THE CHEMISTRY OF THE NUCLEIC ACIDS
     AND NUCLEOPROTEINS
By J. M. GULLAND, G. R. BARKER, AND D. 0. JORDAN
Department of Chemistry, University College, Nottingham, England
This contribution makes no attempt to be a complete compendium
of the literature but is designed as a critical survey of the present posi-
tion in a few main sections of this field. Considerable difficulties and
delays have been encountered in obtaining copies of certain journals
and any omissions on this score are regretted.
NUCLEOPROTEINS AND NUCLEIC ACIDS
Isolation of nucleoproteins.-Many methods for isolating nucleo-
proteins involve a stage, either during the extraction or more generally
in the precipitation process, which is relatively drastic and may pro-
duce an alteration in the chemical and physical properties of the
nucleoprotein ; thus the majority of preparations involve precipitation
of the extracted material with hydrochloric or acetic acids. The ex-
traction processes vary considerably and employ water (1 to 13),
dilute alkaline solution (14 to 17), sodium chloride solution (18, 19,
ZO), or buffer solutions of pH values ranging from 4 to 11 (21 to 26),
followed in each case by precipitation with acid. It has been suggested
(27) that nucleoproteins prepared thus are of variable composition,
the precipitated nucleic acid carrying with it varying quantities of
loosely bound protein, and these methods are now considered unsatis-
factory (28) in view of the possible rupture of the nucleic acid-protein
bond during the acid precipitation. A more controlled extraction of
liver nucleoprotein is that in which the tissue was treated with a solu-
tion containing 0.03M sodium bicarbonate and 0.5M potassium
chloride (29, 30) and the nucleoprotein precipitated by adjusting the
solution to pH 4.2. Even these conditions may, however, be too
drastic, and more recently attention has been concentrated on modi-
fications of the original mild methods (1 to 11, 15), coupled with
precipitation of the nucleoprotein with saturated ammonium sulphate
(12, 31) or calcium chloride (6, 7, 32) solutions. Thus an early
method (32) has been modified (33) whereby fresh pulped calf thy-
mus is extracted with water at 5" C. for twenty-four to thirty-six
hours, and after clarification of the extract the nucleoprotein is pre-
175

176       GULLAND, BARKER, AND JORDAN
cipitated either by the addition of an equal volume of 2 per cent so-
dium chloride solution or of 0.2 per cent calcium chloride solution.
A satisfactory, gentle procedure, apparently of fairly general applica-
tion for the isolation of desoxypentose nucleoproteins (34 to 39),
depends on the rather remarkable changes in solubility of the nucleo-
proteins in sodium chloride solutions of different strengths. They dis-
solve in 1M sodium chloride, forming viscous opalescent solutions, but
are insoluble in 0.14M sodium chloride, although soluble in 0.02M
or less or in pure water. No complete explanation of these phenomena
has so far been put forward. The minced tissues, after being washed
with 0.14M sodium chloride solution to remove cytoplasmic material,
are extracted with 1M sodium chloride (ZM in certain cases) and
after clarification of the extract the nucleoprotein is precipitated in
a distinctly fibrous form by dilution with sufficient water to bring the
sodium chloride concentration to 0.14M. The nucleoprotein may also
be precipitated from the concentrated salt solution by the addition of
alcohol (13), as has been used for the preparation of nucleoproteins
during the isolation of nucleic acids (40,41) ; this method is, however,
more likely than the former process to bring about denaturation of
the protein. Both the desoxypentose nucleoprotein and the pentose
nucleic acid have been isolated from the same tissue sample (42) by
precipitating the aqueous extract of minced rat liver with 0.4 per cent
calcium chloride, and extracting the desoxypentose nucleoprotein from
the solid with 10 per cent or 1M sodium chloride. The pentose nucleic
acid was extracted from the residue with boiling 10 per cent sodium
chloride, a process which was not considered to change the composi-
tion of the pentose nucleoprotein (43).
The isolation of nucleoproteins from cells possessing a resistant
wall has been satisfactorily accomplished (11, 28) by disintegration
of the cell by means of intense audible sonic vibrations (44), the
nucleoproteins being then extracted from the cellular debris with
water and precipitated with 0. 1N hydrochloric acid (11) or ammo-
nium sulphate (28).
Development of the technique of differential centrifugation has
provided an important method whereby the macromolecular nucleo-
proteins, generally in association with lipoid material, may be isolated
from various tissues with a minimum of chemical action. This has
permitted the preparation of the active nucleoprotein fraction of the
Rous sarcoma I (45) and chicken tumour I (46, 47), and of various
fractions from chick embryo (48, 49) and mammalian cells and tis-
       NUCLEIC ACIDS AND NUCLEOPROTEINS     177
sues (50, 51, 52). The method is also used extensively for the isola-
tion of the virus nucleoproteins (53, 54, 55).
Isolation of nucleic acids from tiucleoproteins.-Decomposition of
a nucleoprotein into its constituent nucleic acid and protein is most
easily brought about by alkaline hydrolysis (56), followed by removal
of the protein with colloidal iron and precipitation of the nucleic acid
in acid solution. An obvious objection, however, is that the alkalinity
also causes some degradation of the nucleic acid. This degradation
has no doubt often been considerable (57), but in some cases (see
under viruses) this is apparently the only method which has so far
been available ; an alternative successful procedure is hydrolysis of
the crude nucleoprotein with pepsin (15, 58, 59). A remarkably
simple preparation of the desoxypentose nucleic acids from the nucleo-
proteins of calf thymus and rat liver is that in which the nucleoprotein
solution in water or 1M sodium chloride is saturated with sodium
chloride, the deposit of protein removed, and the nucleic acid, in a
highly polymerised state, precipitated by the addition.of alcohol (8,
42,60,61).
Complete separation of the protein and nucleic acid of nucleo-
proteins may be effected by decomposing the nucleoprotein in 0.5
per cent sodium carbonate solution at 50" C. for one to two hours
and shaking the solution, neutralised to pH 7, with chloroform con-
taining a small amount of a foam-preventing agent such as amyl
alcohol ; the protein concentrates at the interface forming a chloro-
form-protein gel which is easily separated by centrifugation (11).
This method has yielded successfully the nucleic acids from the nu-
cleoprotein of Streptococcus Pyogenes (11) and from a fraction iso-
lated from type III pneumococci (62), and the desoxypentose nucleic
acid of thymus (34).
Procedures used in special cases but yet to be tested in wider
fields are the heat-denaturation of tobacco mosaic virus nucleoprotein
(63), the electrophoresis of tuberculin nucleoprotein (13), and dialy-
sis of thymus nucleoprotein dissolved in 1M sodium chloride against
1M sodium chloride, which results in diffusion of the protein (34).
Direct isolation of nucleic acid.-It has repeatedly been stated that
the methods usually adopted for the direct extraction of nucleic acids
are unsatisfactory (64, 65, 66), and in spite of the fact that nucleic
acids are labile towards alkali, the majority of the procedures employ
sodium hydroxide. By use of this reagent nucleic acids have been
obtained, for example, from yeast (58, 65, 66), pancreas (67) and

178       GULLAND, BARKER, AND JORDAN
various animal tissues (68, 69), onion bulbs (70), malignant tissue
(71,72,73), and bacteria (74 to 77). Extraction with sodium chloride
solution yielded nucleic acid from yeast (18) and liver (41) ; in the
latter case a nucleoprotein was probably isolated and subsequently
converted into the nucleic acid during purification (see below).
