American chemist Har Cobind Khorana, were awarded shares (along with Holley) in the 1968 Nobel Prize for medicine and physiology.
TABLE 13.l
The genetic code. In the left-hand column are the initials of the four RNA bases (uracil, cytosine, adenine, guanine) representing the first “letter” of the codon triplet; the second letter is represented by the initials across the top, while the third but less important letter appears in the final column. For example, tyrosine (Tyr) is coded for by either UAU or UAG. Amino acids coded by each codon are shown abbreviated as follows: Phe—phenylalanine; Leu—leucine; Ileu—isoleucine; Met—methionine; Val—valine; Ser—serine; Pro—proline; Thr—threonine; Ala—alanine; Tyr—tyrosine; His—histidine; Glun—glutamine; Aspn—asparagine, Lys—lysine; Asp—aspastic acid; Clu—glutamic acid; Cys—cysteine; Tryp—tryotophan; Arg—arginine; Gly—glycine.
First
Position Second
Position Third<
br />
Position
U C A G
U Phe
Phe
Leu
Leu Ser
Ser
Ser
Ser Tyr
Tyr
(normal “full stop”)
(less common “full stop”) Cys
Cys
“full stop”
Tryp U
C
A
G
C Leu
Leu
Leu
Leu Pro
Pro
Pro
Pro His
His
Glun
Glun Arg
Arg
Arg
Arg U
C
A
G
A Ileu
Ileu
Ileu?
Met
(“capital letter”) Thr
Thr
Thr
Thr Aspn
Aspn
Lys
Lys Ser
Ser
Arg
Arg U
C
A
G
G Val
Val
Val
Val
(“capital letter”) Ala
Ala
Ala
Ala Asp
Asp
Glu
Glu Gly
Gly
Gly
Gly U
C
A
G
The working out of the genetic code is not, however, a “happy ending” in the sense that now all mysteries are explained. (There are, perhaps, no happy endings of this sort in science—and a good thing, too, for a universe without mysteries would be unbearably dull.)
The genetic code was worked out largely through experiments on bacteria, where the chromosomes are packed tight with working genes that code the formation of proteins. Bacteria are prokaryotes (from Greek words meaning “before the nucleus”), since they lack cell nuclei but have chromosomal material distributed throughout their tiny cells.
As for the eukaryotes, which have a cell nucleus (and include all cells but those of bacteria and blue-green algae), the case is different. The length of nucleic acid is not solidly packed with working genes. Instead, those portions of the nucleotide chain that are used to encode messenger-RNA and, eventually, proteins (exons) are interspersed by sections of chain (introns) that may be described as gibberish. A single gene that controls the production of a single enzyme may consist of a number of exons separated by introns, and the nucleotide chain coils in such a way as to bring the exons together for the encoding of messenger-RNA. Thus, the estimate, given earlier in the chapter, of the existence of 2 million genes in the human cell is far too high, if one is referring to working genes.
Why eukaryotes should carry such a load of what seems dead weight is puzzling. Perhaps that is how genes developed in the first place; and in prokaryotes, the introns were disposed of in order to make shorter nucleotide chains that could be more swiftly replicated in the interest of faster growth and reproduction. In eukaryotes, the introns are not excised, perhaps because they offer some advantage that is not immediately visible. No doubt the answer, when it comes, will be illuminating.
And meanwhile scientists have found methods of participating directly in gene activity. In 1971, the American microbiologists Daniel Nathans and Hamilton Othanel Smith worked with restriction enzymes which were capable of cutting the DNA chain in specific fashion at a particular nucleotide junction and no other. There is another type of enzyme, DNA ligase, which is capable of uniting two strands of DNA. The American biochemist Paul Berg cut DNA strands by restriction enzymes and then recombined strands in fashions other than had originally existed. A molecule of recombinant-DNA was thus formed that was not like the original or, perhaps, not like any that had ever existed.
It became possible, as a result of such work, to modify genes or to design new ones: to insert them into bacterial cells (or into the nuclei of eukaryotic cells) and thus to form cells with new biochemical properties. As a result,
Nathans and Smith were awarded shares of the 1978 Nobel Prize for physiology and medicine, while Berg received a share of the 1980 Nobel Prize for chemistry.
Recombinant-DNA work had its areas of apparent danger. What if, either deliberately or inadvertently, a bacterial cell was produced, or a virus with the ability to produce a toxin to which human beings had no natural immunity? If such a new microorganism escaped from the laboratory, it might inflict an indescribably disastrous epidemic upon humanity. With such thoughts in mind, Berg and others, in 1974, called on scientists for a voluntary adherence to strict controls in work on recombinant-DNA.
