RESEARCH
More Genetic Code Data Bring Solution Closer German findings relate changes in TMV protein to changes in RNA base sequence, agree with workers in U.S. and Britain Evidence continues to pile up in sup port of last month's prediction that scientists might solve the genetic code problem within a year. Dr. H. G. Wittmann of the Max Planck Insti tute for Biology, Tubingen, West Ger many, told the Gustav Stern Sympo sium on Perspectives in Virology in New York that his group has reached conclusions very similar to those of Britain's Dr. F. H. C. Crick, but on a different biological system (C&EN, Feb. 12, page 43). Dr. Wittmann agrees with Dr. Crick that the genetic code is nonoverlapping and probably degenerate, and that it is read from a fixed starting point in triplets. He has studied chemical and spontaneous mutants of tobacco mosaic virus ribonucleic acid
(TMV-RNA) and the effects of these mutations on the amino acid sequence of the synthesized protein. Dr. Crick's work was on bacteriophage T4. Further, Dr. Wittmann told the symposium, the similarity in results from T4, tobacco mosaic virus (TMV), and the polynucleotide-induced pro tein syntheses of Dr. Marshall W. Nirenberg at the National Institutes of Health and Dr. Severo Ochoa at New York University points to another im portant conclusion: The genetic code is universal for all biological systems. Dr. Wittmann also finds that the nucleotide sequence coded for protein synthesis occupies only a part of the RNA chain. He estimates that about 500 of the 650Q nucleotides participate in protein synthesis. The other
nucleotides along the chain have noth ing to do with proteins. Changes in their sequences do not affect protein structure, but appear to alter the host's metabolism instead, he adds. TMV Studies. Genetic code re search at Tubingen has focused on TMV-RNA. In 1958, Dr. Wittmann's colleagues, Dr. Gerhard Schramm, Dr. Heinz Schuster, Dr. Karl W. Mundry, and Dr. Alfred Gierer found that they could deaminate nucleotides on the RNA strand and induce mutations in the RNA with nitrous acid. Nitrous acid converts certain nucleotides into others—for example, cytosine to uracil —thus changes nucleotide sequence in the RNA chain. The extent to which nucleotides are changed can be con trolled by reaction conditions.
Tobacco Mosaic Virus-RNA Mutations Lead to Changes in TMV Protein 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Acetyl-Ser-Tyr-Ser-lleu-Thr-(Pro-Thr)-Ser-GluNH2-Phe-Val-Phe-Leu-Ser-Ser-Ala-Try-Ala-Asp-Pro-!leu-G lieu
32 33 34 35 36 37 38 Gly-AspNHa-GluNHa-Phe-GluNHa-Thr-Glu^ Arg
39
40
41 42 43
44
j
45
46
47 j
48
49
50
24
25
26
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51 52 53 54 55 56
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62 63 64 65 66 67 68 69 70 71 72 7#3 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 Phe-Pro-Asp-Ser-Asp-Phe-Lys-Val-Tyr-Arg-Ty^^ Ser τ Τ Τ
29
30 31
58 59 60 61 Heu
T
92 93 94 95 Τ
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 Val-Glu-AspNHa-GluNHa-Ala-AspNHa-Pro-Thr-Thr-Ala-Glu-Thr-Leu-Asp-Ala-Thr-Arg-Arg-Val-Asp-^ Τ Τ
127
128 129 130 131132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 Leu-lleu-Val-Glu-Leu-lleu-Arg-Gly-Thr-Gly-Ser-Ty^ Thr j Phe Lys j CHANGES. Diagram points out changes that Dr. H. G. Wittmann of the Max Planck Institute, Tubingen, West Germany, has observed in tobacco mosaic virus (TMV) pro tein caused by TMV-RNA mutations. Colored blocks indicate positions on the protein chain where exchange takes place. For example, at No. 5, a threonine group is replaced by an 38
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isoleucine group. Changes at 5, 55, 59, and 63 are caused by nitrous acid mutants. Changes at 33, 129, and 140 are from spontaneous mutants. The change at 138 is from both nitrous acid and spontaneous mutants. The circled Τ indi cates places where trypsin splits the chain. Sequences en closed by parentheses are not completely certain
Building on the opportunities opened by the nitrous acid-induced mutations, Dr. Wittmann has studied a range of such mutant TMV-RNA's and the structures of the proteins synthesized from them. His technique is to treat highly purified TMV, or its RNA, with nitrous acid, and separate the various TMV-mutants by a series of multiplications in specific kinds of tobacco plants. Each mutated virus is inoculated onto about 150 more tobacco plants, where it multiplies. After two or three weeks, the plant leaves are harvested, the virus isolated and purified, the RNA and protein parts separated, and the protein part analyzed by splitting with trypsin. The tryptic peptides are analyzed chromatographically. When a modification in the protein structure is found, the Tubingen scientist then carries out a further analysis to pinpoint the spot at which the amino acid sequence has changed and what the new sequence is. So far, Dr. Wittmann says, he has studied 130 TMV mutants this way, including 114 chemically-induced and 16 spontaneous mutants. In about two thirds of the cases, mutated RNA does not lead to changes in viral protein structure, he finds. This, plus data on the parts of the RNA affected by the nitrous acid, leads him to conclude that not all of the RNA chain gets involved in viral protein synthesis, and that the protein-synthesizing part must be modified to. produce a change in viral protein structure. In the remaining cases where protein differences were found—over 30— only 13 different kinds of amino acid replacement occurred. This is reasonable, he says, considering the limited types of changes in RNA composition that nitrous acid can induce. He also finds that changing a single base in the RNA leads to only one change in amino acid sequence and in only one place in the protein. This leads him to conclude that the genetic code is not overlapping. Taking into account the results of Dr. Nirenberg, Dr. Ochoa, and Dr. Crick, Dr. Wittmann summarizes the present state of knowledge this way. "The base compositions, but not the sequences, of the amino acid determining nucleotide groups are now known for all amino acids. But, since a given amino acid can be determined by more than one triplet, we still need to find out how these triplets relate to one another."