In general, it would seem to be desirable to prepare nucleic acids
by isolation and subsequent decomposition of the nucleoprotein.
Separation of pentose and desoxypentose nucleic acids.-It was
early realised (78) that a tissue could yield both pentose and desoxy-
pentose nucleic acids depending on the method of isolation, but the
significance of the nature of the nucleic acid in this connection seems
to have been lost sight of in subsequent work. The apparently con-
tradictory statements that sodium chloride solution extracts a pentose
nucleic acid from liver (41) and a desoxypentose nucleoprotein from
thymus gland (34) emphasise this point. The fresh minced thymus
glands were first washed with 0.14M sodium chloride solution and
the desoxypentose nucleoprotein then extracted with a one molar
solution. The dried liver powder on the other hand, was extracted
directly with 10 per cent sodium chloride, and the "crude nucleic acid"
purified first by precipitation with barium acetate and then with glacial
acetic acid ; both procedures have previotisly been stated to precipitate
pentose nucleic acids, but not desoxypentose nucleic acids, and also to
remove any protein impurity (40). For the isolation of either a pen-
tose nucleic acid or a desoxypentose nucleic acid, it would therefore
seem that two procedures are possible, (a) fractionation of the nucleic
acids using barium acetate and glacial acetic acid as the precipi-
tant, or (b) fractionation of the nucleoproteins by means of sodium
chloride. The first method has been described in the case of the nu-
cleic acids of pancreas, the pentose nucleic acid being precipitated with
acetic acid and the desoxypentose nucleic acid with alcohol (79) ; the
second method, as has been mentioned, was used for the nucleic acids
of rat liver (42).
PROPERTIES OF NUCLEOPROTEINS
The nucleoprotein isolated by mild methods (33, 34) from calf
thymus has a molecular weight of the order of 2 X lo6 (80) and its
solutions in 1M sodium chloride show the high viscosity and marked
streaming birefringence generally associated with highly asymmetric
macromolecules. Once the nucleoprotein has been dissolved in water
or in 0.02M sodium chloride solution, changes occur which result in
NUCLEIC ACIDS AND NUCLEOPROTEINS      179
-the nucleoprotein being less fibrous when reprecipitated in 0.14M
salt (34), and its solutions in 1M sodium chloride are less viscous
and show less streaming birefringence than those of the original nu-
cleoprotein. These changes are apparently permanent and cannot be
attributed to fractionation of the nucleoprotein. Viscosity measure-
ments (33), however, are only in partial agreement with these obser-
vations ; nucleoprotein extracted with water from calf thymus showed
a much lower viscosity in dilute buffer (0.005M potassium acid phos-
phate plus 0.005M dibasic sodium phosphate) than in the same buffer
containing 5 per cent sodium chloride, which observation would sug-
gest that the viscosity change is reversible. A more detailed compari-
son of the two apparently conflicting observations cannot be made
owing to the lack of experimental data recorded (34).
Nature of the linkage between nucleic acid and protein.-Until
comparatively recently it was not infrequently assumed that the bonds
between nucleic acid and protein are invariably electrovalent. This
form of linkage may have been produced artificially by the relatively
drastic methods of preparation of the materials examined. That sup-
position is supported by the results of a direct comparison of the
nucleoproteins of streptococci (28)) the nucleoproteins having been
precipitated from the aqueous extracts by acid on the one hand and
by ammonium sulphate on the other; in the latter case it is to be em-
phasized that the nucleoprotein had not come into contact with either
acid or alkali at any stage. The examination of the nucleoproteins,
which contained a pentose nucleic acid (1 l), was made b,y four meth-
ods, (a) deproteinisation by shaking with chloroform, (b) fractiona-
tion by ammonium sulphate, (c) precipitation with neutral calcium
chloride solution, and (d) measurement of electrophoretic mobility.
The natural nucleoproteins, unlike their acid-precipitated congeners,
were completely soluble on the acid side of their isoelectric point. The
most interesting results were observed in the precipitation by neutral
calcium chloride ; both the acid-treated nucleoprotein and an artificial
protein nucleate included in the investigation formed an immediate
precipitate, whereas no precipitation occurred with the natural nucleo-
protein. These data imply either that the phosphoric acid groups of
the natural nucleoprotein are bonded in such a manner as not to be
available for reaction with calcium ions, or alternatively that the cal-
cium salt of the natural nucleoprotein is soluble. Whatever the true
explanation, it is clear that acid precipitation of the pentose nucleo-
proteins of streptococci does change their properties.
,

180       GULLAND, BARKER, AND JORDAN
The nucleoproteins of cell nuclei (34), when extracted by mild
methods, give evidence that the bond between the desoxypentose nu-
cleic acid and the protein is electrovalent. Thus dialysis of a solution
of the nucleoprotein of trout sperm in 1M sodium chloride against
1M salt solution resulted in the diffusion of the protein through the
membrane, leaving the highly polymerised desoxypentose nucleic acid
behind. Furthermore, extraction of the nucleoprotein solution with a
chloroform-octyl alcohol mixture caused accumulation of the protein
at the interface whilst the nucleic acid remained in solution (34). The
desoxypentose nucleic acid and protein components of the nucleo-
protein of tuberculin were separated by electrophoresis (13, 81) sug-
gesting the presence of an electrovalent bond, but it should be pointed
out that an acid precipitation was employed in the preparation. There
are indications that the bonds in the pentose nucleoprotein of haemo-
lytic streptococci are stronger than a dissociable salt linkage (25). The
generalisation has been put forward (82) that the bonds between pen-
tose nucleic acid and protein are covalent whereas those between
desoxypentose nucleic acid and protein are electrovalent, but the evi-
dence so far obtained with carefully prepared nucleoproteins is as yet
insufficient to warrant this generalisation, which is certainly not in
agreement with the data obtained from the virus nucleoproteins (see
below).
The problem of the nucleic acid-protein bonds in nucleoproteins
is complicated by experiments which suggest that there is a difference
in properties when the cell nuclei of normal rat liver are isolated at
pH 6.0 to 6.2 and at pH 3.8 to 4.0 (39). In the former case the
nucleoprotein was easily extracted with 5 per cent sodium chloride
solution, whereas in the latter little or no extraction occurred. De-
naturation of the protein, which might conceivably have caused in-
solubility of the nucleoprotein in the sodium chloride solution, was
not responsible for the firmly bound state, because, as was pointed out,
this is found also in the nuclei of chicken erythrocytes prepared at pH
6.8 to 7.0, a treatment considered unlikely to cause denaturation.
Further, denaturation of the protein is generally regarded as liberat-
ing the firmly bound nucleic acid from the protein, as for example in
tobacco mosaic virus (63).
Electrophoretic and other studies.-The interaction of nucleic
acids with various proteins to form complexes or protein nucleates
has been known for some time (see for example 83, 84), but it is
only recently that these complexes have been studied systematically
       NUCLEIC ACIDS AND NUCLEOPROTEINS      181
and their properties compared with those of nucleoproteins isolated
from tissues. The method of investigation generally employed is that
of determining the electrophoretic mobility, and it has been observed
that some interaction occurs between thymus desoxypentose nucleic
acid and seralbumen (85), although it could not be decided from the
data obtained whether several' firmly bound nucleoprotein compounds
or only loose complexes were formed; the latter appeared more prob-
able since it was observed that the electrolytic environment played an
important part in determining the electrophoretic behaviour. Electro-
phoresis pf mixtures of ovalbumen and yeast ribonucleic acid (86)
gave similar results, clear evidence of complex formation being ob-
tained in the isoelectric region of the ovalbumen, although in more
alkaline solutions the two constituents migrated independently. Addi-
tion of the nucleic acid to ovalbumen in solutions more acid than the
isoelectric point brought about partial precipitation of a complex.
