RESEARCH
O b t a i n i n g u n d a m a g e d nucleic acid component of tobacco mosaic virus—accomplished b y H . L. Fraenkel-Conrat (left)—was crucial step in reconstituting the virus from inactive components. T h e University of California research t e a m found their results hard t o believe at first, says N o b e l Laureate Wendell Stanley ( r i g h t ) , b u t finally decided in June t h a t they were correct after several restricted meetings with other eminent scientists in the field. Fragments (m.w. about 100,000) of protein component of T M V combined in acid solution to form doughnut-shaped bodies ( t o p ) . T h e y recombined to form long protein rods ( bottom ) in the form of a helix through which nucleic acid strands run like lead t h r o u g h a pencil
Research Rends Virus Veil Inactive components of tobacco mosaic virus combine in California test tube to form active TMV Same !ab produces crystals of polio virus —first crystals to be obtained of any animal virus
J^ROM THE VIRUS LABORATORY of the University of California comes news of two major achievements—first r e activation of a virus (tobacco mosaic) from its inactive component parts, a n d first crystallization of a virus (poliomyelitis) that affects man or animals. The veil surrounding viruses is t h u s further penetrated, showing them more than ever before to b e a chemist's p r o b lem, an arrangement of chemical components—which viewpoint supports h o ^ e of accelerated progress in virus research. Viruses—at least plant viruses—have long been known to be composed only of t h e two main components of living cells, nucleic acids and proteins. Virus
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reactivation—inducing these components to combine in t h e laboratory to form the original virus—was achieved finally late last spring b y H. L. Fraenkel-Conrat and Robley Williams of the Virus Laboratory. They worked with tobacco mosaic virus, a standard model for such fundamental studies. Barry Commoner and coworkers at Washington University got similar results in part in early August (C&EN, Sept. 26, page 4 0 3 4 ) . Years ago, Nobel Laureate Wendell Stanley, present director of the Virus Laboratory, was able to split virus nucleic acids and proteins, but t h e structural relationship of t h e two remained obscure. Later, t h e nucleic
acids were found to exist as "strands," w h i c h seemed likely to b e b o u n d loosely in the virus structure. And last year the structural problem was cleared u p by Roger Hart, working in Williams' laboratory, with a series of elejj|jfonrnicrographs which showed the partial splitting of virus nucleic acid and protein. Partial splitting was achieved by w a r m i n g the virus for about a minute in very dilute sodium lauryl sulfate w h i c h chewed off part of the protein, exposing the nucleic acid. Hart's photog r a p h s showed that the nucleic acid strands run tlirough the center of a rod, or coat, of protein, like the lead through a pencil. Other electron micrograph and chemical studies showed this protein rod to have the form of a helix, built up of many small subunits. W h e n broken down gently enough, the protein is obtained i n the so-called "native state," fragments too small to be seen even in the electron microscope. These fragments recombine in acid solution to form dougnnut-shaped bodies (cross-sectional view of very short pieces of the protein rods) and under suitable conditions will
finally form long rods which look^like viruses but which are not infectious since they contain no nucleic acids. • N e x t Job—Recombination. At this point entered Fraenkel-Conrat, whose objective was to make t h e protein fragments and nucleic acid strands recombine to form the original virus. To do this, he first needed "native" or undamaged nucleic acid, w h i c h he finally was able to obtain by removing the protein with 1% sodium l a u r y l sulfate—a feat not previously accomplished. Protein, the second component of the proposed partial synthesis, he obtained from different samples of the tobacco mosaic virus by removing the nucleic acid with sodium carbonate solution. Both components were first tested separately for contamination w i t h undegraded tobacco mosaic virus b y rubbing samples on tobacco leaves. Each proved totally inactive. FraenkelConrat then found that a slightly acid water solution (about pHE 6) containing roughly 10 parts of the protein to 1 of the nucleic acid soon acquired the typical opalescent appearance of virus solutions. Samples of this solution, t a k e n at varying periods after it was prepared, produced tobacco mosaic infection when rubbed on tobacco plants. Later analysis showed that samples t a k e n a few minutes after t h e solution w a s prepared were infective, and that t h e infectivity increased as the solution aged up to a maximum of about 2 4 hours. Within 24 hours, then, nearly all of the two virus components, inactive by themselves, had combined to f o r m the original, active virus. The protein and nucleic acid actually combine in a ratio of approximately 20 to 1, leaving an excess of nocleic acid in solution, and the concentration of the two affects their ability to combine. Below certain concentrations recombination does not take place, which supports the idea that t h e process is chemical in nature. Concurrently with Fraenkel-Conrat's chemical and inf ectivity work, Williams was using the electron microscope to clarify the physical nature of the virus and its components at various stages in the process. H e was unable to detect virus particles in e i t h e r of the two components but found that many typical virus rods were formed in the acidified mixture of t h e two. And Hart, also with the electron microscope, found the nucleic acid was located in the center of the newly formed virus rods as it is ir*. natural tobacco mosaic virus. Neither chemical nor electron microscope techniques have detected any differences between the original and reactivated viruses, but the research team does not y e t feel
positive that such differences do not exist. Whether or not the protein combines in its doughnut form with the nucleic acid is still a question, according to Williams, who says the smaller fragments may be involved. The molecular weight of the smaller fragments, determined by H. K. Schachman of the Virus Laboratory, is about 100,000 and that of the nucleic acid about 250,000. Tobacco mosaic virus has a molecular weight of about 50 million and is thus composed of about 500 pieces of protein (the smaller pieces) and 12 of nucleic acid. Protein fragments with a molecular weight of about 20,000 have been detected, but they are not "native," and will not form the larger protein particles under experimental conditions used to date. • Multiple Avenues. T h e achievement opens several avenues of investigation, according to the Virus Laboratory workers: • A study of heredity in living things. If one self-duplicating system can be formed from its inert components, as it has been here, it may be possible to reproduce other such systems—chromosomes, for example, the carriers of hereditary characteristics. • Reconstitution of immunity-producing viruses—of far reaching import in disease prevention. The laboratory is already at work in this field with a major study of the factors that promote and block reconstitution of viruses. If the reconstitution mechanism can be fathomed and if it can then be influenced, it may b e possible to tailor viruses to order. • Development of virus antigens, chemicals which stimulate production in the blood of antibodies which fight disease agents. Particularly interesting, says Stanley, are the sizes of the protein fragments, Those with a molecular weight of about 20,000 are within, the range of chemical and structural analysis and ultimate synthesis. If they can be synthesized in the future it might be possible to make them combine to form the protein units with a molecular weight of about 100,000 that go into viruses.