Viscous Flow Equation Needs New Constant Retardation constant must be added, studies on polytetrafluoroethylene show Studies on polytetrafluoroethylene (in the form of Teflon-TFE fluorocarbon resin) by Dr. John F. Lontz of Du Pont show that a retardation constant must be added to the FrenkelKuczynski expression for viscous flow, he told the Fourth Delaware Valley Regional Meeting of the ACS, held in Philadelphia. The need for this constant is evident from time-dependence studies which show that the viscoelastic nature of Teflon at the transition temperature restrains Teflon's tendency to flow. This restraint is observed throughout the 20- to 120minute period during which the experiment is conducted, and indicates that sintering involves some mechanism other than purely viscous flow, Dr. Lontz says. Polytetrafluoroethylene (in the form of Teflon-TFE fluorocarbon resin), which has a high molecular weight, is also chemically inert and resistant. The resin also has excellent electrical properties, a low coefficient of friction, and nonadhesive qualities. The resin's high molecular weight, however, works against the use of dynamic melt techniques to fabricate the polymer for applications where its properties can be best employed. Instead, the desired item» generally must be preformed under pressure and then sintered above its 327° C. melt transition temperature. In Dr. Lontz's experiment, six 60-mil diameter unsintered Teflon headings are twisted around the seventh identical beading, one twist per inch. This produces what in cross-section is a hexagonal arrangement over the core with 12 contact points for interfacial fusion. Temperatures of 380° to 400° C. ( ± 4 ° to 5° C.) are used. After exposure at the sintering temperatures, the twisted headings are withdrawn from the oven, cooled, and cut perpendicular to the cable length into 2-mil (50-micron) thick sections. For exposures of 30 minutes or less, the thermal lag to the inner core makes it necessary to measure interfacial growth only where the exterior twisted cables contact each other (six points). With longer exposure times to the sintering temperatures, interfacial growth between the inner core and the
twisted cables may also be measured, giving six additional points. Strain Lines. When the junctions formed at these interfaces are examined microscopically under polarized light, biréfringent strain lines are seen that extend well into each of the contacting headings. This, Dr. Lontz notes, is evidence for a gross polymer transport due to segmental diffusion across the interface. The Frenkel-Kuczynski expression for viscous flow is x2/a = 3yf/2^; χ represents the radius of junction growth between two contacting sur faces of radius a (centimeters), γ the surface tension (dynes per square centimeter), t the time of sintering (seconds), and η the viscosity (poises). But a graph of x/a against time yields curves at the sintering temperatures that have almost no slope at all, and certainly not the second-power slopes from the FrenkelKuczynski equation. Furthermore, strict application of the equation using literature values for typical melt vis cosities and surface tension values of 25 dynes per square centimeter does not fit the experimentally observed data. Arbitrarily selecting ranges of ap parent retardation time, Dr. Lontz, recognizing that special techniques may cause these ranges to be even tually revised, solved the FrenkelKuczynski equation using a series of x/a values. The solutions, when com pared to the experimental data, re veal that for the sintering mecha nism, the time-dependent interfacial growth should more properly be de scribed as: x2/a = 3γΐτ/2η(τ-\-ί), where τ is the retardation constant. The best graphical fit between cal culated values using this modified equation and the plotted experimental data is obtained with η equal to 10 s poises and τ in the order of 10 4 sec onds. The equation itself is similar to one described in 1956 by Dr. T. Alfrey and Dr. E. F. Gurnee of Dow Chemi cal. According to Dr. Lontz, the equation, with his suggested retarda tion constant, may be considered as a theory of viscoelastic fusion, quite distinct from that of viscous flow for diffusion. FEB.