It has not always been appreciated that resemblance of electro-
phoretic behaviour does not necessarily imply complete chemical simi-
larity. This point is borne out by the electrophoretic comparison at
pH 7 of (a) a nucleoprotein solution which is not precipitated by the
addition of calcium chloride, and (b) a solution of the same nucleo-
protein which had been treated with acid and could be precipitated by
calcium chloride (28). The mobilities of the two specimens were very
similar, that of the natural nucleoprotein being only slightly higher
than that of the acid-treated sample, but no definite component split
off during the electrophoresis of the latter material. It was concluded
that both specimens were "nucleoprotein," but stress was laid on the
excellence of the calcium chloride precipitation test . for "showing
changes that take place in nucleoproteins." Liver nucleoprotein has
also been observed to migrate as a single component, although in this
case the bond between nucleic acid and protein is such as to permit
both components to be separated by dialysis (34). In contradistinc-
tion to these observations, the nucleoprotein of tuberculin (81) splits
off a highly mobile component, largely nucleic acid, in solutions more
alkaline than pH 5, although at pH 5 and pH 2.2 the nucleoprotein
fraction traveled as a single component. This was held to show that
in these solutions either the electrical properties of the nucleic acid
and protein components were identical below pH 5, or a dissociation
occurred at pH 5, releasing the nucleic acid, The latter explanation
was preferred (81) and it was suggested that an association could
occur between the imino group of histidine in the protein and a sec-

182       GULLAND, BARKER, AND JORDAN
      NUCLEIC ACIDS AND NUCLEOPROTEINS      183
fluenza A (PR8 strain) (97, 98)) influenza B (Lee strain) (98, 99),
and swine influenza (98, 100) which contain nucleic acids of the des-
oxypentose type. The nature of the bond between nucleic acid and
protein is not known but seems to be relatively strong, except in the
case of tobacco ring spot virus, of which part of the nucleic acid at
least appeared to be less firmly bound (54), and of equine encephalo-
myelitis virus (96, lOl), in which the linkage was reported to be
weaker than in rabbit papillorna virus. The conditions necessary for
decomposition of the viruses are, however, not the same. Thus, for
example, only 62 per cent of the nucleic acid was liberated from bushy
stunt virus (102) by treatment with 5 per cent sodium hydroxide for
two hours at 4" C., whereas 68 to 78 per cent of the nucleic acid was
liberated from tobacco mosaic virus when the 5 per cent sodium hy-
droxide was neutralised immediately after it had been added to the
virus (103) ; the yield of this nucleic acid increased to 90 per cent if
the virus was in contact with the alkali for two hours. Both the rabbit
papilloma (96) and vaccinia (95) viruses were only decomposed by
heating with 5 per cent sodium hydroxide for thirty minutes. An
alternative procedure for decomposing tobacco mosaic virus is by
heat-denaturation (63) ; this method is of importance as the nucleic
acid isolated is very probably in, or almost in, its native condition.
The proteins of two strains of tobacco mosaic virus were electro-
phoretically homogeneous and identical (104), and a mixture of the
nucleic acid-free proteins formed a single boundary although a mix-
ture of the intact viruses gave a double boundary. From this it was
concluded that both virus proteins were the same and that the differ-
ence in the two strains was due to the nucleic acid, as had been sug-
gested previously (105) on the basis of the nucleic acid contents of the
two strains. This interpretation, however, is faulty, since it has been
shown that two different proteins can give a single boundary in an
electrophoresis apparatus (106) ; furthermore, amino acid analysis
showed that the difference in two strains of tobacco mosaic virus in-
volves differences in the protein (107). This, however, does not ex-
clude the possibility that differences may also occur in the nucleic
acids. In agreement with this conclusion, the electrophoretic mobili
ties of these mutants of tobacco mosaic virus were not paralleled by
the nucleic acid contents as determined by ultraviolet absorption spec-
tra (108).
X-ray analysis (109, 110, 111) of tobacco mosaic virus did not
indicate a concentration of the comparatively dense nucleic acid in any
ondary phosphoryl group of nucleic acid, both of which dissociate in
the region of pH 5 to 6. It was concluded (87), largely on the basis
of an electrophoretic examination of the nucleoprotein of calf thymus
between pH 3 and 9, that the nucleoproteins are definite compounds
with a constant ratio of nucleic acid to protein, but the preceding ob-
servations suggest to the writers that this conclusion is not entirely
justified. The electrophoretic evidence would only point to such a
conclusion if the natural nucleoprotein and the artificial protein nucle-
ate, prepared by mixing the unaltered nucleic acid and protein of the
original nucleoprotein, were found to behave differently when exam-
ined electrophoretically, the latter splitting into two fractions at a
certain pH value.
Addition of various albumens to solutions of sodium desoxypen-
tose nucleate of thymus reduced the viscosity of the latter to values
between that of the original nucleic acid solution and that of the
protein (88), and at the same time diminished the streaming bire-
fringence of the sodium nucleate solutions. The ability of the pro-
teins to lower viscosity was dependent on their being in the native
state since heat-denatured proteins were considerably less effective.
These phenomena were at first attributed (88) to a polymerisation
of the sodium nucleate although alternative explanations (89), such
as complex formation between the protein and the nucleic acid, are
feasible, and subsequently (90) it'was considered that aggregation of
the nucleate particles, leading to a more globular form, would account
for the loss of streaming birefringence and structural viscosity.
Examination of solutions containing horse serum albumen and
sodium thymus desoxypentose nucleate (90) showed that the osmotic
pressures of the mixtures were in nearly every case almost identical
with the value for the protein component, a result which was inter-
preted as suggesting that the presence of the protein causes aggrega-
tion of the nucleate ions and adsorption of the sodium ions onto the
resulting micelle, thus rendering them osmotically inactive. Some
preliminary experiments have been recorded on the adsorption oE
nucleate ions onto a protein monolayer (34) but no details were given.
Virz4s nucleoproteins.-The isolation and properties of virus nu-
cleoproteins have been described elsewhere (53, 54, 55, 91, 92, 93)
and this review is confined to the nature of the bonds between nucleic
acid and protein, and to the distribution of the nucleic acid in the
virus. All viruses so far isolated contain pentose nucleic acids, except
those of psittacosis (94), vaccinia (95), rabbit papilloma (96), in-

184       GULLAND, BARKER, AND JORDAN
particular part of the nucleoprotein, and it was suggested (104) that
the virus may conceivably consist of either a long protein chain with
nucleic acid side groups, or a regular arrangement of alternate nucleic
acid and protein residues. The latter suggestion was preferred (104)
on the basis of the improbably large molecular weight of the protein
in the former scheme. In contrast with this conclusion, comparison
of the molecular size of the tobacco mosaic virus protein and the nu-
cleic acid obtained from it (63) led to the view that the nucleic acid
exists as a thread-like macromolecule, of which the length is that of
the intact virus. From measurements of absorption of polarised ultra-
violet light by tobacco mosaic virus particles, oriented by streaming
through a quartz capillary, it was considered probable that the pentose
nucleic acid is arranged in an ordered manner and that the planes of
the purine and probably pyrimidine rings are parallel to one another
and perpendicular to the long axis of the molecule (112).
