Key to Genetic Code Seems Nearer Several basic properties of the code emerge from new data gathered by various research groups The key to the genetic code may be at hand. Reasons for this optimistic appraisal—put forth at the Symposium on Informational Maeromolecules. held at Rutgers, The State University of New Jersey—are based on the emergence of several fundamental properties of the code: The code words are nonoverlapping triplets; the function of the triplet coding unit may sometimes depend on the recognition of two out of three of the triplet's nucleotides; and frequently, more than one coding unit conveys the information for the incorporation of an amino acid (degeneracy of the code). Degeneracy of the genetic code is extensive, says Dr. Marshall \V. Nirenberg, National Institutes of Health, Bethesda, Md. Yet the fidelity of in vivo protein synthesis is high. This seeming paradox may be explained by the occurrence of a coding specificity imparted by the nucleotide sequence. That such specificity exists is shown (assuming a triplet code) by the demonstration that both leucine and valine are coded by UUG (uracil, uracil, guanine) ; but the code word for leucine will not code for valine, and vice versa. But, Dr. Nirenberg warns, specificity may not be absolute, since examples of nonspecificity—such as the direction by polyuridylic acid of leucine incorporation into proteinhave been observed. Also, template activities of polynucleotides are dependent on factors other than nucleotide sequence. In the case of polynucleotides containing four bases, the longer chains are more active than the shorter chains. And single-stranded polynucleotides are active while doublestranded and triple-stranded aren't. Polynucleotides having four bases direct so many amino acids into protein that, in this system at least, the code is extensively degenerate. The experiments also show that a high proportion of uracil is not required for messenger ribonucleic acid activity. Additional tests using the three-base polynucleotide poly (adenine, cytosine, guanine) show that polyACG stimulates incorporation of such amino acids as alanine, arginine, glutamic acid, 44
C&EN
OCT. 1, 196 2
histidine, leucine, lysine, proline, serine, and threonine, depending on the base-ratio of the polymer. And, finally, tests using a two-base polynucleotide show that polyAC (adenine, cytosine) stimulates incorporation of aspartic and glutamic acids, histidine, leucine, lysine, proline, and threonine; polyCG (cytosine, guanine) stimulates incorporation of proline, arginine, and serine; and polyAG (adenine, guanine) stimulates incorporation of lysine, glutamic acid, and arginine. Data from the four-base, three-base, two-base, and one-base polynucleotide tests reveal that the code word for proline incorporation may contain only cytosine, and that for lysine only adenine. The data also show that some amino acids can be coded by qualitatively different polymers. Dr. Nirenberg concludes that the recognition of the widespread degeneracy and the determination of its molecular basis may be helpful in deciphering the genetic code. And others feel that this recognition of widespread degeneracy is a key to the final cracking of the code. Reading Order. One of the problems needing solution if the code is to be broken is the order in which the code is read. According to Dr. Sol Spiegelman and T. Kano-Sueoka of the University of Illinois, Urbana, the genome (genetic book) is read in an ordered fashion. But there are two processes: the transcription into polynucleotides and the translation into a protein. Reading occurs nonrandomly at that part of the RNA that corresponds to the free carboxyl end, and continues to that part corresponding to the amino end of the polypeptide chain. The reading process is in the opposite direction of polypeptide chain growth, where the sequential adaptation of amino acid residues proceeds from the terminal amino end to the free carboxyl end. The ordered nature of the genome's transcription is made clear by sampling radioactive RNA messages in time and noting their discordant radioactive curves, Dr. Spiegelman says. To ensure the sensitive detection of chromatographic differences (if such
differences occur), double labeling of the RNA preparation is used (C&EN, Sept. 17, page 5 3 ) . One RNA preparation is tritium-labeled, the other labeled with carbon-14. When a mixture of the two is loaded on a methylated-albumin-kieselguhr column, the radioactive elution profiles are the same if the preparations are the same. The profiles differ if one contains one or more components absent in the other. The technique, Dr. Spiegelman says, shows that RNA molecules synthesized by phage-infected bacteria during the first three to five minutes of infection are different from those formed 13 to 15 minutes after infection. Actually, other periods have been examined; each shows different RNA patterns. If the time intervals are more finely divided, it may be possible to identify individual messages for particular proteins, he adds. Relative Order. The determination of the relative order of the ribonucleotides in the coding units for six amino acids has been achieved by Dr. Charles Yanofsky and co-workers at Stanford University, Stanford, Calif. Earlier workers assigned the ribonucleotides UGA (uracil, guanine, adenine) to glutamic acid, UGG to glycine, UGC to alanine, and UGU to valine. C represents cytosine. The Stanford group shows that all these amino acids are related by single mutational changes. Thus the nucleotide difference between the different messages must be in the same position. From these data and other amino acid replacements, the ribonucleotides in the arginine coding unit, UCG, must have a different order from those in alanine. Arginine has the same code letters as alanine but is derived from glycine by a single code letter change. So, too, the relative order for serine, UCU, must be related to the UCG of arginine, since serine is in turn derived from arginine, Dr. Yanofsky says. Therefore, when A23 mutants—having arginine (UCG) instead of glycine (UGG) in the A protein of tryptophan synthetase—are crossed with A46 mutants having glutamic acid (UGA) replacing the same glycine (UGG) residue, glycine recombinants are obtained. The finding supports the conclusion that the relative positions of the nucleotides in arginine (UCG) and glutamic acid (UGA) coding units is correct. An additional recombinant having the coding unit UCA is also expected,
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46
C&EN
OCT.
