i@zflections on biochemistry

  Early nucleic acid chemistry
-+, ,zy- L Fritz Schlenk
         ?   1

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The contemporary biochemist, perusing
the publications of the past century, may
easily be misled into some arrogance
when sorting out the erroneous data and
leads, the slow progress and the primi-
tive reports of that early period. How-
ever,  when recalling the limited
techniques and the lack of facilities at the
early stages of biochemistry, he will
change his mind. The difficulties encoun-
tered by the investigators of nucleic acid
structure were many. To begin with, it
was difficult to ascertain the degree of
uniformity of the starting material.
There were no established methods for
studying macromolecules,  and no
guidelines for the isolation of structural
units. Fortunately for the identification
of new compounds, reference material
or closely related substances had already
been synthesized by organic chemists in
experiments that were usually unrelated
to nucleic acid problems. Much informa-
tion on purines already existed, and
some pyrimidines had been synthesized.
Emil Fischer's work on monosaccharides
provided the basic information for the
identification of ribose and deoxyribose.
Thus, nucleic acid research of that period
owes much to organic chemistry.

Beginnings

The history of nucleic acid research
begins with Friedrich Miescher's search
for chromatin in pus. Miescher was
trained in Basel, in the laboratory of his
uncle, the renowned anatomist Wilhelm
His, Sr. After receiving his medical
degree in 1869, he joined F. Hoppe-
SeylerinTuebingen. Extraction ofsoiled
bandages from infected wounds led to
the isolation of material that he consi-
dered to be a protein, mire acid in
nature than any earlier known protein.
Miescher's publication was delayed, and
when it appeared two years later in
an irregular collection, Hoppe-S&T's
Medizinixh-Chemisehe Umuchungm,

his mentor had added two papers of
other co-workers in which similar acid
protein material from different sources
was reported'.
This delay in publication was not
unusual for the period. The practice of
publishing research findings as they
occurred was unknown prior to the
establishment of the Zeimhrift fier
Physiologirche Chemie in 1877. Its foun-
der*, Felix Hoppe-Seyler, along with T.
Schwann, was a pupil of the physiologist
Johannes Mueller. At the University
of Tuebingen, which was established
around 1500, Hoppe-Seyler had research
quarters in the picturesque old castle; a
tablet at the entrance to a side wing com-
memorates his activity there from 1862
to 1872. Thereafter, he accepted a posi-
tion at the University of Strasbourg.
Miescher returned to Base1 in 1870, to
start his academic career and to continue
the investigation of chromatin. His study
on `Protamine, a New Organic Base
from Salmon Sperm" achieved better
uniformity of sc~urce material, extraction
technique, results and speed of publica-
tion.

The latter remark pertains to protamine-
like compounds; Miescherwasnot aware
of the presence of purines and
pyrimidines. However, on his suggestion
Jules Piccard*, the head of the chemistry
department in Basel, did more work on
nuclein and found 688% of the previ-
ously know. purine bases guanine and
hypoxanthine (sarkin) as compona&.

He suggested that `the presence of such
significant quantities of these rare com-
pounds in a readily accessible material
may be of some interest to chemists and
physiologists'. The quality and speed of
Piccard's publication suggests that he
would have isolated all nucleic acid bases
in the course of a few years, had he only
continued the investigation of nuclein.
Instead this task took several decades in
the hands of others.

Slow progress

The term `nucleic acid' was used first
in 1889 by R. Altmanns, who studied the
phosphate-containing material from
thymus, egg yolk and salmon sperm; his
results confirmed Miescher's observa-
tions. However, the term `nuclein' pre-
vailed until the turn of the century. No
attempt was made by Altmann to con-
firm Piccard's discovery of guanine.
Likewise, A. Kossel in his numerous
publications made only passing refer-
ence to Piccard's work. He may have felt
that the latter had preempted his
domain, because as late as in 1910, he
wrote: `Guanine has been known for
some time in various animal tissues, and
was found, for example, in the sperm-
atozoa of salmon by Piccard, although
indeed this investigator had no suspicion
that it had any genetic relationship with
nuclein' (Ref. 6, p. 396).