STRUCTURE OF NUCLEIC ACIDS AND THE TETRANUCLEOTIDE
              HYPOTHESIS
Originally the term "tetranucleotide" indicated the occurrence of
the four appropriate nucleotides or the corresponding nitrogenous
derivatives in equimolecular proportions in the decomposition prod-
ucts of a nucleic acid, and was-thus used to describe certain of the then
known nucleic acids at a period when these were regarded as having
a molecule so simply composed. Now that the complex nature of
nucleic acids as polynucleotides is established, the term is still appli-
cable in this sense to such polynucleotides as conform to the tetranu-
cleotide ratio in their nucleotide contents.
Later, as a development arising from the recognition of this com-
plex character, the name has been used to denote a unit, consisting of
one molecule of each of the four nucleotides, which by recurrent com-
bination with itself forms the polynucleotide ; it seems to be implied
that the mode of this union is uniform throughout the polynucleotide.
Finally, it has been postulated that in each of these units the four
nucleotides are always combined in a fixed manner in an unchangeable
sequence. As a logical outcome of these later hypotheses there has
arisen the conception of certain polynucleotides as "polytetranucleo-
tides."
The term "tetranucleotide" has thus a graded series of implica-
tions ranging from a statistical expression of analytical results to a
definition of an exact chemical structure, and in order to avoid con-
      NUCLEIC ACIDS AND NUCLEOPROTEINS     185
fusion the sense in which the name is being used must be defined;
the terms "statistical" and "structural" tetranucleotide are suggested
for this purpose. It is opportune to review and assess the evidence
on which these interpretations are based, all the more so because the
importance of polynucleotides in the biological fields makes it essen-
tial that the possibilities of resemblance or divergence between poly-
nucleotides from different sources should be clearly recognised so
that conjectures involving nucleic acids may be based only on accepted
chemical knowledge, The question of the existence of structural tetra-
nucleotides has been discussed briefly (89, 113).
Present information allows polynucleotides-to be classified in two
main groups depending on the nature of the component sugars as
pentose or desoxypentose. The existence of hybrid polynucleotides
containing both types of sugar has been postulated (114)) but it has
been pointed out that in the absence of experimental evidence the
suggestion is at present purely speculative (115). The literature also
records the isolation of materials which on decomposition yielded the
fragments of both types of acid, but it is probably correct to regard
these as mixtures, in so far as their nucleic acid components are con-
cerned, until the reverse has been proved. Where examination has
been adequate, polynucleotides are characterised up-to-date by the
presence of the nitrogenous radicals guanine, adenine, and cytosine,
together with uracil in the pentose polynucleotides and thymine in the
desoxypentose polynucleotides. Tuberculinic acid A (74, 116, 117,
118) seems so far to be unique in containing 5-methylcytosine (65,
119), but the presence there of this pyrimidine does suggest the need
for close identification of pyrimidine components, perhaps more thor-
ough than hitherto.
Origins of the tetranucleotide hypothesis and the newer concep-
tion of molecular sizes.- Although Miescher formed a picture of "nu-
clein" as a multi-basic, phosphorus-containing acid of molecular weight
so large as to prevent dialysis, those properties were seldom stressed
during the earlier part of the present century (60, 120, 121), but in-
stead the development of the subject followed increasingly problems
of detailed structure. This concentration of effort led to the wide as-
sumption, chiefly from five causes (89), that the molecules of both
types of nucleic acid, typified by the desoxypentose acid of thymus and
the pentose acid of yeast, consisted of simple tetranucleotides. Recent
data have shown that nucleic acids are considerably larger than tetra-
nucleotides (for summaries and references, see 89, 122) ; the sizes

186       GULLAND, BARKER, AND JORDAN
vary with the type and source of the acid and with the method of iso-
lation and purification, but the general assumption of polynucleotide
character for all nucleic acids seems to be justified. Nevertheless, the
extent to which information obtained on isolated samples of polynu-
cleotides can be applied to consideration of them in their native states
is more obscure. Some methods employed in isolation may bring about
considerable alteration in properties (59, 123)) and although the sug-
gestion (124) is probably correct that polynucleotides of lower size
are formed by transverse breakage of larger molecules, the doubt
remains that alterations of the polynucleotide may also be caused by
processes of de- and repolymerisation, analogous to those observed
in the case of thymus desoxypentose nucleic acid (61, 88, 125). Thus,
measurements (126) of the effect of pH changes on the viscosities of
solutions of a sodium desoxypentose nucleate of high molecular weight
from thymus suggested that in neutral solution this salt acts as a linear
polymer which is rapidly depolymerised to a considerable extent as
the pH is altered from neutrality. The effect was complete at pH 2.6
and 11.6, and was reversed when these solutions were neutralised, the
nucleic acid material then repolymerising slowly. Sedimentation ve-
locity and diffusion measurements showed that both the degraded and
the repolymerised polynucleotides have wider distributions of molecu-
lar weights than the original substance and that some of the molecules
in the repolymerised acid are much larger than those present in the
original solution. The sensitivity of these polynucleotide molecules to
their concentration in solution, to the presence of added salts, and to
changes of pH emphasises the difficulties inherent in studies of the
structures of the acids in their natural condition.
The recognition of the complex character of nucleic acids un-
doubtedly opened afresh the whole question of their structure, but it
is surprising to observe how readily there has occurred a mental super-
position of the newer knowledge of molecular sizes onto the older ideas
of the simple tetranucleotides, bringing with it the concept of the poly-
tetranucleotide. Had the sizes of nucleic acid molecules been realised
at an earlier date, it is doubtful whether the hypothesis of the struc-
tural tetranucleotide would have gained such a firm hold as is appar-
ently the case. The relevant data are discussed below.
Desoxypentose poZynucZeotides.-Examination (124, 127) of the
molecular sizes of samples of the polynucleotides from thymus pre-
pared by different methods showed that treatment with alkali or en-
zymic preparations, or the action of heat, causes diminution as com-
      NUCLEIC ACIDS AND NVCLEOPROTEINS     187
pared with that of material obtained by a milder procedure (60), in
which only neutral solutions and temperatures near 0" C. were used.
Enzymic degradation appears to proceed in two stages, possibly
by two distinct enzymes, although the demarcation is indistinct and
it is not clear whether the preparations causing the more deep-seated
fission are not merely the more active. The a-nucleic acid of thymus,
the large polynucleotide of which the soluble salts show gel-forming
properties, is converted by the nucleogelase of commercial pancreatin
into the o-acid which no longer forms gels but is still precipitable by
acids from solutions of its salts (120, 121). On the other hand, labo-
ratory-made extracts of fresh or dried pancreas contain an enzyme,
thymopolynucleotidase, which causes a more fundamental hydrolysis
of the a-acid, either previously isolated or while still present in the
minced thymus gland (128,129, 130) ; presumably it attacks the P-acid
similarly. An enzyme causing similar effects has been recorded as
present in a variety of animal and plant tissues and named thymo-
nucleodepolymerase ( 13 1, 132).
