AN ANALYSl.S OF THE HLSTORY OF INFECTIOUS NUCLEIC ACIDS :. -. ..' .:: by Mark Weidenbaum, University of Connecticut This work is based on a Directed Studies Summer Program carried out at Stanford University in the Summer of 1975 under the direction of Dr. Joshua Lederberg. TABLE OF CONTENTS Abstract Part I - History Prel imi nary ideas Quant$tati.ve Studi.es Proof of Infectious Nucleic Acids Part 11 - Ana1ysi.s Francesco Sanfel ice Lncl us i,on Bodi.es Cytochemical Staining of lnclus A Mistake in Interpretation Problems in Communication ion Bod ies Part 111 :. - The Graphical Ci tati.on Index .J. ; a,/ !: lnt roduct i on s Chronological Perspective Clustering Combinati.on of Techni.ques P roh 1 ems Summa ry Bib1 iography (To be used in conjunction tii th accompanying chart entitled, "Citation Index for Infectious Nucleic Acids ."I Acknowledgments Page i i 1 1 2 4 5 5 6 7 8 8 10 10 10 1 I 12 12 13 14 20 ABSTRACT This review traces the significant developments in virus chemistry from Bei jerinck's (1898) recognition of viruses as distinct bodies to the demonstration of their infectious nucleic acid nature by Gierer and Schramm ( 1956) . The study demonstrates how cytochemi cal staining methods applied to inclusion bodies served,asa useful meth,od fo r investigating the early years of virus chemistry. The investigation a 1 so analyzes the effects of Stanley's misinterpretation of proteins as infectious agents /' and ,his dismissal of nucleic acids as being important in the viral infec- : .* ,. ,.." . j tion process. The study reveals that Sanfelice's (1918) observations _I. c were lost from central thought but anticipated many later developments. . The review includes a discussion of graphical citation indexing as well as a graphical citation index surveying th'e history of infectious nucleic acids from 1873 to 1960. -i L * Prel iminary Ideas PART I - HISTORY In I892 D. lwanowski 44 recognized that the juice of Turkish Tobacco plants having the tobacco mosaic disease remained active after being passed through a Chamber-land f i lter (a standard microorganism f i 1 ter) . Via this work, lwanowski had demonstrated the existence of what is now known to be a vi rus. Yet, he chose to regard the infectious agent as bacterial in nature. Six years later M. Beijerinck4 repeated lwanowski `s work but inter- preted the results in terms of a "contagious living fluid." Therefore, he was the first to recognize the fundamental difference between the "f i 1 ter passing agent" 4 .I and ordinary bacteria. After Beijerinck's &work several diseases were determined to be caused by "filter passing agents," which had been termed viruses. In + . `(' `?) 1898"Loef f ler and Frosch 49 fi.rst discovered an animal virus, 62 r that causing hoof-and-mouth di,sease in cattle, In 19,ll Walter Reed 62 de te r- mi.ned that yellow fever w.as`also a viral disease. The identi.fication of another major family of viruses, namely those affecting bacteria,_ fol lowed when Twort 82 and d'Here1 le 40 recog- nized the "bacteriophages .`I Al though vi ruses were thus recognized as themselves, their structure and mechanism rema As W. Stanley 76 stated, ". . . the general nature organisms dist inct in ined unknown at this time. of the vi ruses was unknown and they had been regarded variously as invisible forms of ordinary bacteria, as a new kind of invisible living organism, as protozoa, as unusual products of cellular metabolism, as enzymes, and as different ki nds of i nan i mate them i ca'll s ubs tances . " In general, the problems involved in isolating pure virus samples and obtaining conclusive data prevented significant progress. As the reviews by Roux 63 , Wolback 88 , Twort83, and Bayon demonstrate no part i cu- lar theory predominated. Quantitative Studies Until 1935 the ideas concerning the nature of viruses were largely based on conjecture. However, in that year W. Stanley 74 successfully crystallized Tobacco Mosaic Virus (TMV) protein via ammonium sulfate precipitation. He then confirmed that this virus multiplied only within the cells of certain definite hosts, thereby differentiating from normal pathogenic bacteria. By thus isolating a crystal line "protein" having . TMV activity, Stanley facilitated direct measurements of infectivity by correlating the amounts of TMV protein present with the degree of virus actr'vi ty. : +. *i li a After measuring chemical and physical constants of the crystallized "protein," Stanley concluded that `I.. . the high molecular weight proteins . carrying virus activity a.re characteristic of virus-diseased activity." 