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Method Determines Lipid Phosphorus in Bacteria Cells
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as much nonlipid Ρ Lipid phosphorus in bacterial cells may be determined quantitatively even in the presence of 20 to 30 times as much nonlipid phosphorus. This lipid phosphorus is located within the bac terial membrane where it represents but a few per cent of the total cellular phosphorus. Thus a measure of the lipid phosphorus content of bacterial cells as affected by cultural conditions may, in turn, indicate the extent to which membrane formation is affected by these conditions. For example, Joseph J. Kolb, Insti tute for Cancer Research, Philadelphia, pointed out at the Fourth Delaware Valley Regional Meeting of the ACS in Philadelphia that based on lipid protein determination, cell wall ma terial makes up about 25% of the gross weight of bacterial cells during their most rapid growth phase. On the other hand, the cell wall material of bacteria (Streptococcus faecalis) grown under conditions in which threonine is limiting, makes up 44% of the gross weight of the cells. Un like threonine-limited cells, cells that are limited by one or another of the essential amino acids, or by other limiting conditions imposed by Mr. Kolb in his experiments, show no major change in their lipid phos phorus content during the phase of most active growth. However, should the bacteria not yet be in the most rapid growth phase or beyond it, the lipid phosphorus con tent is markedly different from that of the actively growing cells. The method used by Mr. Kolb is capable of detecting as little as 0.2 microgram of lipid phosphorus in samples containing 100 to 200 micro grams of bacterial substance. It has five major steps: • The lipid is split from the proteolipids and lipoproteins. T h e sample is dried and then refluxed in 95% meth anol. It is then centrifuged and the supernatant saved. After one reflux washing, the combined supernatants are evaporated in nitrogen nearly to dryness, the final drying being done in vacuum over silica gel. • Petroleum ether is used to sepa rate lipids from nonlipids.
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Preferential solvation of the neutral ization products is the major factor in determining the effects of solvents on the acidity and basicity of solutes according to studies by Dr. Orest Popovych, an Esso Research scientist. T h e equilibria between an indicator acid, bromophenol blue, and a series of heterocyclic amines in hydrocarbon and hydroxylic media have been de scribed for the first time by a series of equations and constants. These can be evaluated by a combination of visible spectrophotometry (as represented by the above illustration) and electrolytic conductance. The ion-pair formation constant K B = (BH+A")/(B)(HA) was
adopted as the measure of basicity of an amine Β relative to a reference acid HA. It was found that while the relative strengths of the bases remained inde pendent of the solvent, the absolute values of the K B ' s were a sensitive function of the medium. T h e acid-base reactions were most favored by polar and hydroxylic media. For example, adding isopropyl alcohol to toluene caused a rapid initial rise in reactivity which reached a plateau at about 5 % of added alcohol.The addi tion of 0 . 5 % water to a 50-50 isopropyl alcohol-toluene mixture produced a similar effect.
These indications together with other data make it clear that stabilization of ion pairs and ions by solvation seems to be the major factor in determining variation of acidity and basicity as a function of solvent. This new knowledge will permit more meaningful interpretation of acid-base phenomena and a more realistic approach to all research fields where nonaqueous media are involved. . . . adapted from the scientist's notes at
Esso Research and EngineeringCompany (P. O. Box 45B, Linden, New Jersey)
scientific affiliate of Humble Oil & Refining Company
• Water soluble material is removed from the lipid fraction by a water wash. • The petroleum ether fraction is centrifuged and the supernatant liquid is removed and evaporated to dryness. Sulfuric acid and water are added to the residue to hydrolyze hydrophosphates. • The digests are treated with niolybdate hydrazine reagent and heated. The resulting colored solutions are read against blanks at 830 millimicrons and the readings compared to known standards. Provision is also made in this technique to adjust optical density because S. faccalis growing in the medium doubles its number of cells and consequently its optical density each 30-minute period. When graphed, curved lines result due to light scattering. When adjusted, straight lines are obtained. These adjustments in optical density are determined experimentally.
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BRIEFS Adding cerium raises the operating temperatures of silicone lubricants
from 400° to 575° F., according to studies at the Naval Research Laboratory. Findings at NRL show a striking improvement in the quality of silicone lubricants by using metal ions— particularly cerium—to inhibit oxidation and gelation. NRL also tested two complex amines, disalicylalethylenediamine and disalicylalpropylenediamine with lightly phcnylated silicone. The two amines proved to be good antioxidants and extended gelation time eightfold. These developments may extend the use of silicones to jet-engine lubrication, aircraft hydraulic fluids, and heat transfer liquids in high-temperature mechanisms, NRL savs.
Research in the physics of fine particles is under way at a new laboratory set up by the U.S. Department of Agriculture. The lab is at Wooster, Ohio. Dr. Ross D. Brazee of USDA's Agricultural Research Service has been assigned to the lab. He will be responsible for research in the formation and behavior of finely divided liquid and solid particles.
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