  The results of thymopolynucleotidase action were a fall in viscosity
and rise in conductivity of the solution and the liberation of one acid
equivalent (titrated to pH 9) for each four atoms of phosphorus pres-
ent ; the products were oligonucleotides corresponding to 3.9 nucleo-
tides in size (129, 130). Oligonucleotides are amorphous powders and
differ from the a- and pacids in being soluble in hydrochloric acid
and in having sodium and magnesium salts which dissolve in their
own weight of water to form solutions which are viscous but do not
gel. They differ from nucleotides in being precipitated, as are the
higher polynucleotides, by molybdate in acid solution. Estimations of
guanine and adenine indicated that both purines were present in the
proportions of one molecule for each four atoms of phosphorus.
Treatment of the a-acid, free or in the thymus, with hot alkali
produced a similar series of changes and resulted in mixtures of oligo-
nucleotides and material resembling the p-acid ; the size of the latter
when prepared in this way was 8.5 to 10.5 (from the gland) or 18
to 19 (from the a-acid) nucleotides, whereas the former corresponded
to 3.3 to 3.6 and 3.2 to 4.0 nucleotides respectively. The materials
prepared directly from the gland still contained at least 5 to 6 per cent
of pentose-containing substances. Drastic alkaline extraction and pu-
rification of the desoxypentose nucleic acid of beef spleen (133) yielded
material considered to be closely similar to the oligonucleotides. This
product and the corresponding deaminated acid and thymic acid were

188      GULLAND, BARKER, AND JORDAN
water-soluble penta-basic acids with approximately the correct ana-
lytical compositions and weights for molecules composed of four nu-
cleotides (133, 134).
It has been postulated (113, 129, 133) that the oligonucleotides
(I) are true "structural tetranucleotides" from which the polynucleo-
tides are formed by polymerisation and that the tetranucleotide has
the structure given to it by Levene (135). Definite positions were as-
signed (113) to the nucleotides of adenine and thymine, the relative
orientation of the others being undetermined, on the basis of the fol-
lowing experiments. A mixture of mononucleotidase and oligonucleo-
tidase (diesterase) from intestinal mucosa rapidly split off 25 per cent
of the total phosphorus from the oligonucleotides. Further dephos-
phorylation occurred, and when this had reached 50 per cent the phos-
phorus-free fraction contained only one molecular proportion of nu-
cleoside, consisting of a mixture of about equal parts of two nucleo-
sides, those of adenine and thymine. Neither the guanine nucleoside
nor mononucleotides were present in the solution.
       NUCLEIC ACIDS AND NUCLEOPROTEINS      189
obtained by the two methods are the same or different. Fourth, all
measurements made on the oligonucleotides, as on all polynucleotide
material, give average results only. In a mixture of oligonucleotides,
therefore, each molecule need not have the same composition or weight
as its neighbours ; it is stated (113) that fractional dialysis has so far
failed to effect separation into fractions of lower and higher molecular
weight, but it is doubtful whether the sensitivity of such a method
would permit the recognition of small differences. A massing of any
particular nucleotide in the polynucleotide chain is therefore not ex-
cluded. Fifth, even if the oligonucleotides were tetranucleotides, there
is no reason why their nucleotides should be arranged uniformly in
each molecule, and in the writers' opinion the experimental results
could be explained in other ways which leave open the question of a
regular sequence. There does not seem to be evidence that the ade-
nine and thymine nucleosides both arise from the same oligonucleo-
tide ; the same statistical result would have ensued had each nucleo-
side been removed in its entirety from two different molecules. Fur-
thermore, consideration and experience of enzyme experiments in
this field suggest that their use in the interpretation of complex struc-
tures demands full information as to the constitutions of all the prod-
ucts and the specificity of the enzymes. In a fission of (I) to give the
observed result the oligonucleotidase ,of intestinal mucosa must attack
at approximately equal rates a 3- and a 5-phosphoester linkage.
Knowledge whether the enzyme specificity discriminates between
these is necessary for the interpretation of the results, but is unknown,
since the positions of the phosphoryl groups in the desoxypentose
nucleotides have not yet been determined ; these were prepared by the
action of the enzymes of intestinal mucosa on thymus desoxypentose
nucleic acid (136, 137, 138). Sixth, Levene's structure (135) for a
tetranucleotide cannot be accepted with certainty on the basis of the
existing data. The proposal of (I) as the structure of desoxypentose
nucleic acid (135,139) was partly dependent on a supposed parallelism
between that acid and yeast ribonucleic acid as regards the general
form of their internucleotide linkages. The structure formerly proposed
(140) for yeast ribonucleic acid has now been shown (141) to be at
variance with the experimental facts in this respect (see below), and
without drawing unwarranted parallels between these two acids it may
eventuate that ideas on the internucleotide linkages of desoxypentose
nucleic acids may also need revision.
On a wider issue the question arises whether desoxypentose nu-
    /PO(OHh
Ada&-sugar \
Cytosine-sugar /WOW
Guanine-sugar >WOW
    `PO(OH)
Th yminc-sugar '
I
If such postulates could be substantiated, an important advance
would have been made. Whatever may come to light in the future,
there is at present in the writers' opinion no evidence to justify the
recognition of the existence of the "structural tetranucleotide," as will
be seen from the following discussion.
First, only 65 to 70 per cent of the organically combined phospho-
rus was actually isolated as oligonucleotides in the alkaline or enzymic
preparation, and there was thus ample possibility of the formation of
other products. Second, the nature of the linkage attacked by thymo-
polynucleotidase is unknown ; the enzyme is highly specific and does
not attack yeast ribonucleic acid or a variety of representative poten-
tial substrates (128, 132). Third, the nature of the linkages attacked
by alkali is unknown, and it is not clear whether these are the same
as those split by the enzyme and thus whether the oligonucleotides

190       GULLAND, BARKER, AND JORDAN
cleic acids from different sources are one polynucleotide, uniform
throughout nature, or whether there are a number of chemically dis-
tinct individuals. It seems frequently to be either assumed or im-
plied that the former is the case (see however 142). The writers are
not aware of facts which favour either alternative, but attention is
drawn to the considerable possibilities of variation in structure of
polynucleotides which could arise from divergences of molecular size,
relative orientation and proportions of nucleotides, and nature of the
sugar components.
Several instances are recorded (e.g., 68, 143) in which hydrolysis
of nucleic acids from different sources yielded the nitrogenous bases
in amounts differing considerably from those expected on the basis
of the tetranucleotide ratio. Such results, however, need to be con-
firmed or refuted through improved technique, or other methods, and
it may be that a beginning has been made (82) in the use of the di-
phenylamine colour reaction (144, 145) to determine the ratio of total
purine to total pyrimidine nucleotides. The method does not distin-
guish between individual purine or pyrimidine nucleotides. The in-
terpretation of even this simple estimation may be complicated, since
it is stated on the one hand to be positive specifically for the sugar of
the purine nucleotides (82, 146) and on the other to be given by all
the sugar of a desoxypentose nucleic acid (147, 148) ; these diver-
gences may depend on the exact conditions since the glycosidic link-
ages vary in stability. According to a recent report (82) all the des-
oxypentose nucleic acids so far examined, but from sources as yet
unnamed, agree in having equimolecular proportions of total purine
and total pyrimidine nucleotides.