74 Hence, his evidence pointed towards the existence of "infectious proteins." 74 Although Stanley had assumed that his TMV protein preparations were -6 sufficiently pure, Bawden and Pirie2 showed that liquid crystals of TMV contained 0.5% phosphorus and 2.5% carbohydrates. They noted that these materials were nucleic acids of the "ribose type.`12 They also postulated that the TMV proteins consisted of long fiber-like particles that aggre- gated to form longer threads. Despite this work, Stanley maintained that his preparations were good enough and that the phosphorous "impurities" to which Baden and Pirie referred were of litgle significance. He based this conclusion -2- `. on his earlier findings that phosphorus was unnecessary for virus activity. Thus, he maintained that different viruses led to the synthesis of different proteins and that nucleic acids were of minor importance. By 1942 Cohen and Stanley 17 had determined that RNA particles had an average molecular weight of 300,000 and were highly assymetrical. More importantly, this time Stanley also recognized that Bawden and Pirie's "nucleic acids" were far more important than he had earlier suspected. Cohen and Stanley concluded from thei.r data that vi ral nucleic acids existed as threadlike molecules, the length of whi ch corresponded to that of the intact virus molecule. During the mid-1940's emphasis shifted away, from proteins and t. .' towards nucleic acids as the possible infectious elements of viral in- fection. Avery then contributed his finding that in Pneumococcus the ; subftance which induced transformation of one bacterial type to another - ,: + . w i *.. "appeared to be.. . sodi urn deoxyribonucleate, inducing the synthesis of non- nitrogenous polysaccharide composed of glucose-glucuronic acid linked . in glycosidic union." 1 Although Avery was working with bacteria and not viruses, his discovery of DNA as the "Transforming Principle" stimulated interest in -. nucleic acids as vectors of infectivity. He had verified that the inducing substance (DNA) and the substances it induced (high molecular weight proteins) were chemically distinct and biologically specific. These findings heightened the possibility that nucleic acids themselves were responsible for infectivity. In 1952 Hershey and Chase 41 employed radioactive sulfur and phos- phorus tracers to prove that the viral protein she1.J attaches to the ccl 1 .,. -3- , wall of its target but does not enter the cell itself. Instead, it injects its nucleic acid core into the ccl 1. Despi te these f indi ngs Hershey and Chase could not explain how the vi rus rep1 icated once inside the .I nfected ce 11 . In an attempt to explain nucleic acid structure, Paul i ng and Corey57 postulated that the nucleic acids might form an alpha-helix. Although this prediction was wrong, the idea of a helix contributed to Watson and Cri ck's86 demonstration in 1953 that DNA was composed of a double helix held together by hydrogen bonding and van der Waals forces. Later that year Watson and Crick 87 proposed that the double he1 ix contained a pair of complementary template strands which could pull apart. Each strand could'then form a new complementary chain. They suspected that the sequence of bases attached to the sugar-phosphate :. backbone making up the strands was the code that carried genetic infor- ,L 1,' .: , matidn. - The Watson and Crick model resulted in the interpretation that viral nucleic acids carried their own replication instructions. Once inside their host, they could replicate and take control of the cell math inery for prote i n syn thes i.s . The viral nucleic acids could then -.. L use this machinery for the synthesis of the high molecular weight protei~ns necessary for their protective she1 1s. Thus, the viral nucleic acid could itself be the infectious agent responsible for disrupting cell metabolism. Proof of infectious Nucleic Acids The final proof that nucleic acids were themselves capable of infection came in 1956 when Gierer and Schramm 32 showed that bare -: -4- RNA, purified from TMV, was itself capable of inducing infection in the tobacco plant. They stated : "We are thus led to conclude that the in- fectivi ty is due to the nucleic acid itself. ,132 Gierer and Schramm's work stimulated subsequent investigation for infectious nucleic acids in animal and bacterial viruses. In 1957 Spizizen73 attempted to establish that T2 bacteriophage DNA could infect E. co1 i bacteria protoplasts, -- but his preparations were impure and his results inconclusive. 