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

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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:
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. .
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-

-    .~ -- -_.. - -