It seems to be widely accepted that the sugar (thyminose) is d-2-
desoxyribofuranose in all nucleotides. This may be the case, but it
should be realised that the assumption of the furanose structure is
based solely on the demonstration of its presence in the thymine nu-
cleoside (149, 150, 151), and that the identification of the sugar itself
applies only to the guanine nucleoside of the acid from thymus (152,
153,154) ; the sugars of the other nucleosides (135,155) and of nucleic
acids from other sources do not seem to have been prepared. A de-
tailed study of the identification and estimation of the sugars in sev-
eral nucleic acids (147) by the Dische carbazole colour reaction as-
sumes that the sugar is all desoxyribose, although authentic samples
of desoxyribose and its guanine nucleoside gave inferior colours. Not
infrequently the classification of a nucleic acid as "desoxyribose" nu-
      NUCLEIC `ACIDS AND NUCLEOPROTEINS      191
cleic acid rests merely on a positive result in the :Feulgen (156)) Dische
diphenylamine (81, 144), Thomas (157), or Kiliani (158) colour
reactions; these are commonly regarded as specific for thyminose,
whereas in reality they demonstrate the presence of a desoxy-sugar,
and according to the experimental conditions (see above) may refer
only to the purine nucleotides.
Yeast ribonucleic a&.-This is the only pentose nucleic acid which
has been considered seriously from the standpoint of a "structural
tetranucleotide." The constitutions (159) and syntheses (89) of the
component nucleotides have been reviewed, and since purines and
pyrimidines are present in approximately the tetranucleotide ratio
(160 to 165), the acid is a "statistical tetranucleotide." The original
conclusion (56) that the sugar radicals are d(-)-ribose has been con-
firmed (166, 167, 168), at any rate as regards some, by identification
as the ribonic phenylhydrazide and benziminazole derivative. Small
quantities of 1-lyxose benziminazole were also isolated, together with
some d-arabobenziminazole ; the formation of the latter has been ob-
served as the result of epimerisation during the alkaline oxidation of
ribose (169), but this may not explain its presence in products formed
from the nucleic acid (168), and the possible presence of sugars other
than ribose in nucleic acids should remain open for the present. In
this connection it may be noted that the colours developed by ribose,
arabinose, and lyxose in the Dische carbazole reaction for pentoses
were so similar in intensity that these sugars could not be distinguished
(147). Not infrequently a nucleic acid is classified as a "ribose" nu-
cleic acid (e.g., 170, 171) solely by a negative colour reaction for des-
oxypentose and a positive result in one of the methods for detection
or estimation of pentoses, which depend on the formation of furfural.
Theories of the biogenesis of ribose have been reviewed (89).
As with the desoxypentose nucleic acids, alkaline and enzymic
fission reduce the molecular size of yeast ribopolynucleotide without
dephosphorylation. Milder conditions of hydrolysis than those causing
complete fission into nucleotides converted an already partially de-
graded acid into a product considered to be a structural tetranucleo-
tide (134). This and the corresponding deaminated material were
water-soluble, approximately penta-basic acids of the correct analyti-
cal compositions and molecular weights of 1177 and 1121 (calculated
1304 and 1307). It has been pointed out (141) that these materials
cannot have been true tetranucleotides ; the deaminated tetranucleotide
should have been hexa-basic since the hydroxyl of xanthine titrates

192      GULLAND, BARKER, AND JORDAN
over the range pH 6 to 8 and its presence was demonstrated in de-
aminated yeast ribonucleic acid by electrometric titration. Moreover
the low molecular size of the product was not confirmed (172), and it
was concluded (141) that these fission products were really mixtures
of small polynucleotides and that there is no evidence on these grounds
for the existence of a "structural tetranucleotide."
At one time it seemed possible that a study of the enzyme ribo-
nuclease (ribonucleinase, ribonucleodepolymerase) (see 122, 173)
might provide evidence of a "structural tetranucleotide." Further
data, however, show that the enzyme causes deeper fission ; the aver-
age size of the dialysable products of the action of preparations of heat-
stable pancreatic enzymes corresponded to that of a dinucleotide (128),
and nucleotides were isolated from the products of digestion by crys-
talline ribonuclease (174). Nevertheless, the exact effect of heat-stable
pancreatic enzymes is not clear; enzymes isolated from whole pan-
creas provide small and variable yields of the four nucleotides, whereas
the claims that depolymerisation took place without formation of ap-
preciable quantities of nucleotides arose from experiments with en-
zyme preparations made from commercial pancreatin. The possible
parallel with somewhat analogous observations in the fission of thymus
desoxypentose polynucleotide with pancreatic enzymes is worthy of
note.
The results, on which exact positions of certain nucleotides relative
to each other in a structural tetranucleotide were defined from en-
zyme experiments (175) or aqueous pyridine hydrolyses (176), seem
to the writers to indicate merely that certain linkages in the poly-
nucleotide are more labile than others. It is thus clear that there is
at present no evidence for the "structural tetranucleotide" as a unit of
yeast ribonucleic acid.
It is generally agreed that the action of the heat-stable pancreas
enzymes renders about half of the phosphorus of the acid non-precipi-
table by the uranyl-trichloracetic acid reagent (177) although still or-
ganically combined ; the remainder resembles the original acid in being
precipitated by the reagent and also, although less readily (178), by
hydrochloric acid. It should not be automatically inferred that the
precipitable material is unchanged acid. It has been pointed out (122)
that yeast ribonucleic acid must contain at least two different types
of linkage, one labile, the other resistant, to the action of ribonuclease,
and several alternatives are possible, in that the liberated nucleotides
may not come from every nucleic acid molecule, but may result from
       NUCLEIC ACIDS AND NUCLEOPROTEINS     193
transverse fission of the polynucleotide chain, or may be lopped from a
main polynucleotide trunk bearing nucleotides as branches (141) (see
below). In addition, hints are not lacking that all samples of the acid
are not identical in structure; lability to alkaline hydrolysis is a vari-
able property (179;180), ribonuclease activity alters considerably with
the sample of nucleic acid and is also sensitive to the age of the sub-
strate solution (lSl), and only certain preparations of the acid give
guanine-uridylic acid on hydrolysis (176, 182, 183).
New possibilities have been brought to light for the internucleotide
linkages of yeast ribonucleic acid. In view. of the complete agreement
that the polynucleotide exhibits four phosphoryl dissociations and
since hydrolysis results in the appearance of secondary phosphoryl
groups, it might be assumed that the polynucleotide is a polymer of
the structural tetranucleotide proposed by Levene (140) and that in
consequence each of its phosphor-y! groups, except the terminal one,
should exhibit one primary dissociation. This view seems to be in-
compatible with the experimental facts (141). Samples of the acid,
and of the deaminated acid prepared by a method which avoided any
fall in molecular weight, were titrated electrometrically between pH 2
and 12. Comparison of the experimental curves with those constructed
theoretically showed that for each four atoms of phosphorus there were
three primary and one secondary phosphoryl dissociations. A hypo-
thetical structural tetranucleotide forms a useful standard for the as-
sessment of results (89), and if these observations are considered on
the basis of a polytetranucleotide from this standpoint only, each indi-
vidual unit of the polymer would contain one triply esterified phos-
phoryl group ; the shape of the titration curves agrees best, but not ab-
solutely, with a formulation in which this is the group of uridylic acid.
Two dissociations would be primary and associated with doubly es-
terified phosphoryls, and one phosphoryl group would be singly linked
to a sugar radical and would exhibit both primary and secondary dis-
sociations. Possible formulae in agreement with the facts, including
the presence of titratable, and hence unsubstituted, purine and pyrim-
idine hydroxyls, are II and III; polymerisation would occur either
through the group A or 13, the other member of the pair remaining
singly linked.