30 Later that year Fraser et al. performed the same -- experiment and presented more evidence that naked bacteriophage DNA could induce infection. However, i.t remained for Guthrie and Sinsheimer to prove conclusively in 1960 that "protoplats of E., coli can be infected -- with the DNA of 0Xl74."37. (0X174 is another bacteriophage.) Concurrent with this work on bacteriophages, Colter et al. 18 -- :. demonstrated that infectious nucleic acids existed in animal viruses. `P . . `ii He stated: "Ribonucleic aci.d isolated from Ehrlich asci tes tumour I cells infected with Mengo encephalitis virus is infectious, and the ribonucleic acid component, rather than residual intact virus particles, is responsible for this activity." 18 * -- PART I I - ANALYSIS Francesco Sanfel ice While studying Epithelioma Contagiosum (a viral skin disease observed mainly in birds) in 1914, Francesco Sanfelice 65 noted that "it is most interesting to see how a disease can be produced with the nucleoproteide which was extracted from the diseased tissue." In 1928 Bronfenbrenner 11 that Sanfel ice had extracted a substance which per- . -5- petuated disease as if i t "were a 1 ivi ng vi rus .`I 11 Unfortunately, the 1 imitations of optical, physical, and chemical techniques prevented Sanfelice from obtaining more than speculative evidence concerning the nature of the Epi the1 ioma Contagiosum virus mechanism. Nevertheless, Ssnfelice was apparently the first to suggest that viral infection was due to something other than attack by the intact virus. Rather, he speculated that the "nucleoproteide", not the complete virus particle, was the infectious element. Thus, he anticipated much of what has since been determined concerning the mechanism of viral attack. inclusion Bodies One of the earl iest clues concerning the nature of vi ruses centered on the observation that some diseases induced development of cellular inclusions, referred to as inclusion bodies. In 1881 Rivolta61 \. observed such inclusion bodies in the cells of chickens having fowl pox. . . Boll inger7 made similar observations while working with fowl diseased with Moluscum Contagiosum. In 1894 Guarnieri 36 discovered typical in- clusions in cells having vaccinia (small pox virus). At first these inclusion bodies were mistaken for protozoa. This mistake gave rise to the term "Chlamydozoa" 59 . During the period of approximately 1910 through 1930 this term was used to describe inclusion bodies. Although inclusion bodies had been recognized since 1873, their specific connection with virus activity was not demonstrated until Paschen56 did this in 1917. Subsequently, two fundamental theories arose concern ing the relat onship of inclusion bodies to viruses. One theory -6- proposed that the inclusions were products of the react on of the infected ccl 1 to the virus. The other theory, which has since been proven, was that inclusion bodies were virus colonies themselves. Cytochemical Staining of lnclusi,on Bodies Once inclusion bodies were associated with viruses, the former were tested cytochemically for a variety of substances, including nucleic acids. Consequently, it has been possi.ble to use cytochemical staining methods as a means of exploring the early years of virus chemistry. The cytochemical test most useful in studying these years has been It is still the lysis of the alde- the Feulgen React ion, developed by Feulgen 26 in 1924. single most definitive test for DNA. 1 t involves hydro > hydes .in the nitrogenous bases of DNA which is then fol lowed by rosanil ine staining. `. ; *" i -Another method for testing nucleic acids (usually - 1~ * _I \= 10 1 ' differential staining. In 1940 Brachet discussed the WI. RNA) i nvol ved use of ribo- nuclease to cleave "pentosenuclei c aci d" into so!uble rnononucleoti des as . a specific test for RNA. .The material to be tested was stained via the Feulgen react ion before and after the act ion of the ribonuclease in order to make sure that no DNA had contributed to the observed results. Cowdry 21 was apparently the fjrst to apply the Feulgen test directly * to inclusi.on bodies. In 1928 he stated, I'.. . the Feulgen reaction showed both types of inclusions contain 1 i tt le or no thymonuclei c acid." 21 Haagen and Kodama3' used the Feulgen reaction in 1937, getting positive results (indicating the presence of DNA) for "inclusi negative results for "elementary bodi.es." 38 (There i today about the precise meaning of th.is statement as .` \ -7- on bodies" and s some confusion the terms "inclusion .". ,/,, body" and "elementary body" are now considered vi rtual 1 y synonymous. The first proof that inclusion bodies contained DNA came in 1940 when Smadel and Hoagland 71 used a positive Feulgen reaction to prove the . presence of DNA in vaccinia-induced inclusion bodies. A Mistake in interpretation Si.nce little was possible before the crystallization of TMV, W. Stanley's work was a major step in turning virology into a disciplined and quantitative science. Hmever Stanley erred seriously in insisting that viruses were pure protein and in initially dismissing Bawden and Pirie's emphasis on "impuri ties .`I Ironically, the very "impurities" to which they referred had, in fact, accounted fo'r the infectious activity which Stanley had measured and mistakenly attributed to viral proteins. Due to his prowess at the time, Stanley's misinterpretation of the possible role of nucleic I. . *.;;. acids directed virus research in the late thirties and mid-forties toward . the study of proteins and away from investigation of nucleic acids. Problems in Communications 1. With regard to Sanfelice, why was his original and provocative work, done in 1914, lost from the focus of the scientific world? Since no ideas during his time could be substantiated, why were his ideas ignored while others flourished? A possible hypothesis is that his use of scientific German was very poor. Thus, his contemporaries probably had so many problems with his ill-constructed sentences that they did not seriously consider the content of his work. Hence, Sanfelice's valuable insights did not fluorish. -8- : .* 2. It has often been necessary to use critical reviews in order to determine how well gi.ven ideas were accepted in the scientific community. In particular, considerable controversy `arose concerning the distribution of credit between Spizizen, Fraser, and Guthrie and Sinsheimer for first credit for proving infectious bacteriophage DNA, Kozloff 46 and Co1 ter and Ellem2' did not agree. While Kozloff and Colter demanded more proof, Ravin and Schramm accepted Fraser's evidence as sufficient proof. it appears that Ravin's and Schramm's interpretations were incorrect since proving the existence of infectious phage DNA. The critical reviews on this topic highlight some differences in approach among investigators . Whi le Ravi n 60 and Sch ramm 66 gave Fraser they were based on premature assumptions. Ravi n's statement "these I findings, albeit preliminary, on the infection of protoplasts by viral DNA raise enormous possibilities for the future..." implies that infectious ix, DNA had already been proven. Actual ly, the f i rst accepted proof of II 7 infectious DNA came with DiMayorca 24 in 19.59, a year after Ravin wrote I his review. .3. The need for improved. communication has clearly been demonstrated in reviewing the literature. An example of this is in the article by Bland and Robinor! wherein they state, L'S0 far as we are aware, Haagen 38 (1937) is the only investigator who has applied this reaction (Feulgen reaction) to a vi rus. He stated that the inclusion bodies of vaccinia gave a positive Felgen react ion, but that the elementary bodies are negative." 