The recognition of two distinct types
of nucleic acid, DNA and RNA, occur-
red about 25 years after Miescher's dis-
covery, and another 40 years passed
before the first biological function was
described. During this early period, the
limited amount of nucleic acid research
was mainly in the hands of investigators
with a medical background. Organic
chemists at that time did not take much
interest in these ill-defined, almost
untractable compounds; in their own
field, the methods of organic synthesis
provided easy access to new substances
that could be purified readily and often
had great commercial value.

Neither Miescher (1844-1895) nor
Hoppe-Seyler (1825-1895) contributed
much to the biochemistry of `nuclein'
beyond their initial work. In 1871,
Miescher was appointed to head the
department of Physiology in Basel. He
then devoted his efforts mainly to
cytological problems, but this work was
intermpted repeatedly by boutsoftuber-
culosis'.


6P

TIBS IS - February IYXX

A new approach

Biochemical investigations of nucleic
acids came to be one of the main
activities of another of Hoppe-Seyler's
students, Albrecht Kossel (185s-1927).
Heidelberg was the principal site of his
academic career, and he attracted many
co-workers and guest investigators to his
laboratory. E. Kennaway~ has given us
a charming set of recollections of his
sojourn with Geheimrat Kossel, and of
the commotion and torchlight parade of
the students when the award of the
Nobel Prize to Kossel was announced in
19 10. Kossel was probably the first inves-
tigator to surmise that m&in may be
involved in growth and differentiation?

The most valid basis for this conclusion
seems to have been Miescher's observe-
tion on salmon sperm, which is formed at
the expense of tissue reserves; the fish
does not take nutriment during the
period of spawning. Kossel studied this
problem with a few starving chickens,
but the quantitation of nucleic acid by
the methods of that time was rather
uncertain.

Components identified

Progress in clarifying the structure and
composition of nucleic acids was pain-
fully slow. Gamine was known prior to
its isolation from nuclein by Piccardd; it
had been discovered already in 1846 by
Unger"' in guano, the bird excreta
imported as fertilizer to Europe from the
South American west coast. Guanosine
was isolated by Schulze and Bosshard in
1886 from plant materially. A relation to
nucleic acids was not suspected, but the
identity was established in 1910 by com-
parison with the m&aside that Levene
had obtained from guanylic acid'*.
Adenine was isolated from thymus gland
by Kossel'~; he derived the name from
the Greek term for gland: aden, adenos.
The name purine was coined by Emil
Fischer14 to indicate the unaltered, pure
nature of the basic ring system. Kossel
and Neumann's discovered thymine and
came to the conclusion that a carbohy-
drate group is present in thymus nucleic
acid. Kossel's co-workers, , ,scoli and
Steudel, discovered cytosine and uracil.
Pinner'6 synthesized pyrimidine and
suggested its name in analogy to pyridine.
Sometimes, degradation products of
nucleic acid bases were obtained and, for
a period, hypoxanthine and xanthine
were believed to be nucleic acid compo-

nents. Also, a variety of nucleic acids was
assumed, each having only one kind of a
base. It is impossible to enumerate all the
misconceptions and the work necessary
to undo them. Review articles were not
customary at that time, but the Nobel
Lecture of A. Kossel in 1910 gives an
idea of all the vagaries of early nucleic
acid biochemist@.

Ribose and deoxyribose were the last
principal nucleic acid components to be
identified. The investigations of Emil
Fischer and his school on carbohydrates
provided the fundamental informa-
tionl'. Xylose and arabinose were
known then as naturally occurring pen-
toses, and Fischer projected the config-
uration of the other two pentoses and
suggested the names lyxose and ribose
by rearrangement of some of the letters
of xylose and arabinose, respectively'K.
How different would the development of
nucleic acid biochemistry have been, if
Fischer had made it one of his main
enterprises!