Without necessarily assuming a uniform tetranucleotide, it was
considered that structural relationships of this nature may occur in the
polynucleotide, and three points are noteworthy. First, the literature
shows a marked tendency for the phosphorus analyses to be low. In a

194      GULLAND, BARKER, AND JORDAN
polymer of nucleotides all united successively through their phosphoryl
groups, a deficiency of phosphorus could only occur by removal of the
singly linked terminal phosphoryl, but the extent of the deficiency was
often greater than could be accounted for in this way. In a polymer
       NUCLEIC ACIDS AND NUCLEOPROTEINS     195
ally through phosphoryl at C, and C, of the pentose. Union of such
polynucleotides through phospho-amide groups, e.g., guanine-uridylic
acid, might form a large polynucleotide in its native state, and the
ultimate presence or absence of alkali-labile phospho-amide groups in
a given sample of yeast ribonucleic acid might depend on their survival
or destruction in the alkali at present used to extract the acid from
yeast.
0 ther pentose poZynucZeotides.-Cold alkali converted the higher
polynucleotides of tobacco mosaic virus into particles of molecular
weight of 15,000, and it was specifically stated that there is at present
no evidence that a unit smaller than this but larger than a nucleotide
exists as a fundamental unit of the virus acid (63). Alkaline fission
yielded an insoluble trisodium guanylate apparently identical with that
from yeast ribonucleic acid, and a uracil nucleotide isomeric with
uridylic acid ; the purine content was 20 per cent lower than that re-
quired by the tetranucleotide ratio (103).
4 pentose nucleic acid (41), believed to occur in the cytoplasm,
was isolated from sheep liver; part of the sugar was identified as
d-ribose and the amounts of total purine and easily hydrolysable phos-
phorus were consistent with the tetranucleotide ratio.
The pentose polynucleotide of pancreas (26,189, 190) contains the
four nucleotides (67, 191, 192) in the ratio, it has been proposed, of
one molecule of adenylic acid and each pyrimidine nucleotide to vary-
ing proportions of guanylic acid, ranging frbm two to four molecules
(40, 43, 79, 193). The pentose nucleic acid of rat liver appears to
resemble, pancreas pentose nucleic acid in its high nitrogen : phospho-
rus ratio, which exceeds that required by a tetranucleotide (42).
No conclusions can be drawn as to the structure of pentose nucleic
acids which have been characterised only by detection of some or a!!
of the nitrogenous components, or by the demonstration of the pentose
nature of the sugar (for example, 47, 76, 170, 171, 194 to 197) or by
the isolation of incompletely identified nucleotides (198).
In view of the wider possibilities of isomerism of nucleic acids rec-
ognised now as compared with formerly, claims that two acids are
identical, as for example yeast ribonucleic acid and triticonucleic acid
of wheat germ (58), require confirmation.
Terminology of nuckic acids.-Some discussion has taken place
on this subject. On the one hand (199,200), it was proposed that the
name chromonucleic acid should be used instead of thymus nucleic
acid as the biological term for the substance described chemically as
Base-sugar-0-PO(OH). A
  LO 1         Base-w ar-0-PO(OH), A
        k Ol
Base-sugar-O-PO      Basc-sugar-O-PO- 0 1
           Base-sugar-0-PO(OH) Base-sugar-0-PO(OH), B
II                      III
of II or III it would be possible for up to 25 per cent of the total
phosphorus to be absent without upsetting the main polynucleotide
structure. Second, a structure can be visualised in which nucleotides
form side chains and could be removed by the action of ribonuclease,
leaving a molecule sufficiently large to resemble the original acid in its
failure to dialyse and in its precipitation reactions. Third, mixtures of
enzyme preparations containing a phosphomonoesterase and a phos-
phodiesterase produced only 75 per cent dephosphorylation of yeast
ribonucleic acid (184), and these mixed enzymes caused only 75 per
cent dephosphorylation after pretreatment of the acid with a boiled
extract of pancreatin (185) ; these results are in agreement with the
presence of one triply linked phosphoryl for each four atoms of phos-
phorus.
Russell's viper venom, containing a phosphodiesterase (186, 187)
but only very weak nonspecific phosphomonoesterase activity, de-
phosphorylated yeast ribonucleic acid ultimately to 50 to 55 per cent
(188). If during their action the enzymes were inactivated with cya-
nide ions, dephosphorylation continued slowly in the alkaline medium
or more rapidly in 1 per cent sodium hydroxide, thus demonstrating
the existence in the acid of alkali-labile phosphoryl linkages. These
labile groups were assigned tentatively to the hydroxyls at C, of the
sugar radicals, and a working hypothesis was suggested that the poly-
nucleotides of intermediate size which do not suffer loss in molecular
weight when deaminated, and hence are free from phospho-amide
groups (113, 182, 183), may be composed of nucleotides joined mutu-

1%      GULLAND, BARKER, AND JORDAN
desoxyribcse nucleic acid, and that plasmonucleic acid should be sub-
stituted for yeast nucleic acid as the biological equivalent of the chemi-
cal term ribose nucleic acid. On the other hand (159, 201), the ne-
cessity was stressed in the light of present information of defining a
nucleic acid by. referring both to its origin and its type (pentose or
desoxypentose). This would avoid confusion until such time as it
can be stated with certainty either that two (or more) individual
nucleic acids exist and that all examples of each are identical in chemi-
cal constitution, or that two (or more) types of nucleic acid exist and
that each type comprises a number of examples of related but different
constitutions ; closer specification of the type would be permissible
when justified on chemical grounds. The suggestion (123) has dso
been made that in the characterisation of different nucleic acids the
treatment in the course of isolation, such as the degree of tissue autoly-
sis, reagents used, etc., as well as the chemical and physical properties,
should be described.
LOCATION OF NUCLEIC ACIDS IN CELLS
In an extensive survey of the nucleic acids of embryonic and adult
tissues (202), the dried powders, freed from acid-soluble and lipoid
phosphorus compounds, were extracted with 10 per cent sodium
chloride and the total nucleic acids precipitated with lanthanum ace-
tate. Analyses for phosphorus, pentose, and desoxypentose confirmed
the belief (202) that pentose nucleic acids are of general occurrence in
animal cells, and indicated that rapidly growing tissues are character-
ised by high concentrations of both types of acid.
In the light of recent reviews (82, 122, 203), the remainder of this
section is restricted to surveys of the criticism levelled at the widely
accepted theory of the part played by nucleic acids in the nucleus and
to the progress made in elucidating the mode of occurrence of cyto-
plasmic nucleic acids.