6 Indeed, Bland and Robinow were not aware that Cowdry had applied the Feulgen test to inclusion bodies in 1928. From the above, it is apparent that as the volume of work grows, new and more effective means of communication are neebed. One useful approach which will be described is the Graphical Citation Index. /. `. Part III - The Graphical Citation Index A. Introduction The accompanying citation index provides a visual means for tracing developments in virus chemistry from 1873-1960. Continuing analysis of the history of virus chemistry is particularly warranted in view of the close relationship between molecular genetics and the study of infectious nucleic acids. This study centers on the events leading up to the demonstration of the infectious nucleic acid nature of viruses. Such analysis clarifies and readily exposes historically significant developments by pointing out changes of ideas in addition to new experimental proceedings. Moreover, such interpretation serves as a". working tool for re-evaluating early insights and possibly minimizing repetitive experimental work. :.The index has several features which are briefly outlined below. .J. *, *; '. . I. 8* Chronological Perspective . .A broad overview of the index clearly shows periods of high activity during 1935-1942 an-d 1953-1960. These time periods, which show much higher "publication density" than the periods 1873-1935 and 1943-1953, generally follow some critical investigation that made availAble new materials or concepts: For example, Stanley's 1935 TMV crystallization (reference 74) essentially sparked the high "publication density" that ensued from 1935-1942 since it provided a previously unavailable material, the crystallized Tobacco Mosaic virus. Similarly, Hershey and Chase's labelling experiments in 1952 (reference 41) together with Fraenkel-Conrat's work on TMV structure in 1955 (reference 28) stimu%ated the high "publication density" from 1953-1960 by providing new empirical and theoretical input concerning *.the mechanism of viral attack. 0 0 Although there may be some omissions, this graphical method physically displays the general periods of activity as well as those of relative passivity. c, Clustering The central power of this index lies in that it reveals which authors commonly cited the same references and thereby determines the common-reference-clustering-pattern for each article. Such clustering patterns may then be used to determine the relatedness of different articles according to their degree of common citation. If two articles cite a common reference, they are probably related to each other. Otherwise, they would not have cited the same work. If,two or more articles cite two common references, they are almost certain to be closely related. Hence, as their number of corrqhon citations increases, the probable relatedness of two (or mor6') articles also increases. .r: The determination of relatedness by use of the graphical display is of great use in searching the literature. Using the index one first determines the clustering pattern for a given article. Having then found several articles with at least one reference common to that (and probabl * initially given y many more) one can directly examine these and bypass much of the mass o'f unrelated material that usually accompanies a literature search, This method clearly depends on the completeness of the citation index used for the search. The full value of this technique thus increases directly relative to the completeness of the index, Although not exhaustive, the accompanying index provides point for surveying the literature on infectious nucleic It is recommended that this work be more fully expanded. _ a starting acids. D. Combination of Techniques . ' c.9 / A third service provided by the index is that it highlights I$@ different fields overlap and physically shows where isolated techniques have been combined. New applications of existing techniques has visibly been critical to many of the investigations reviewed here. Some examples include (1) Smadel and Hoagland's demonstration of viral DNA by application of the Feulgen stain to inclusion bodies (reference 71) and (2) Stanley's use of ammonium sulfate precipitation with globulins to precipitate TW (reference 74). Thus, by following the techniques cited on the chart, it is possible to decipher where and how different methods came together. This process confirms that important results often follow when two isolated findings are pooled and also brings out the more common :$nstances of lack of communication between investigators. E. Problems 4. . .r: rj 1 Presently, there