The isolation of the carbohydrate of
pentose nucleic acid was tried repeatedly,
and its identity with arabinose, xylose,
and lyxose was suggested by various
investigators. Success in the identitica-
tion of ribas@ and deoxyribos&2' in
1909 and 1930, respectively, was
achieved by P. A. Levene and his co-
workers, mainly W. A. Jacobs, E. S.
London, T. Mori, and S. R. Tipson. In
both instances, the isolation of the nu-
cleosides was a prerequisite to provide
the starting material. Here again,
Levene did the pioneering work; the
term nucleoside was coined by him for
the reason that they link carbohydrate in
a glycosidic union to the nucleic acid
bases. His work with Tipson and with
Stiller also led to the recognition of the
furanoid structure and of positions 3 and
5 of the pentoses as the sites of esterifica-
tion of phosphoric acid.

Levene's numerous contributions
were summarized in collaboration with
L. W. Bass in 1931, in NucleicAci&~, the
first monograph of consequence cover-
ing the entire field, with emphasison the
chemistry of nucleic acid constituents; of
necessity, biological data were minimal
and altogether speculative at that time.
In all, Levene has done far more for
chemical nucleic acid research than any
of his predecessor+. His work is often
underestimated by biochemists and
biologists of a more recent period, who
did not forgive him his erroneous conept
of the `tetranucleotide structure' and his
reservation about a possible macro-
molecular structure of nucleic acids.
Several other leading specialists of that

period, including Steudel and Feulgen,
likewise favored the concept of a tetra-
nucleotide structure, and nobody con-
tested it. In his famous monograph
(lY31), Levene devoted only a few pages
to `Nucleic Acids of a Higher Order'. He
summarized his opinion?

Thus, in conclusion, it must be admitted that
judgment as to the existence of nucleic acids
of a higher order should be postponed until the
work is repeated on a larger scale. On the other
hand. the presence oi a ribqwlymucleotide in
the animal fissues must now be regarded as
well-established.

In the late 193Os, however, Levene
accepted the macromolecular structure
of all nucleic acids, which by then had
been firmly established by ultracentrifw
gation and dialysisexperiments. Still, the
repetitive occurrence of tetranucleotide
units in these macromolecules persisted
in the mind of many investigators of that
period. J. A. wtkowskiZ6 recently has
reviewed the reasons for such simplifying
concepts of macromolecular structure of
nucleic acids as well as proteins.
Numerology, psychology and inadequate
analytical data played a role.

Organic chemistry also played a key
role in other developments of nucleic
acid research. As described recently by J.
Brachet*`, the production of special
stains and the development of specific
color reactions greatly aided progress in
nucleic acid cytochemistry. Thus, during
the second quarter of this century, cell
biologists showed an increasing interest
in nucleic acids and lifted them from the
status of biochemical oddities. A fortu-
nate interplay among scientific disci-
plines resulted.

The beginnings of a new era

The tit unequivocal demonstration of
a specific biological activity of DNAwas
provided in 1944, by 0. T Avery, C. M.
MacLeod and M. McCarty?s. They suc-
ceeded in demonstrating that the trans-
forming principle isolated from smooth
cultures of pathogenic Pnewmxocc~ is a
specific deoxyribonucleic acid. Recogni-
tion of their results, however, was not
immediate. As described by M.
McCarty*9, one of the first comprehen-
sive presentations of the data was given
by him at an exclusive meeting of top
scientists, among them seven Nobel
Laureates, and a small group of younger
scientists. Unfortunately, the proceed-
ings of this conference in 1945, at the
resoti of Hershey, Pennsylvania, were
not published. I remember the excellent
presentation given by Maclyn McCarty;
unfortunately, it did not arouse much


excitement at the time. One of the NeS-  tions of DNA and RNA. However,     143-151
tars of the group, Linderstrtim-Lang.  chemistry continued to provide valuable   1.3 K"sre1.A. (1X85) Chern. Rm 18. 1928-1930
restricted his summarizing remarks  contributions.The most important -now  14 Fischer. E. (1X97) Ckm. Ber 30. **21r2*s1
about promesS eS%ntiaUy fo the mysteries historical - account of this transition   1s Kosrel.A, and Newnan". A. (,X94) Cho,,. Ber
                                                                    ~- n,E l-_ll