Nucleic acids of the nucleus.- For some time it has been consid-
ered as established that desoxypentose nucleic acids are located princi-
pally in the nucleus and that pentose nucleic acids characterise the cyto-
plasm. This concept is largely founded on the cytological application
of ultraviolet photomicrography to demonstrate the presence of purine
and pyrimidine rings and on the use of Feulgen staining to detect
desoxypentose nucleic acids (for representative papers, see 204 to 209,
and for reviews, see 82, 203, 210, 211). The hypothesis that nucleic
acids play an essential part in the reproductive cycle of the cell and in
     NUCLEIC ACIDS AND NUCLEOPROTEINS     197
the genie perpetuation of inherited characteristics (212) is largely de-
pendent on results achieved by means of these techniques, and hence
the specificity of the Feulgen reaction in denoting the site of desoxy-
pentose nucleic acids is a vital factor. Question of its validity might
necessitate reconsideration of a large mass of conjecture and experi-
mental conclusions. Criticism has been made and refuted (213 to 216),
and it has been pointed out that the reaction is specific for desoxy-
pentose only in the absence of lipoids (96). Serious doubts have now
been thrown on its reliability as an indicator both of the total amount
of desoxypentose polynucleotide material (113) and also of the loca-
tion of such substances in the cell (113,217).    '
Cellular nuclei contain, besides nucleic acid and histone, an acidic
protein, chromosomin (217), postulated as the characteristic material
of chromatin. It may be identical with, or related to, a new protein of
cellular nuclei, reported eailier by other workers (57). Chromosomin
has the property of taking up the basic, water-soluble dye produced by
the interaction of decolorised magenta and hydrolysed desoxypentose
nucleic acid, and hence the appearance of this dye in the nucleus of the
cell does not, it was maintained, necessarily indicate the presence there
of the desoxypentose nucleic acid. Furthermore, it was possible to
stain chromoSomes directly with the "developed nucleal stain" (218).
It was suggested that desoxypentose nucleic acid is present in the
nuclear sap, rather than in the chromosomes, and may form the mitotic
spindle, and that chromosomin is "the chemical basis of inheritance"
(219). Histone, on the other hand, was regarded as filling the role of
regulating mitosis (220). Previously, desoxypentose nucleic acids
were regarded as essential to the duplication of the chromosomes
(211), and although no concrete proposals were made, there was a
tentative assumption that the genes consist of desoxypentose nucleic
acids combined with protein (210).
These postulates as to the part played by chromosomin and the
location of desoxypentose nucleic acids outside the chromosomes have
been adversely criticised (221, 222). Enzymic destruction of nucleic
acid in the nucleus rendered the chromosomes not stainable by Feul-
gen's method, whereas hydrolysis of the protein component by trypsin
did not alter the selective ultraviolet absorption of the nucleus in the
region characteristic of nucleic acids. The parts of the chromosomes
which can be stained by the Feulgen technique contained a high pro-
portion of phosphorus, in accord with the presence there of nucleic
acid, and isolated chromosome threads were found to contain 40 per

198       GULLAND, BARKER, AND JORDAN
      NUCLEIC ACIDS AND NUCLEOPROTEINS      199
differentiation of the cell, and from which, for instance, the secretory
granules of the pancreas are derived.
An interesting aspect of the nucleic acids of bacterial cells has been
revived recently. For some time it has been known that Gram-positive
pneumococci can become Gram-negative, and this change was brought
about by extracting the cells in neutral solution (20) and by an en-
zyme, apparently identical with the pancreas enzyme which acts upon
yeast ribonucleic acid (226). The material released into the solution
during the former process contained pentose nucleic acid and a nucleo-
protein (20, 227). A similar change has now been effected in yeast
cells and Gram-positive bacteria by extraction with a solution of a bile
salt (228), and it was also possible to restore the Gram-positive re-
action by replacing the responsible material, an essential component
of which appears to be the magnesium salt of a pentose nucleic acid;
other salts of nucleic acid could not be plated back in this way. In
agreement with the previous workers, the stainable material could be
progressively extracted, that part on the surface of the cell being re-
moved first.
INDUCED TRANSFORMATION OF PNEUMOCOCCAL TYPES '
Among micro-organisms, the most striking example of the repro-
ducible and controllable induction of inheritable and specific altera-
tions in cell structure and function is the transformation of specific
types of pneumococcus. This type of change has been brought about
both in tivo and in vitro, and analogous transformations have been
carried out in the field of viruses (for references, see 62). Avery,
MacLeod & McCarty (62) have now isolated from type III pneu-
mococci a biologically active fraction which in exceedingly minute
amounts is capable under appropriate conditions of inducing the trans-
formation of unencapsulated R variants of pneumococcus type II
into fully encapsulated S cells of type III. Other variants are not
transformed in this way. Examination of the active extract indicated,
within the limits of the methods employed, that protein, unbound
lipoid, and serologically active polysaccharide were absent, and that it
consisted principally, if not solely, of a sodium salt, in homogeneous
viscous form, of a desoxypentose nucleic acid of molecular weight of
the order of 500,000. It is possible, as the authors suggest, that the
biological activity of the material is not an inherent property of the
nucleic acid but is due to minute amounts of some other substance so
intimately associated with it as to escape detection. If, however, as
cent of desoxypentose nucleic acid (for references see 221, 222).
Analyses of different tissues (220)! which contain varying quantities
of nuclear sap but show little variation in the amounts of nucleic acid,
were quoted in opposition to the hypothesis that the nucleic acid is
present in the nuclear sap. The fact that mitotic spindle fibres are
positively birefringent with respect to their length, whereas desoxy-
pentose nucleic acid is negatively birefringent, was held to be in ap-
parent opposition to the suggestion that the spindle consists of a gel
of desoxypentose nucleic acids (222). It was also stated (223) that the
assumption of chromosomin as an integral constituent of the nucleus
in no way affects the views which, chiefly with the aid of ultraviolet
absorption measurements, have been advanced in regard to the protein
and nucleic acid metabolism and the distribution of those substances
in the nucleus. Replies to these criticisms have been made (219, 224).
In view of this controversy, many may hesitate to acknowledge as yet
definite functions or dominant roles for these cellular constituents.
Nucleic acids of the cytoplasm.-Comparatively little attention has
been paid to the organisation of cytoplasmic nucleic acids until recent
years. By high-speed centrifugation, fractions consisting of submicro-
scopic particles were obtained from chicken tumour I agent (46) and
normal chicken embryo (48) ; these were concentrated separately from
the nuclear material and toward the centripetal end. The particles from
the tumour agent had high tumour-producing activity whereas those
from normal embryo were inactive, and both contained lipoid together
with a pentose nucleoprotein. From the tumour agent, a pentose
nucleic acid was isolated, having a broad absorption band with a maxi-
mum at rather shorter wave-length than 2600 8,; this was regarded
as indicating the presence of a high proportion of guanine, and guanine
nucleotide was isolated after alkaline hydrolysis (47, 50, 225). These
observations constitute a marked advance in the study of the chicken
tumour and in knowledge of the location of pentose nucleic acids in
cytoplasm. Similar particles (microsomes), obtained from various
tissues, had a constant composition irrespective of the source (51, 52),
consisting of phospholipids and pentose. nucleoproteins associated in
definite proportions. Nucleoproteins have been found in conjunction
with lipoid material by other workers (for summary, see above and
203). The part played by microsomes in the structure of the cytoplasm
has been discussed (52) but no fully substantiated proposal has yet
been put forward. They have been regarded as fragmented mito-
chondria (50) and as independent cellular structures associated with

GULLAND, BARKER, AND JORDAN
the evidence strongly suggests, the transforming principle is a sodium
salt of a desoxypentose nucleic acid, this type of polynucleotide must
be regarded not merely as structurally important but as functionally
active in determining the biochemical activities and specific character-
istics of pneumococcal cells. This would appear to be the first occasion
on which specific transformation has been experimentally induced
in vitro by a chemically defined substance, and its implications are of
the greatest importance in the fields of genetics, virology, and cancer
research.
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DEPARTMENT OF CHEMISTRY
   UNIVERSITY COLLEGE
  NOTTINGHAM, ENGLAND