are very few graphical citation indices Gvailabie. This limits the amount of investjgation that can be done with them. Therefore, an important task now is to develop and provide more complete graphical citation systems, In addition, there se some *herent difficulties which result from the fatiure by some authors to directly cite original references. Instead, some authors cite seccdary references or even none at all if the tectiique whi& the-y izrvolve is v-cry common. This introduces the possibility that the r&'erences urpsn which the index is built may themselves :tie inzzrmplete `bLbXicgraph.ies.. One solution to this problem would be to adopt the convention whereby a f~rmaI.`bLbli~hi-c Xist.ing wouldnot :be necessary to . warrant an tidex ,connectidn.. IRather,, Limply menti.oning a method *. . c) r3 . br concept in the body of the paper would suffice for indexing purposes. Another problem arises from the possibility that authors may cite the same reference for different purposes. As a result, the relatedness of citing articles would not be guaranteed simply by their clustering patterns. I This difficulty mainly affects those articles commonly citing only one reference. As long as one or two additional common citations exist this problem is insignificant. F. Summary Despite kts problems listed above, the graphical citation index is a powerfu$ tool for analyzing developments in their proper historical framework and for facilitating rapid and accurate literature searches. This method is universally applicable to all areas ,of study and i; provid& an immediate picture of how past events have shaped a given field. Hence, it is an excellent teaching tool , A 'complete catalogue of citation indices covering specific topics and sub-topics in well defined disciplines could be one of the most useful investigative 3001s available. As all branches of investigation become increasingly complex, w corresponding problems arise concerning how to maintain the necessary levels of communication. In such light, the citation system outlined above becomes increasingly important since it highlights particular developments, places them in proper perspective, and facilitates rapid information transfer between different sources. 5 I BL I OGRAPHY --. . Note: The following sources are graphically displayed in the accompany- ing Citation Index. The key is as follows: 0 denotes general reference 0 denotes review article or text A denotes key (i.e., highly significant) article Those articles 1 isted in the Bibliography with an f: are not included on the Citation Index. cl 1. Avery, 0. T., C. M. MacLeod and M. HcCarty, "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types." J. Exp. Med. 79: 137 1944. ---a- - Bawden, F. C., N. W. Pirie, J. D. Bernal and I. Fankuchen, "Liquid Crystalline Substances ,from Virus-Infected Plants." Nature 138: 1051 ?936. I 3. Bayon, H. P., "Virus Diseases of Bacteria, Plants, and Verte- brates.li J. Trop.' Med. 29: 17 1926. ---- Beijerinck, M. W., Verd. Akad. - -.-- Nr. 5:l 1898. Bland, J. W. W., and C. F. Rob Method to the Study of Vi ruses Wetensch. Amsterd. 11 6, - -' inow, "Application of the Feulgen . `I Nature 142: 721 -- - 1938. cl 6. Bland, J. 0. W., and C. F. Robinow, "The Inclusion Bodies of Vaccinia and Their Relationship to the Elementary Bodies Studied in Cultures of the Rabbit's Cornea." J. Path. Bact. 48: 381 1939. ---- 0 7. Boll inger, O., "Ueher Epithilioma Contagiosum beim Haushuhun und die Sogenannten Pocken des Geflugels." Virch. Arch. f. Path. Anat. 58: 349 1873. --- I -- Borrel , A., "Epi the1 ioses I nfectieuses et Epi the1 ioma." Ann. I. Past. 17: 81 1903. -- -- 0 9. Borre! , A., `Sur Les Inclusions de 1'Epithelioma Contagieux des Oiseaux." C. R. Sot. Biol. 57: 642 1904. -m--p__ Brachet, J., "La Detection Histochemique Des Acids Pentosenucleiques." C. R. Sot. Biol. 133: 88 1940. w---- Bronfenbrenner, (Jordan and Fal k) , The Newer Knowled e of Bacteriolo and Immunology. --.+-F Unjv. of Chicago Press, Chicago, 192 , p. 5 .I , I 12. Buist, J. B., Vaccinia and Variola: -II_- London, Churchi I1 Pres A Study of Their Life tli;tory, 18vTv - --. - _I_ * .