_

of proteins. He bemoaned theircdmplex-
ity and stated that the primary structure
of proteins probably never could be
resolved, and that synthetic efforts at
best would lead 10 caricatures of the cel-
lular products. Proteins preoccupied the
affection of most investigators at that
time to such an extent that their surmized
role as carriers of genetic information
was not readily abandoned'g.&?th Some
delay, however, the DNA experiments
were extended, and other transforma-
tions were found. DNA became very
nnnular.

period is the compendium on Nudeic
Acids, Chem&rry and Biology, edited by E.
Chargaff and J. N. Davidson~". In its 46
chapters nearly all contributors to the
progress of that period are represented.
Asimilar effort now (three decades later)
would involve many hundred con-
tributors and result in a treatise filling a
small library.

  i /, LLlP`L`L
16 Pi""W.A. (IRXS) Chrm. Bw 1% 795-763
17 Bergmann. M.. Schorte. 13. and Lechinsky.W
(1922) Chm Ber 53. 1Sic172

r-r~~~~
The effects of E. Chargaff to elucidate   ,-j'                  24 Levene, P. A. and Bass. L. w. (1931) NUCki<
the base composition of DNA from vari-   3 Miescher. F (1X74) Chem. Bm Z 376379      Acids. Chem. Catalog
ous species led to the recognition of indi-  4 Piccard, 1. (1874) Chm Ber 7, 1714-1719     25 lipion. R. S. (1957) P, A. Levene (obituary)
viduality and of the A-T and G-C equiva-   5 Ahmann, R. (1X89) Arch. Ana PhysrOl    Adv. Carbohydr Chem. I?, I-I?
lence; from there it was only a short step    (Physid Aht.) s2`Ls36             26 Witkowrki. J.A. (19X5) Pm& Bkxhrm. SC;. IO.
                       6 Kossel. A. (1910) Nobel Leaures: Phy~io,, and     13e141
lo the double helix. The recognition of   Med. lw1-1Y*1. pp. 39245, Elrevicr      27 &ache,. J. (1987) 7rends Bk,ch~m~ SC;. 12.244
various kinds of RNA was not lone in   7 Miescher. F (Conenary) (1944) He/v. Phyriol.     246

coming; H. G. Khorana synthe&d    Ptuvmacol. `km, Suppl. u,           2x *very. 0. T.. MacLcod. c. M.. an* McCa*y.
polynucleotides, and the establishment   8 Kennawa): E. (lY52)Arr". Pi. x, 3YM97      M (1944)J Exp. Mrd, 71). 137-m
of the genetic code by M. W. Nirenberg   9 Kossel. A. (1882) Z. Phyriol. Chem. 7.742    29 McCarty, M. (1985) The ,iam,oming Principir.
                                ,n ,,n.pr R ,,X4hi4n                      Nnrtnn

was the crowning event.                   ,. . ..n. Chem. (Liebig, 59, s&6x
                                 11
The limelight now shifted to the ever   Schulz. E. and Bosshard. E. (1886) Z. Physiol.  30 Chargaff, E. and "avidwn. J. N. (edr) (1955:
                           Chem. 10,8(u(Y                 1YM) Nurluic Aridr, C:h<misq and Biulog, c.i
increasing studies of the biological func-  12 Schuke. E. (191~1911) Z. Phyiol. Chm 70.    ""ls,. Academic Pnx