1,
1962
but hasn't been found—probably because only three recombinants have been examined so far, Dr. Yanofsky says. Similar crosses with arginine (UCG) and valine (UGU) mutants yield glycine (UGG) recombinants and serine (UCU) recombinants. From these data, Dr. Yanofsky concludes that the relative positions of the code letters for the six amino acids -glutamic acid (UGA), glycine ( U G G ) , alanine (UGC), valine ( U G U ) , arginine (UCG), and serine (UCU)—are confirmed. The data also show that intracoding unit recombination is indistinguishable from mutation, thus may be a significant source of heritable change. Mutationally altered genes that reverse the effects of a primary mutation in another gene are called suppressor mutations. Such suppressor mutations appear to lead to a change in transfer RNA molecules, resulting in the transfer RNA accepting the wrong amino acid, according to work carried out by Stanford's S. Brody. Thus the primary structures of all proteins are subject to mutational events outside the structural gene, Dr. Yanofsky points out. The mutant form of the suppressor gene is detrimental in many of the cases studied, he says. This indicates that a mistake in incorporation, which leads to an active form of a previously inactive protein, may at the same time inactivate some molecules of other proteins. Sequence. The sequence for a triplet can be determined by making a polynucleotide with a known triplet at one end. Earlier workers showed that tyrosine is coded by a triplet having two uracils and one adenine. Dr. Severo Ochoa of New York University school of medicine says that the order is AUU. He made a polyuridylic acid starting with an AUU triplet (some AAU triplet was present as a contaminant) . With the mixed polyuridylic acid, phenylalanine is incorporated into acid-insoluble polypeptides. Some of the phenylalanine is at the amino end of the polypeptide. This suggests that polyuridylic acid can be used to start the synthesis of polypeptide chains, he notes. Tyrosine wasn't found at the amino end; rather, some tyrosine was found at the carboxyl end. The AUU primer at the beginning of the mixed polyuridylic acid thus corresponds to the last amino acid added to the polypeptide, Dr. Ochoa says. Similar tests show that the code that works for
cysteine is GUU; these tests establish the sequence of the nucleotides in two code words. And from these, results from amino acid replacements can be used to establish the sequence of the other triplets, notes Dr. Thomas H. Jukes of American Cyanamid, Princeton, N.J.
Enzyme Studies Get New Tool An electrochemical technique is now available for measuring the kinetics of thiocholine ester hydrolysis. And it allows rapid determination of the highly toxic, organophosphorus anticholinesterase compounds in nonagram ( 1 0 - i ) ) amounts. The method, developed by Dr. G. G. Guilbault, Dr. D. N. Kramer, and P. L. Cannon, Jr., at the U.S. Army Chemical Center, Md., depends on voltage changes occurring between platinum electrodes caused by ester hydrolysis. It requires no significant amounts of extraneous reagents that might affect enzyme activity. The depolarization rate of a platinum anode by thiocholine iodide is a measure of the hydrolysis of thiocholine iodide esters by cholinesterase or acetylcholinesterase, Mr. Cannon told the Division of Biological Chemistry during the 142nd ACS National Meeting. Hydrolysis is carried out at pH 7.4 in a 0.1M tris ( hydroxymethyl ) amino methane buffer system. A vacuum tube voltmeter measures the voltage at the anode against a standard calomel electrode. The technique is a general one. It applies to any reaction of the type: A (mediator)-» Β + C if A or the mediator and either Β or C are electroactive. In the case of thiocho line iodide ester hydrolysis, iodide ion is needed as a mediator because neither cholinesterase nor acetylcholin esterase is electroactive. A peroxideperoxidase system needs no mediator because peroxide is electroactive, Mr. Cannon notes. The same technique can be used to measure small amounts of anticholin esterase organophosphorus compounds such as Systox, Sarin, parathion, and malathion, Dr. Guilbault told the Division of Analytical Chemistry. It is both rapid and accurate. Anticholin esterase causes a decrease in the rate of depolarization of the platinum elec trode, because of the decreased rate of hydrolysis of thiocholine iodide.