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Animal Virus Crystallizes I n 1935, Stanley crystallized tobacco mosaic virus, the first virus of any kind to b e crystallized. A half dozen or so other plant viruses have been crystallized since then, but viruses that affect men or animals have resisted until this year, when C. E. Schwerdt and F. L. Schaffer of the Virus Laboratory finally achieved success with a strain of polio virus. Schwerdt and Schaffer worked with
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Under t h e optical microscope, t h e crystals appear to be tetragonal with pyramidal ends. Detailed, measure ments of t h e crystal faces h a v e not yet been made., but the crystals themselves are about 3 0 microns long a n d contain perhaps a billion virus particles. Apart from the fact that i t is a scien tific "first," this achievement by Schwerdt a n d Schaffer re-emphasizes certain basic similarities of animal and plant viruses. Also, t h e ability of a virus sample to crystallize is believed generally to be an important measure of its purity and homogeneity, proper ties of extreme importance t o the Virus Laboratory, whose broad objective is to determine t h e basic physical and chem ical properties of viruses.
A Success Story University of California researchers Schaffer (left) a n d Schwerdt examine crystals of polio virus from the first
First antifungal antibiotic said to be s a f e f o r human
or animals
to N e w York scientists 1 H E SUCCESS STORY of nystatin, Eliza
M E F - 1 , Type I I polio virus whose physical and chemical properties have been under investigation at the Virus Laboratory since 1952. Their starting point was a standard monkey kidney tissue culture of t h e virus containing something like 1 gram per liter of pro tein of which about O.l mg. was actual virus. In brief, Schwerdt and Schaffer pre cipitated the protein from the tissue culture fluid by adjusting the ρ Η to about 4 and adding methanol. T h e precipitate was redissolved in 1M sodium chloride solution from .which protein contaminants were extracted with butanol arid t h e solution was con centrated b y ultracentrifuging. Next, nucleic acid contaminants were di gested b y nuclease enzymes and the sample was ultracentrifuged again. At this point the original tissue culture sample h a d been concentrated about 10,000-fold, volume wise. After further purification (physical), the sample was held at about 4° C. for 2 4 hours, during which time roughly 40% of t h e purified virus crystallized. As a n experimental check, the re covered crystals—about 0.001 gram in weight—were redissolved and infective virus was recovered. 4796
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beth Hazen, and Rachel Brown started in 1946 when the two undertook a search for an antifungal antibiotic. The need then for an antibiotic for fungus diseases was especially striking in contrast to the widespread use of these drugs against bacteria. The story culminated last week in Albany when the scientists from the N e w York State Department of Health received the $5000 Squibb Award in Chemotherapy.
The search for t h e antibiotics started with a program of screening soils for actinomycetes antagonistic to fungi pathogenic for man. Hazen, a micro biologist, finally centered the studies on a strain found in a soil sample from Warrenton, Va. She collected t h e sam ple while vacationing on a friend's farm. She named the n e w species Streptomyces nouresi, in honor of her host. • Two, Not O n e . Trial extracts of whole culture, mycelium, or filtrate were made with many solvents. Brown, a biochemist, found that there must b e at least two agents; in 1948, she found that nystatin was a relatively insoluble product in the mycelium and active against both test fungi Candida albi cans a n d neoformans. T h e other agent was too toxic for human use. Brown tentatively arrived at an em pirical formula of C 4 6 H S 3 N 0 1 S . T h e molecule has weakly basic a n d acidic properties and forms unstable salts with acids and with metals of t h e alkali and alkaline earth groups. Ultraviolet absorption spectra in dicate a conjugated tetraene structure. This structure is of great interest in that several new antifungal agents derived from t h e streptomycetes give UV a b sorption spectra which indicate a con jugated polyene chromophore. T h e positions of the absorption maxima place these compounds into four groups corresponding to four, five, six, or seven double bonds in t h e polyene moiety. This structure is n e w among antibiotics, although related unsatu rated structures have been recognized
Nine years of research brought N e w York scientists Hazen ( left ) a n d Brown the first antifungal antibiotic safe for h u m a n use and the Squibb Award in Chemo therapy. Nystatin's structure is said to b e new among antibiotics