As it happened, though, further experience showed that there was little danger of anything untoward happening. Precautions were extreme, and the new genes placed in microorganisms produced strains that were so weak (an unnatural gene is not easy to live with) that they could barely be kept alive under the most favorable conditions.
Then, too, recombinant-DNA work involves the possibility of great benefits. Aside from the possibility of the advancement of knowledge concerning the fine details of the workings of cells and of the mechanism of inheritance in particular, there are more immediate benefits. By appropriately modifying a gene, or by inserting a foreign gene, a bacterial cell might become a tiny factory that is manufacturing molecules of something needed by human beings rather than by itself.
Thus, bacterial cells, in the 1980s, have been so modified as to manufacture human insulin, with the unattractive name of humulin. Hence, in time, diabetics will no longer be dependent upon the necessarily limited supplies available from the pancreases of slaughtered animals and will not have to use the adequate, but not ideal, insulin varieties of cattle and swine.
Other proteins that can be made available by appropriately modified microorganisms are interferon and growth hormone-with, on the horizon, unlimited possibilities. It is not surprising that the question has now arisen whether new forms of life can be patented.
The Origin of Life
Once we get down to the nucleic-acid molecules, we are as close to the basis of life as we can get. Here, surely, is the prime substance of life itself. Without DNA, living organisms could not reproduce, and life as we know it could not have started. All the substances of living matter—enzymes and all the others, whose production is catalyzed by enzymes—depend in the last analysis on DNA. How, then, did DNA, and life, start?
This is a question that science has always hesitated to ask, because the origin of life has been bound up with religious beliefs even more strongly than has the origin of the earth and the universe. It is still dealt with only hesitantly and apologetically. A book entitled The Origin of Life, by the Russian biochemist Aleksandr Ivanovich Oparin, brought the subject to the fore. The book was published in the Soviet Union in 1924 and in English translation in 1936. In it the problem of life’s origin for the first time was dealt with in detail from a completely materialistic point of view. Since the Soviet Union is not inhibited by the religious scruples to which the Western nations feel bound, this, perhaps, is not surprising.
EARLY THEORIES
Most early cultures developed myths telling of the creation of the first human beings (and sometimes of other forms of life as well) by gods or demons. However, the formation of life itself was rarely thought of as being entirely a divine prerogative. At least the lower forms of life might arise spontaneously from nonliving material without supernatural intervention. Insects and worms might, for instance, arise from decaying meat, frogs from mud, mice from rotting wheat. This idea was based on actual observation, for decaying meat, to take the most obvious example, did indeed suddenly give rise to maggots. It was only natural to assume that the maggots were formed from the meat.
Aristotle believed in the existence of spontaneous generation. So did the great theologians of the Middle Ages, such as Thoma
s Aquinas. So did William Harvey and Isaac Newton. After all, the evidence of one’s own eyes is hard to refute.
The first to put this belief to the test of experimentation was the Italian physician Francesco Redi. In 1668, he decided to check on whether maggots really formed out of decaying meat. He put pieces of meat in a series of jars and then covered some of them with fine gauze and left others uncovered. Maggots developed only in the meat in the uncovered jars, to which flies had had free access. Redi concluded that the maggots had arisen from microscopically small eggs laid on the meat by the flies. Without flies and their eggs, he insisted, meat could never produce maggots, however long it decayed and putrefied.
Experimenters who followed Redi confirmed this finding, and thc belief that visible organisms arise from dead matter died. But when microbes were discovered, shortly after Redi’s time, many scientists decided that these forms of life at least must come from dead matter. Even in gauze-covered jars, meat would soon begin to swarm with microorganisms. For two centuries after Redi’s experiments, belief in the possibility of the spontaneous generation of microorganisms remained very much alive.
It was another Italian, the naturalist Lazzaro Spallanzani, who first cast serious doubt on this notion. In 1765, he set out two sets of vesselscontaining a broth. One he left open to the air. The other, which he had boiled to kiJl any organisms already present, he sealed up to keep out any organisms that might be floating in the air. The broth in the first vessels soon teemed with microorganisms, but the boiled and sealed-up broth remained sterile. This proved to Spallanzani’s satisfaction that even microscopic life could not arise from inanimate matter. He even isolated a single bacterium and witnessed its division into two bacteria.