% But-net, F. M., "Contribution a 1'Etude de 1'Epithelioma Contagieux des Oiseaux." Ann. I. Past. 20: 742 1906. ---I L-4 14. Burnet, F. and W. M. Stanley, The Viruses, Vol. 1 (General Virology), Academic Press, New York 1959. - G 15. Caspersson, T. and J. Schultz, "Pentose Nucleotides in the Cyto- plasm of Growing Tissues," Nature 143: 602 1939. /\ 16 Chargaff, E., "Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic Degradation," Experientia 6: 201 1950. n 17. Cohen, S. C. and W. M. Stanley, "The Molecular Size and Shape of the Nucleic Acid of Tobacco Mosaic Virus," J. Biol. Chem. 144. 589 1942. - -,- - -* A 8. Colter, J. S., H. H. Bird and R. A. Brown, "Infectivity of Ribo- nucleic Acid from Ehrlich Ascites Tumour Cells Infected with Mengo Encephalitis," Nature 179: 859 1957. .- 0 19. Colter, J. W., H. H. Bird, A. W, Moyer and R. A. Brown, "infectivity of Ribonucleic Acid. Isolated from Virus-Infected Tissues," Virol. k: 522 1957. ;420. Colter, J. S. and Ellem, "Structure of Viruses," Ann. Rev. Microb. 15: 219 1961. -- :. - '- 9. 0 q: 21. : Cowdry, E. V., "The,Microchemistry of Nuclear Inclusions in Virus _ Diseases," Science 68: 40 1928. -- - 0 22. Cowdry, E. V., "A Comparison of the lntranuclear Inclusions Pro- duced by the Herpetic Virus and by Virus III in Rabbits," Arch. Path. 10: 23 1930.' -- ~23. Diener, T. O., "The Smallest Known Agents of infectious Disease," J. Ret. SOC. 15: 322 1974. --PC DiMayorca, G. A., B. E. Eddy, S. E. Stewart, W. S. Hunter, C. Friend and A. Bendich, Prqc. Natl. Acad. Sci. USA 45: 1805 1959. -P-P-`- 0 25. Dubos, R. J. and R. H. Thompson, "Decomposition of Yeast Nucleic Acid," J. Biol. Chem. 124: 501 m-p- 1938. /\ 26 Feulgen, R., Zeit. f. Physiol. Chem. 135: 203 1924. _I__- -- 0 27. Findlay, G. M. and R. J. Ludford, "The Ultra-Microscope Viruses," Brit. J. 223 1926. -- Exp. Path. 7: -- :.. \ -15- 0 28. Fraenkel-Conrat, H. and R. C. Williams, Proc. Nat]. Acad. Sci. USA 41: 697 1955. -----' "29. Fraser, D. and E. A. Jerrel, "The Amino Acid Composition of T3 Bacteriophage," J. Biol. Chem. 205: 291 1953. ---- cl 30. Fraser-, D. , H. R. Mahler, A. L. Shug and C. A. Thomas, "The infection of Sub-Cellular E. coli, Strain B, With a DNA Preparation from T2 Bacteriophage," -PrKNatl. Acad. Sci. USA 43: 939 1957. ---- ;k31. Fruton, J. S., Molecules and Life, Wi ley-Interscience Press. New York. 1972. -- A 2. Gieret, A. and G. Schramm, "Infectivity of Ribonucleic Acid from Tobacco Mosaic Virus," Nature 177: 702 1556. a 33. Goodpasture, E. W. and C. E. Woodruff, "The infectivity of Isolated Inclusion Bodies of Fowl Pox," Am. J. Path. 5: 1 1929. ---- 0 34. Goodpasture, E. W. and C. E. Woodruff, "A Comparison of the Inclusion Bodies of Fowl Pox and Molluscum Contagiosum," Am. J. Path. 7: 1 ----- 1931. 0 35. Griffith, F., J. Hy,g. 27: 113 1928. 0 36: Guarnieri, G., Zbl. Bakter. -- 1 16: 255 1854. " c> 37: Guthrie, G. D. and %R. L. Sinsheimer, "Infection of Protoplasts of :. E. coli by Survival Particles of Bacteriophage 0X174." J. Mol. a_ `, -.' .: Bion2: 257 1960. -- -- Haagen, E. and M. Uodama, "Zur Frage der Enstehung der Einschlub- korperchen ,I' Arch. Exp. Zelf. 19: 425 1937. -- ,O 39. Hayden, H., "The Nucleoproteins in Virus Reproduction," Co. Sp. Ha. Sym. Quan. Biol. 12: 1947. -- 105 --- 0 40. d'Here1 le, F., "Sur un Microbe Invisible Antagoniste des Bacilles Dysenteriques," C. R. Acad. Sci. 165: 373 1917. --- -- A 41 Hershey, A. D. and M. Chase, "Independent Functions of Viral Protein and Nucleic Acid in Growth 06 Bacteriophage," 1952. J. Gen. Phy. 36: 35 -- 0 42. Hoagland, C. L., C. I. Lavin, L. E. Smadel and T. M. Rivers, "Constituents of Elementary Bodies of Vaccinia II Properties of Nucleic Acid Obtained from Vaccine Virus," J. Exp. Med. 72: 139 1940. ---- "43. Hua, S., R. P. Mackal, 6. Werninghaus and E. A. Evans, "Infectious DNA Preparations from T2 and T4 Bacteriophage," Viral. 46: 152 1971. - > 0 44. *45. cl 46. 0 47. 0 48. A 49. El 50: 0 51. -- ;$52. `) 0 53. 1.. 3% . *; 7 . :k54. 0 55 * 0 56. 0 57. ;`;58 0 59. l-l 0 lwanowski , D., Bull. Acad. imp. Sci. St. Petersburg, N. 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Blochem. them. 6: 86 1956. -- -19- . . II ACKNOWLEDGEMENTS Chairman, Department of Genetics, Stanford Univers 1 would also like to thank Ruth Redse for translat from German. 1 would like to express my appreciation to Dr. Joshua Lederberg, is guidance. I articles i ty, for h ing severa . . -2o- - .~ -- -_.. - -